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250-351 Administration of HA Solutions for(R) Windows using VCS 5.0

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250-351 exam Dumps Source : Administration of HA Solutions for(R) Windows using VCS 5.0

Test Code : 250-351
Test denomination : Administration of HA Solutions for(R) Windows using VCS 5.0
Vendor denomination : Symantec
real questions : 253 real Questions

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Symantec Administration of HA Solutions

Symantec's Veritas Cluster Server 5.0 for VMware ESX provides extravagant Availability and calamity restoration for physical and digital Server Environments | killexams.com real Questions and Pass4sure dumps

source: Symantec

November 07, 2006 08:00 ET

Veritas Cluster Server for VMware ESX Simplifies Cluster Administration and Automates Failover for VMware digital Servers throughout Heterogeneous Networks

CUPERTINO, CA -- (MARKET WIRE) -- November 7, 2006 -- Symantec Corp. (NASDAQ: SYMC) these days unveiled Veritas™ Cluster Server (VCS) 5.0 for VMware ESX, bringing towering availability and catastrophe recovery to heterogeneous records facilities operating digital server utility. VCS for VMware ESX automates remote failover for calamity healing and offers management of clustered digital and physical servers. ultimate to avoid downtime in case of application, digital computer, community link, or server failures, VCS for VMware ESX centralizes cluster administration in a separate ESX server or throughout a campus or WAN. VCS is a key component of Veritas Server foundation, a set of items which permits enterprise shoppers to discover in ingredient what is operating on the servers in their statistics middle, actively manage and administer those servers, and corroborate that mission censorious purposes running on those servers are every the time obtainable. Symantec could be demonstrating VCS for VMware ESX on the VMworld 2006 convention being held in la this week.

"VMware directors are seeking tools that not simplest automate calamity recovery but aid them reduce the vulnerabilities associated with operating distinctive digital servers on the identical physical server," renowned Poulomi Damany, director of product management for Symantec's information heart management group. "Veritas Cluster Server for VMware ESX solves these issues with the aid of combining catastrophe restoration and towering availability, and consolidating control of each digital and actual servers and their dependencies."

VCS for VMware ESX complements Symantec's clustering solutions for windows, Linux and UNIX platforms. Symantec is the market chief in move-platform server clustering, based on the 2006 edition of the IDC international Clustering and Availability application record(1). With brought assist for VMware ESX, the market's most widespread digital server platform, VCS for VMware ESX gives a separate retort to consolidate management of VMware virtual servers in heterogeneous records core environments.

comprehensive extravagant Availability and calamity recuperation

VCS for VMware ESX gives towering availability and catastrophe healing for actual and digital servers. by using simplifying and automating far flung failover for VMware digital server environments, VCS for VMware ESX gives introduced insurance procedure against digital computer or application disasters, together with:

-- software and useful resource monitoring, as well as server monitoring, which offers an improved degree of availability; -- automatic healing from application, community storage, virtual useful resource, virtual server, and actual server disasters; -- Centralized management of virtual and actual resources and servers from a separate console; -- complete trying out for catastrophe recuperation integrating both utility failover and statistics replication to enable corporations to test catastrophe recuperation with out disrupting construction environments. "because it managers are attempting to rein in server sprawl and enhance aid utilization throughout the business, they are confronted with the problem of deploying diverse data availability and administration solutions to manage and give protection to an ever-starting to be population of virtual servers," talked about Brian Babineau, Analyst, business mode neighborhood. "With VCS for VMware ESX, Symantec has simplified the task for VMware valued clientele by course of proposing a separate platform that can avoid downtime of mission essential purposes working in digital and actual server environments throughout any distance and any platform."

New waiton for VMware ESX

VCS for VMware ESX additionally allows for customers to maximize the advanced points of VMware through recognizing and seamlessly interoperating with VMware's VMotion and disbursed resource Scheduler (DRS). If a virtual laptop is moved from one server to an additional for planned protection the usage of VMotion, the coast can be recognized via VCS and VCS will remove the essential motion to update the cluster fame for this reason. it's moreover compatible with allotted aid Scheduler (DRS), VMware's workload optimization characteristic.

computerized catastrophe restoration trying out

exciting to VCS is fire Drill, a characteristic of VCS that provides an delivered layer of protection for digital servers. With fireplace Drill, organizations can examine their catastrophe recuperation procedure and configuration without impacting the creation environment. In virtual environments the station server places exchange frequently, fireplace Drill helps array screen and tune mobile servers, their configuration and dependency hyperlinks.

rate and Availability

Veritas Cluster Server for VMware ESX is scheduled to be launched within the first quarter of 2007. Pricing for VCS for VMware ESX starts at $1,995 per server.

About Symantec

Symantec is the world chief in presenting solutions to champion individuals and companies assure the security, availability, and integrity of their counsel. Headquartered in Cupertino, Calif., Symantec has operations in forty countries. greater assistance is obtainable at www.symantec.com.

(1) IDC, global Clustering and Availability software 2005 dealer Shares, Doc #203676, October, 2006

word TO EDITORS: if you want additional information on Symantec enterprise and its products, please visit the Symantec intelligence latitude at http://www.symantec.com/information. every prices referred to are in U.S. dollars and are sound only within the united states.

Symantec and the Symantec brand are emblems or registered emblems of Symantec company or its associates in the U.S. and other nations. different names can be logos of their respective owners.


DialogicONE three.0 offers visible carrier introduction and speedy Prototyping, permits Acceleration of CSP IoT options | killexams.com real Questions and Pass4sure dumps

Dialogic, a cloud-optimized functions and infrastructure options company for service suppliers, corporations, and builders, introduced these days the prevalent availability of DialogicONE 3.0, an immense unlock of the company’s utility integration and orchestration platform, adding a number of chopping-part elements that enable service providers to generate functions at a rapid tempo with IoT platforms and for 5G networks.

Two mainly massive features were delivered with the 3.0 unencumber visual carrier introduction and speedy Prototyping. each enable non-developers to capitalize entry to tools for innovation and introduction of unique purposes in hours and days, as adverse to weeks or months.

visible provider introduction permits integration with any API and development of services running consolidated facts fashions, through applying suggestions and movements to create common sense and circulation of triggers in an experience-pushed retort architecture.

fast Prototyping, built-in with visual carrier introduction, makes it viable for convenient creation of aboriginal cellular purposes linked to capabilities working on the DialogicONE platform. iOS and Android mobilephone and pills are supported for administration of proof of ideas, prototypes, and affliction solutions.

“visual carrier advent and hasty Prototyping in DialogicONE three.0 are key milestones in their imaginative and prescient to provide agile, contemporary, scalable, and extremely creative construction and construction environments for provider suppliers around the globe,” pointed out Peter Kuciak, neighborhood vice chairman of DialogicONE. “Our goal is to be a ample enabling platform for provider suppliers in search of acceleration of current options as they set up IoT capabilities and 5G networks.”

DialogicONE (dialogic.com/one) offers a unique platform to transform many carrier provider departments into self serving innovation hubs by course of giving every and sundry entry to visual intuitive tools that can deliver current ideas to lifestyles in days and at a fraction of budgets in the past required to construct affliction options with groups of developers.

“Dialogic’s tradition lies in state-of-the-art tools that helped energy the VoIP revolution and the advent of numerous provider provider options,” talked about bill Crank, CEO of Dialogic. “DialogicONE continues this legacy into next-era technologies corresponding to IoT, AI, and AR/MR, plus allows carrier suppliers to readily test many current concepts for the 5G networks being deployed by means of Tier1 service providers everywhere.”

About Dialogic

Dialogic (dialogic.com) is a leading cloud-optimized options issuer for real-time communications media, purposes, and infrastructure to provider suppliers and developers world wide. based in Parsippany, NJ with workplaces international, Dialogic helps forty eight of the world’s proper 50 mobile operators, and practically 1,000 software developers construct and install on agile networks.

Dialogic and DialogicONE are both registered logos or logos of Dialogic business enterprise or a subsidiary thereof (“Dialogic”). other emblems mentioned and/or marked herein belong to their respective homeowners.


Symantec proclaims Integration of recent Anti-spam know-how in Mail safety and Antivirus enterprise edition solutions | killexams.com real Questions and Pass4sure dumps

Symantec publicizes Integration of current Anti-junk mail technology in Mail safety and Antivirus commercial enterprise edition solutions

CUPERTINO, Calif. -- December 15, 2004 -- Symantec Corp., a leader in tips security, today introduced Symantec top rate AntiSpam, an not obligatory add-on subscription provider for Symantec Mail security and Symantec AntiVirus commercial enterprise edition clients, scheduled to be available this month. Symantec premium AntiSpam, powered via Brightmail technology and response, offers most beneficial-of-breed spam prevention devoid of requiring extra administration or application setting up.

raising the bar for commercial enterprise spam prevention, Symantec premium AntiSpam add-on subscription provider provides the optimal aggregate of effectiveness and accuracy in the market nowadays. Multi-layered spam prevention leverages greater than 20 filtering applied sciences, including unsolicited mail signatures, heuristics, popularity filters, language identification and proprietary strategies, with automated updates happening inside each 10 minutes. The service allows for organizations to obtain extravagant detection rates of 95 percent and the industry’s highest accuracy charge towards erroneous positives (ninety nine.9999 p.c), enabling clients to securely delete junk mail with out review. No current software or hardware is required, and the service requires no further IT administration or tuning once deployed.

“Symantec top class AntiSpam showcases Symantec’s ongoing commitment to offering its consumers with essentially the most resourceful and technically expert mail safety options available on the market,” mentioned Enrique Salem, senior vice president, community and gateway solutions, Symantec Corp. “Symantec premium AntiSpam is a transparent demonstration of Symantec’s skill to directly integrate market-main expertise, obtained from Brightmail, into their existing product line.”

Symantec Mail protection for Domino™, Symantec Mail security for Microsoft® exchange and Symantec Mail safety for SMTP supply excessive-performance, integrated mail insurance policy against virus threats, unsolicited mail, and other unwanted content. Symantec’s award-successful extensible NAVEXTM antivirus expertise defends in opposition t current and regular viruses, while attachment and discipline line blockading supply hour-zero responses against rising threats. Symantec Mail security options consist of spam tools comparable to antispam heuristics, custom filtering rules, assist for third-birthday celebration real-time blacklists, as well as customized allow and blocks lists.

Symantec AntiVirus enterprise edition, which includes the Symantec Mail protection product line, gives virus insurance plan, content filtering, and spam prevention for the groupware server and gateway, and virus insurance procedure for the laptop in a single, handy-to-install answer. It combines award-winning technologies and Symantec’s international response infrastructure to give advantageous coverage at every community tier.

Availability

Symantec top rate AntiSpam might be obtainable in December 2004 and bought on a subscription model via Symantec’s global network of price-brought licensed resellers, distributors and programs integrators. organizations may moreover be connected with Symantec’s resellers and distributors of their areas by using travelling the Symantec retort company locator at https://partnernet.symantec.com/internet/locator/Locator01.asp.

About Symantec

Symantec is a worldwide leader in guidance protection offering a wide latitude of software, appliances and functions designed to waiton people, petite and mid-sized organizations, and big firms secure and maneuver their IT infrastructure. Symantec's Norton company of products is a pacesetter in customer protection and issue-fixing solutions. extra advice is attainable at http://www.symantec.com.

Symantec and the Symantec brand are emblems or registered emblems, within the united states and likely other international locations, of Symantec corporation. extra company and product names can be logos or registered logos of the individual businesses and are respectfully recounted.


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Administration of HA Solutions for(R) Windows using VCS 5.0

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RIM introduces BlackBerry Mobile Fusion - The Next Generation Enterprise Mobility Solution for BlackBerry, Android and iOS | killexams.com real questions and Pass4sure dumps

RIM

RIM

 

Press Release 

RIM Announces BlackBerry Mobile Fusion - The Next Generation Enterprise Mobility Solution for BlackBerry, Android and iOS Smartphones and Tablets 

Simplifies Management of Smartphones and Tablets for business and Government

Waterloo, ON - Research In Motion (RIM) (NASDAQ: RIMM; TSX: RIM) today introduced BlackBerry® Mobile Fusion - the Company's next-generation enterprise mobility solution and RIM's entry into the multi-platform Mobile Device Management (MDM) marketplace. structure on years of leading enterprise mobility management solutions from RIM, BlackBerry Mobile Fusion will simplify the management of smartphones and tablets running BlackBerry®, Google® Android® and Apple® iOS® operating systems.

"We are pleased to interlard BlackBerry Mobile Fusion - RIM's next generation enterprise mobility solution - to get it easier for their business and government customers to manage the diversity of devices in their operations today," said Alan Panezic, Vice President, Enterprise Product Management and Marketing at Research In Motion. "BlackBerry Mobile Fusion brings together their industry-leading BlackBerry Enterprise Server technology for BlackBerry devices with mobile device management capabilities for iOS and Android devices, every managed from one web-based console. It provides the necessary management capabilities to allow IT departments to confidently oversee the expend of both company-owned and employee-owned mobile devices within their organizations."

RIM is the leading provider of enterprise mobility solutions with over 90 percent of the Fortune 500 provisioning BlackBerry devices today. The enterprise market for smartphones and tablets continues to grow in both the company-provisioned and employee-owned (Bring Your Own Device or BYOD) categories. BYOD in particular has led to an extend in the diversity of mobile devices in expend in the enterprise and current challenges for CIOs and IT departments as they struggle to manage and control wireless access to confidential company information on the corporate network. This has resulted in increased require for mobile device management solutions.

BlackBerry Mobile Fusion brings together the market-leading BlackBerry® Enterprise Server (version 5.0.3) for BlackBerry smartphones; current management capabilities for BlackBerry PlayBook tablets built on BlackBerry Enterprise Server technology; and mobile device management for smartphones and tablets running Android and iOS operating systems.

BlackBerry Mobile Fusion will provide the following mobile device management capabilities for every supported mobile devices*:

• Asset management• Configuration management• Security and policy definition and management• Secure and protect lost or stolen devices (remote lock, wipe)• User- and group-based administration• Multiple device per user capable• Application and software management• Connectivity management (Wi-Fi®, VPN, certificate)• Centralized console• towering scalability

BlackBerry smartphones will continue to capitalize from the many advantages of the end-to-end BlackBerry solution including the same advanced IT management, security and control available with BlackBerry Enterprise Server 5.0.3, which is fragment of BlackBerry Mobile Fusion. These advanced features embrace BlackBerry® BalanceTM technology supporting the expend of a separate device for both drudgery and personal purposes without compromising the organization's requisite to secure, manage and control confidential information; over 500 IT policies; over-the-air app and software installation and management; towering availability; and much more. BlackBerry Mobile Fusion will moreover interlard current self-service functionality for employees to secure lost or stolen BlackBerry smartphones and BlackBerry PlayBook tablets.

BlackBerry Mobile Fusion is currently in early beta testing with select enterprise customers. RIM is now accepting customer nominations for the closed beta program which will start in January, and general availability is expected in late March.

For more information, visit www.blackberry.com/mobilefusion.

* Device security, manageability and controls will continue to vary according to the inherent capabilities of the individual device operating systems.

About Research In Motion

Research In Motion (RIM), a global leader in wireless innovation, revolutionized the mobile industry with the introduction of the BlackBerry® solution in 1999. Today, BlackBerry products and services are used by millions of customers around the world to tarry connected to the people and content that matter most throughout their day. Founded in 1984 and based in Waterloo, Ontario, RIM operates offices in North America, Europe, Asia Pacific and Latin America. RIM is listed on the NASDAQ Stock Market (NASDAQ: RIMM) and the Toronto Stock Exchange (TSX: RIM). For more information, visit www.rim.com or www.blackberry.com.


Architecture of the complete oxygen-sensing FixL-FixJ two-component signal transduction system | killexams.com real questions and Pass4sure dumps

Signal relay through a two-component system

In two-component systems, a sensor histidine kinase undergoes autophosphorylation and transfers a phosphate group to its cognate response regulator, which then mediates cellular responses by binding to DNA, performing enzymatic reactions, or interacting with other proteins. In the FixL-FixJ two-component system of the plant root nodule symbiont Bradyrhizobium japonicum, the histidine kinase FixL undergoes autophosphorylation only when it is not bound to oxygen. This ensures that its cognate response regulator FixJ stimulates the expression of genes required for nitrogen fixation only under low-oxygen conditions. Wright et al. combined high- and low-resolution structural analyses with modeling techniques and functional analysis to generate a model of signal relay through the FixL-FixJ two-component system. The model shows how the dissociation of oxygen from FixL stimulates FixL autophosphorylation and phosphotransfer from FixL to FixJ.

Abstract

The symbiotic nitrogen-fixing bacterium Bradyrhizobium japonicum is censorious to the agro-industrial production of soybean because it enables the production of towering yields of soybeans with slight expend of nitrogenous fertilizers. The FixL and FixJ two-component system (TCS) of this bacterium ensures that nitrogen fixation is only stimulated under conditions of low oxygen. When it is not bound to oxygen, the histidine kinase FixL undergoes autophosphorylation and transfers phosphate from adenosine triphosphate (ATP) to the response regulator FixJ, which, in turn, stimulates the expression of genes required for nitrogen fixation. They purified full-length B. japonicum FixL and FixJ proteins and defined their structures individually and in knotty using small-angle x-ray scattering, crystallographic, and in silico modeling techniques. Comparison of vigorous and dormant forms of FixL suggests that intramolecular signal transduction is driven by local changes in the sensor domain and in the coiled-coil region connecting the sensor and histidine kinase domains. They moreover institute that FixJ exhibits conformational plasticity not only in the monomeric situation but moreover in tetrameric complexes with FixL during phosphotransfer. This structural characterization of a complete TCS contributes both a mechanistic and evolutionary understanding to TCS signal relay, specifically in the context of the control of nitrogen fixation in root nodules.

INTRODUCTION

Two-component systems (TCSs) are widely distributed in bacteria, fungi, and higher plants. They facilitate cellular adaptation in response to environmental change and are considered superior targets for the development of novel antibiotics and plant growth modulators because of their conspicuous absence in metazoans (1–3). TCSs are generally composed of two types of multidomain proteins: sensory histidine kinases (HKs) and response regulators (RRs). The TCSs can be classified into three classes, based on their domain architectures. Class I HKs consist of an N-terminal stimulus-specific sensor domain and a C-terminal HK module. The latter comprises the dimerization and histidine phosphotransfer (DHp) and catalytic adenosine triphosphate (ATP)–binding (CA) domains. Class II HKs, which are specific for chemotaxis (4, 5), contain an N-terminal histidine-containing phosphotransfer (HPt) domain and a C-terminal HK module. Class III HKs gain features of both class I and class II HKs, combining the class I HK’s sensor domain and the HPt domain and HK module of the class II HKs (5, 6). RRs contain a conserved N-terminal receiver (REC) domain, which is connected to diverse C-terminal effector domains. In response to an environmental stimulus sensed by the HK sensor domain, the CA domain catalyzes the autophosphorylation of a specific histidine residue in the DHp (class I) or HPt (class II and class III) domain. That phosphoryl group is subsequently transferred to a conserved aspartate residue in the REC domain of the cognate RR. This phosphotransfer activates the RR to promote DNA or RNA binding, enzymatic reactions, or protein interactions that are mediated by the C-terminal effector domain (7). When autophosphorylation activity is turned off, HKs often act as a phosphatase of their cognate phosphorylated RRs (8), thus contributing to shutting down the pathway.

The key questions of TCS signaling are how HKs are activated by stimuli and how they interact with the RRs. The answers to these questions gain been hampered by the need of molecular- and atomic-level structural information on intact, full-length HKs in both the kinase-active and kinase-inactive forms and in knotty with RRs. Here, they narrate the structural characteristics of the oxygen (O2)–sensing FixL and FixJ (FixL-FixJ) TCS of the rhizobium species Bradyrhizobium japonicum, a root nodule, nitrogen-fixing bacterium that forms symbiotic relationships with leguminous plant such as soybean. B. japonicum FixL is a class I HK that senses the O2 tension in the cytoplasm through a heme-containing Per-Arnt-Sim (PAS) domain and transfers phosphate from ATP by sequential autophosphorylation and phosphotransfer reactions in its C-terminal effector modules to the RR FixJ (Fig. 1). O2 association to and dissociation from the heme-PAS domain of FixL trigger intra- and intermolecular signaling mechanisms such that the deoxy (O2-unbound) form of FixL is vigorous for autophosphorylation and phosphotransfer, whereas the oxy (O2-bound) form does not undergo autophosphorylation and exhibits phosphatase activity toward FixJ (9, 10). As a result, the rhizobial FixL and FixJ system stimulates the expression of genes required for nitrogen fixation only when O2 concentrations in the plant root nodules are low because the vigorous heart of nitrogenase is O2-labile (11).

Fig. 1 Schematic representation of the domain structures of full-length and truncated versions of B. japonicum FixL and FixJ.

Full-length Bradyrhizobium japonicum FixL comprises two N-terminal Per-Arnt-Sim (PAS) domains, PAS-A and PAS-B, and C-terminal dimerization and histidine phosphotransfer (DHp) and catalytic adenosine triphosphate (ATP)–binding (CA) domains. His200 in the PAS-B domain is censorious for binding to heme; His291 in the DHp domain is the site of autophosphorylation; and Asp431-Val467 of the CA domain constitutes the ATP-binding site. Full-length B. japonicum FixJ contains an N-terminal receiver (REC) domain and a C-terminal effector domain that binds to DNA. FixJ is activated by FixL-mediated phosphorylation at Asp55. Structures of the truncated FixL and FixJ proteins FixLPAS-PAS and FixJN used in this study are indicated. The residues that define the boundaries of these domains are noted. Domain structures were generated using the SMART appliance (http://smart.embl-heidelberg.de/).

Although most TCS HKs, including FixL homologs from most other species, are integrated into the membrane for sensing extracellular stimuli, B. japonicum FixL is a water-soluble, cytoplasmic sensor. The difficulty of purifying integral membrane TCS HKs in combination with their multidomain configurations and structural flexibility has impeded structure-function studies. Thus, every previous structural studies of TCS HKs, and even those of FixL, were performed with truncated, rather than full-length, proteins. They gain isolated full-length B. japonicum FixL and FixJ proteins at towering purity and obtained structural information on FixL in both the kinase-active (deoxy) and kinase-inactive (oxy) forms, FixJ, and the FixL-FixJ knotty by combining size exclusion chromatography–integrated small-angle x-ray scattering (SEC-SAXS), x-ray crystallography, and molecular modeling techniques. This analysis provides insights into how microorganisms and plants conform to environmental change at the molecular flat and elucidates details of a microbial signaling pathway that facilitates the agro-industrial production of soybeans for human food, livestock feed, and biofuel with only limited requisite for nitrogenous fertilizers.

RESULTS Autophosphorylation and phosphotransfer activities of full-length FixL and FixJ

We prepared recombinant full-length B. japonicum FixL and FixJ with very towering purity (fig. S1, A and B). B. japonicum FixL is a naturally occurring soluble FixL, in contrast to membrane-anchored FixLs, such as that from Sinorhizobium meliloti. B. japonicum FixL comprises N-terminal tandem PAS domains, PAS-A and PAS-B, followed by C-terminal DHp and CA domains (Fig. 1). The PAS-B domain senses O2 through a heme b cofactor (10, 12). The role of the N-terminal PAS-A domain in B. japonicum FixL is not clear, but some biological studies of the water-soluble FixL from Rhizobium etli intimate that the PAS-A domain influences the oxygen affinity of the heme in the PAS-B domain (13).

We tested the phosphotransfer activity of their highly purified full-length FixL in several heme iron oxidation and ligation states. These included oxy (Fe2+-O2), deoxy (Fe2+), ferric cyanide–bound (cyanomet; Fe3+-CN−), and ferric ligand–free (met; Fe3+) forms (fig. S2, A and B). Phosphotransfer from FixL to FixJ was suppressed in the oxy and cyanomet forms, whereas the activities of these reactions were fully restored in the deoxy and met forms (Table 1). These results expose that their preparation of full-length FixL and FixJ was successful and that signal transduction by the FixL and FixJ system could be controlled by ligand (O2 or CN−) binding to the sensor domain of FixL, irrespective of the ferrous or ferric oxidation situation of the heme iron. Because of the low affinity of O2 for FixL (Table 1) and relatively hasty autoxidation (τ1/2 ~ 15 min) of oxy FixL (12), they used the met and the cyanomet forms of the full-length FixL as analogs for the deoxy and oxy states, respectively, as samples for the SAXS measurements. They moreover institute that CN− binding to the heme iron of the FixL sensor domain in the met form suppressed the autophosphorylation activity (fig. S3A) but promoted the phosphatase activity toward phosphorylated FixJ (fig. S3B). On the basis of these experimental data, they expected that the tertiary and quaternary structures of the full-length FixL are equivalent between ferric CN−– and ferrous O2–bound forms.

Table 1 Phosphotransfer activities of full-length FixL to FixJ by ATP-NADH coupled assay (42). Molecular architecture of FixL

Very petite amounts of aggregated protein can adversely influence SAXS measurements (14). Despite the towering monodispersity of full-length B. japonicum FixL, petite amounts of aggregation often resulted in indigent SAXS data from static measurements (table S1). To overcome this effect, they performed SEC-SAXS, where the data collection was performed in-line with protein purification, both at the RIKEN beamline BL45XU (15) at the SPring-8 synchrotron in Japan and at the sway beamline (16) at the SOLEIL synchrotron in France. The SECs of FixL in the met form with SAXS parameters plotted together with absorbance at 280 and 398 nm showed that the aggregated species were eliminated before the SAXS measurements (fig. S4A). Using this method, they obtained high-quality SAXS data from full-length FixL in both the met and the cyanomet forms. The plot of log q versus log I(q) obtained by SEC-SAXS at BL45XU (Fig. 2A) and the SEC-SAXS parameters are summarized in Table 2 and table S2. The data measured at SPring-8 and SOLEIL showed a towering degree of reproducibility. Radii of gyration (Rg) of 49.7 ± 0.1 Å and 48.4 ± 0.2 Å for met and cyanomet FixL, respectively, were lower than those collected by static SAXS measurements (Table 2 and table S2) and reflect the capacity of SEC-SAXS to seclude scattering from the species of interest. Molecular mass estimations from experimental SEC-SAXS data call protein masses of 140.1 ± 1.9 kDa and 136.3 ± 2.3 kDa for the met and cyanomet forms, respectively, indicating that FixL is homodimeric in solution. These observations are consistent with the structural characteristics of other HKs, in which two helices of the DHp domain from each monomer interact to form a stable four-helix bundle in the homodimer (17, 18). The distance distribution function, P(r), for each FixL situation gave information on the maximum dimension (Dmax) and the mediocre electron distribution (Fig. 2B). The Dmax was calculated at 163 ± 4 Å and 158 ± 3 Å for the met and cyanomet forms, respectively. CN− binding to the heme group of FixL suppressed the kinase activity, which was reflected in a slight abate in Rg (48.4 Å versus 49.7 Å; Table 2) and Dmax (163 Å versus 158 Å; Table 2).

Fig. 2 SEC-SAXS profiles of full-length FixL, FixJ, and FixL-FixJ complexes.

(A) Log-log plots of x-ray scattering for the met and cyanomet forms of full-length FixL, full-length FixJ, the FixL-FixJ complex, and the FixL-FixJN complex, q = 4πsinθ/λ, where 2θ is the scattering angle and λ is the wavelength of incident x-rays. I(q) is the measured scattered intensity at a given value of 2θ. (B) Corresponding pair distance distribution functions for the indicated proteins and protein complexes. P(r) is the frequency of r intramolecular distances. (C) Dimensionless Kratky plots, which compare the compactness of a protein, for full-length FixL (met form), the truncated FixLPAS-PAS (met form), full-length FixJ, and the full-length FixL-FixJ complex. qRg = q, as defined above, multiplied by the radius of gyration of the protein. A compact protein has a peak maximum at √3 and 1.2 on the abscissa and ordinate, respectively. Movement of the peak further into the positive quartile of the graph indicates unfolding and conformational flexibility. Data were collected on the BL45XU beamline at the SPring-8 synchrotron. n > 3 independent protein preparations and data collections.

Table 2 Structural parameters for FixL and FixL-FixJ complexes determined by SEC-SAXS experiments at BL45XU in SPring-8.

Using the FixL SEC-SAXS data, they constructed an ab initio model of full-length FixL in the met state. FixL exhibits an extended and club-like form (fig. S5, A and B). Correspondingly, dimensionless Kratky plot analysis of full-length FixL describes a protein that does not adopt either a compact or globular conformation (Fig. 2C). For comparison, they moreover collected SEC-SAXS data for a truncated form of FixL that contains only the PAS-A and PAS-B domains (FixLPAS-PAS; Fig. 1 and fig. S6, A and B). The Kratky plot analysis of FixLPAS-PAS is much different from that of full-length FixL and shows a towering degree of globularity (Fig. 2C). Using this information as a guide, they constructed a pseudoatomic model of full-length FixL, incorporating a combination of previously published crystallographic domain structures [Protein Data Bank (PDB) identifiers (IDs): 3MR0 for PAS-A domain, 1DRM (9) for PAS-B domain, and 4GCZ (19) for HK module], homology modeling, a priori structure prediction, and refinement against the SAXS data, and compared this model with the space-filling model (Fig. 3A). The length of the dimer axis in the model is identical to the experimentally determined Dmax value (Table 2), and its calculated SAXS profile (fig. S7, A and B) has a χ value of 2.57 when compared with the experimental data, indicating the internal consistency of their proposed model and that it is a reasonable snapshot of FixL in solution.

Fig. 3 Space-filling and pseudoatomic models of full-length FixL.

SAXS-based models of the met form of full-length FixL showing the overall shape and domain arrangement.

Our model for FixL displays an elongated structure in which the tandem PAS domains (PAS-A-A and PAS-B-B) and the DHp domains homodimerize. There is no interaction between the PAS-A dimer and PAS-B dimer or between either the PAS-A or PAS-B dimer and the CA domain. They note that the PAS-B sensor domain connects with the kinase module only through a coiled-coil linker region. This architecture of full-length FixL is comparable to an “in-line” model proposed for transmembrane and membrane-associated HKs (17, 18, 20).

Our constructed model has an asymmetric DHp domain (Fig. 3A). To test the possibility that FixL homodimers gain a symmetric four-helix bundle, they assembled models of FixL based on the HKs VicK [PDB ID: 4I5S (21)] (fig. S8A), DesKC [PDB ID: 3GIE (22)] (fig. S8B), and CckA [PDB ID: 5IDJ (23)] (fig. S8C). This approach did not bow a better lucky to the experimental data than that provided by their proposed structure of intact, asymmetric full-length FixL.

Phosphotransfer activities of FixL mutants

In their proposed model, they institute that the sensor PAS-B domains are followed by the coiled-coil linker region and then the four-helix bundle, but there is no direct interaction between the PAS-B and the catalytic kinase domains. From this observation, as one of the viable mechanisms for intermolecular signal transduction of the O2 sensor FixL, they could propound that conformational change in the PAS-B domain is propagated to the DHp domain through the coiled-coil linker region (see Discussion). To examine the weight of the coiled-coil linker region in transducing the signal from the sensor domain to the kinase domain to stimulate autophosphorylation, they prepared 12 proteins presence substitution mutations of the coiled-coil region by site-directed mutagenesis (R254A, T257A, E258A, E258Q, Q261A, T262A, T262S, Q263A, R265A, L266P, Q267A, and L269P) and measured their autophosphorylation activities in both the met and cyanomet forms (fig. S9, A and B). Except for the E258Q mutation, which was tolerated and responded to the CN− binding, each mutation significantly reduced the phosphorylation activity in the met form, indicating that the dimerization of the coiled-coil region might be answerable for activation of the kinase activities of the HK module in FixL. In addition, they moreover renowned that the activity of R254A mutant FixL showed a fivefold lower activity in the met form and temper impairment in the cyanomet form. The Arg254 residues on the coiled-coil helices near the discontinuance of the PAS-B domains might form a hydrogen bonding interaction between the helices to yoke CN− binding to kinase activity (fig. S9).

Crystal and solution structures of full-length FixJ

One of the most intriguing features of TCSs is the intermolecular communication that facilitates the transfer of a phosphoryl group from the kinase domain to the RR. They analyzed full-length FixJ lonely with SEC-SAXS and crystallographic techniques. Full-length FixJ was crystallized in space groups C2221 (one chain in the asymmetric unit) and P212121 (five chains, A to E, in the asymmetric unit) (Fig. 4, A and B, and table S3). Crystallographic analyses revealed that FixJ is a two-domain protein, comprising an N-terminal α/β-type REC domain with a phosphorylation site (Asp55) and a C-terminal every α-type effector domain with a helix-turn-helix DNA binding motif connected by a helical linker (Fig. 4A). The N-terminal REC domain and the C-terminal effector domain each exhibits the same fold as those reported for other DNA binding RRs (24). The linker region contained two α helices (α6A and α6B) in the C2221 data. Chains A to D in the P212121 data represented similar overall conformations with one another, whereas the chain E adopted a different conformation in the linker region compared to chains A to D. Helices α6A and α6B observed in the C2221 data were fused to a separate helix, α6, in the chains A to D of the P212121 data, whereas the helix α6A was transformed to a tight curve structure in the chain E of the P212121 data, indicating that the helix α6A is conformationally flexible. Structural comparison of FixJ crystal structures with those of other full-length RRs shows that the α6A helix in the linker region is labile and acts as a hinge enabling TCS RRs, including FixJ, to adopt a variety of overall molecular shapes.

Fig. 4 Crystal and SAXS solution structures of full-length FixJ.

(A) Crystal structure of full-length FixJ (the C2221 data) showing the relative positions of the N-terminal REC (pink), linker (cyan), and C-terminal effector (green) domains. The phosphorylation site, Asp55, is indicated as a stick model with carbon and oxygen atoms indicated in yellow and red, respectively. (B) Comparison of the crystal structures of FixJ in space groups C2221 (blue) and P212121 [yellow (chain A as a representative of chains A to D) and magenta (chain E)]. The view is in the same orientation as (A). (C) Space-filling and pseudoatomic models of full-length FixJ. The ribbon model was colored by temperature factors (b factors). Low and towering temperatures are represented in colder and warmer colors, respectively. (D) Comparison of FixJ crystallographic and SAXS models against experimental SAXS data. Experimental SAXS data are shown in black; the calculated scattering curve of the SAXS FixJ model in (C) is shown in red; the calculated scattering curves of the crystal structures of FixJ are shown in other colors as indicated. q = 4πsinθ/λ as defined in Fig. 2.

The SEC-SAXS parameters of B. japonicum FixJ (Table 2), that is, Rg = 22.3 ± 0.2 Å, Dmax = 66 ± 2 Å, and molecular mass = 24.5 ± 0.7 kDa, are comparable with those of the S. meliloti ortholog (25) and betoken that FixJ in the nonphosphorylated situation is monomeric in solution. An ab initio model constructed from experimental scattering data showed that FixJ in solution exhibits an ellipsoidal shape (Fig. 4C and Table 2), and dimensionless Kratky plot analysis (Fig. 2C) moreover indicated that FixJ does not comfort in an extended conformation in solution. No crystallographic conformer fitted the experimental SAXS data well, indicating that the mediocre conformation of FixJ in solution is not fully represented by the crystal structures. They were able to refine the FixJ structure against the SAXS data to create a pseudoatomic model that fits the experimental data with a χ value of 2.5 (Fig. 4D). In solution, the N-terminal REC domain and the C-terminal effector domain are able to fold back upon one another because of the kinked linking α helix, thus allowing a more compact structure. The estimated Dmax (66 Å) from the SAXS data is smaller than the longitudinal length of ~78 Å for a dumbbell-like shape of the crystal structures. This incompatibility may arise from the conformational flexibility in the linker region, which confers conformational plasticity in solution.

Solution structure of the full-length FixL-FixJ complex

To promote FixL-FixJ knotty formation, they performed SEC of FixL in a buffer containing 40 μM FixJ. This concentration is 10-fold greater than the FixL-FixJ dissociation constant (Kd), which has been reported as 0.8 to 4.0 μM in the absence of ATP analogs and Mg2+ (26). Under this condition, the FixL-FixJ knotty is expected to predominate over that of homodimeric FixL in solution. When the FixJ concentration in the loading buffer was reduced to 20 μM, the FixL-FixJ knotty was not well separated from homodimeric FixL by SEC.

FixJ-saturated FixL elutes earlier than FixL homodimers (fig. S4B). Under these conditions, they collected SEC-SAXS data for the FixL-FixJ knotty with the met form of FixL (Fig. 2). Size parameters were institute to be 53.1 ± 0.1 Å for Rg, 160 ± 5 Å for Dmax, and 185.4 ± 1.5 kDa for molecular mass (Table 2). The Rg value was larger than that of FixL alone, and the molecular mass was increased by 45.3 kDa, indicative of two molecules of FixJ (24.5 kDa) binding to each FixL homodimer. These parameters betoken that phosphotransfer from FixL to FixJ is facilitated through homodimeric FixL binding to two FixJ molecules to form a heterotetramer. In addition, the SAXS data did not betoken big domain rearrangements upon knotty formation, and dimensionless Kratky plot analysis (Fig. 2C) showed that the noncompact FixL conformation is conserved after forming a knotty with two molecules of FixJ.

On the basis of this observation, they docked two FixJ monomers onto their FixL pseudoatomic model (Fig. 3B). In this construction, the phosphodonor 3-phospho-His291 in the FixL DHp domain and the phosphoacceptor Asp55 of FixJ are within phosphotransfer distance (~3 Å). The overall lucky of this model to the experimental SAXS data is good, with a χ value of 2.71 (fig. S7B), thus validating the proposed model. To their knowledge, this is the first complete model of the knotty formed by an intact full-length sensor HK and its full-length cognate RR that mediates TCS transduction.

To compare the kinase-active FixL-FixJ knotty with the dormant complex, they moreover measured the SEC-SAXS of the knotty in the cyanomet form, whose parameters are shown in Table 2. Their values, especially Rg and Dmax, were the same as those of the vigorous met FixL-FixJ complex, suggesting that the ligand (CN−) binding to the PAS-B sensor domain of FixL, which suppressed the kinase activity, does not strongly influence the overall molecular shape of the FixL-FixJ complex.

In the proposed FixL-FixJ complex, the N-terminal REC domain of FixJ, as determined by their FixJN structure, fits well within the SAXS envelope model, whereas the C-terminal effector domain of FixJ, as determined by their structure of FixJC, does not. They hypothesize that, given the conformational flexibility of monomeric FixJ, the C-terminal domain is moreover able to adopt multiple conformations while in knotty with FixL due to the dynamic nature of the linker region observed in the crystallographic structure of FixJ (Fig. 4B). To test this possibility, they moreover measured and analyzed the SEC-SAXS of FixL complexed with FixJN. The SAXS parameters of the FixL-FixJN knotty showed that the Dmax (170 ± 3 Å) of this knotty is similar to that of the FixL-FixJ complex, whereas the molecular mass (153.6 ± 1.4 kDa) and Rg (48.9 ± 0.1 Å) values were smaller, as expected (Table 2). The absence of the FixJ C-terminal domain was reflected in the changes in mass and Rg but did not influence the maximum dimension of the FixL-FixJ complex. In addition, ab initio models of the FixL-FixJ and FixL-FixJN complexes were very similar (fig. S5B). These results intimate that the C-terminal domain of FixJ does not contribute to the FixL-FixJ interaction and is instead free to coast in solution.

DISCUSSION

Intra- and intermolecular signal transduction through TCSs occur in every domains of life except metazoans. These systems facilitate survival by allowing rapid adaptation to environmental changes. To understand the molecular mechanism of TCSs in detail, structural information about TCS HKs, RRs, and the complexes they form gain been generated. However, many structural studies on TCSs gain relied on breaking the protagonists down into biochemically tractable domains. Here, they gain investigated the structure of a complete TCS using the full-length forms of components of the B. japonicum O2-sensing system: FixL, FixJ, and the FixL-FixJ complex. The information gained for the kinase-active and kinase-inactive forms of full-length FixL and of full-length FixL in knotty with FixJ is particularly valuable because it enabled us to investigate the modular structures that facilitate signal relay in this agriculturally essential TCS. To validate their model of full-length FixL, which used the asymmetric four-helix bundle structure of the FixL DHp domain conjoined to a light sensor domain as a template (19), they created homology models of FixL based on other HK DHp domains. These structures exhibited consistently worse lucky to the experimental SAXS data than did the FixL model they proposed.

With respect to the intramolecular signal transduction from the sensor domain to the CA domain of FixL, two viable mechanisms gain been proposed so far. Sousa and co-workers proposed a “globular” model based on biochemical studies of the full-length, cytoplasmic R. etli FixL protein (13), which has a similar domain organization as B. japonicum FixL. In the R. etli FixL, interactions between this protein’s cytoplasmic PAS domains (PAS-A and PAS-B) in homodimers or between the PAS homodimers and the CA domain are colorable in the globular form. Therefore, it was proposed that changes to these interactions would be involved in the intramolecular signal transduction stimulated by the association of O2 with or dissociation from the sensor PAS domain. On the other hand, the crystal structure of the transmembrane Thermotoga maritima ThkA, the separate PAS domain of which exhibits 24% primary structure similarity to that of FixL PAS-B, displays no interaction between the PAS domains in the homodimeric form, but rather direct interaction of the PAS domains with the CA domains through hydrogen bonding (27). These models, proposing interactions between the PAS domain and the CA domain, were based on the assumption that the mechanism of the intramolecular signal transduction is not necessarily similar between the water-soluble and membrane-integrated TCS HKs. The former HK functions as a sensor of a stimulus in the cytoplasm, whereas the sensor domain of the later HK detects an extracellular stimulus and transfers information into the cytoplasm across the membrane for cellular adaptation. However, the proposed architectures of R. etli FixL and ThkA are incongruent with their present SAXS data of full-length B. japonicum FixL, deduced parameters, and low-resolution models. These structural inconsistencies intimate that neither the globular model nor the direct transfer of the signal from the sensor to the CA domains is possible.

Our results lead us to propound an in-line model for full-length FixL with dimerization through PAS-A–PAS-A, PAS-B–PAS-B, and DHp-DHp interactions, but no direct interaction of the heme-containing PAS-B domain with the CA domain (Fig. 3A). Their model of intact full-length FixL provides us with a structural basis from which to argue the molecular activation mechanism of TCS HK modules by autophosphorylation in response to stimuli. The full-length FixL met (active) and cyanomet (inactive) forms (Table 2) expose that the overall molecular conformation is not largely changed upon activation. Correspondingly, their SAXS envelope models are moreover unaltered by CN− binding (fig. S5A). Thus, it is likely that the conformational changes that control kinase activity after ligand binding are localized, causing subtle changes in the interaction between the sensor domain and the HK domain. They propound that, upon O2 dissociation from the heme iron in the PAS-B domain, a local conformational change in this domain, previously observed in high-resolution structures and spectroscopic characterization of FixL sensor domains (28–32), is propagated to the DHp domain through the coiled-coil linker region. These structural changes antecedent ATP bound to the CA domain and adjust the position of the His291 residue of DHp, resulting in phosphotransfer (Fig. 6).

Our thought was supported by the results of site-directed mutagenesis experiments, in which mutations in the coiled-coil linker region inhibited the phosphotransfer activity of FixL (fig. S9). The results are consistent with their previous drudgery on chimeric sensor proteins, in which the FixL PAS-B sensor domain was fused with the HK domain of T. maritima ThkA at the coiled-coil region (33). The kinase activities of the chimeric proteins were impaired, maintained, or enhanced by ligand binding, depending on where in the coiled-coil region the two functional domains were fused. every these results champion their proposal that the coiled-coil linker region between the PAS-B and DHp domains functions as a key modulator of the autophosphorylation activity of FixL.

This proposal, the so-called in-line mechanism, is comparable to those given from previous studies of membrane-integrated or membrane-associated TCS HKs such as VicK and DesKC. The structures of truncated forms of these HKs, in which the transmembrane region or the extracellular domain or both were deleted (18, 20–23, 34), were the basis of this proposal. However, there was no structural comparison between the kinase-active and kinase-inactive forms of either. The only full-length structure of an HK in the literature is an engineered, chimeric HK called YF1, which is vigorous in the dismal and dormant by stimulation with blue light (19). YF1 was constructed by fusing the FixL HK module (DHp + CA domains) with the light-sensitive light-oxygen-voltage (LOV) domain of Bacillus subtilis YtvA (19). On the basis of the structures of YF1 and the vigorous and dormant forms of the isolated LOV domain, it was suggested that the coiled-coil linker region between the sensor and the DHp domain promotes the symmetry and asymmetry transitions of the four-helix bundle of the DHp domain to adjust the orientation between the DHp and the CA domains. The effects of site-directed mutagenesis of the coiled-coil linker region on the kinase activity of YF1 (19) are comparable to their observations of the effects of similar mutations on the kinase activity of FixL (fig. S9). Therefore, their present study using the intact and full-length FixL establishes the intramolecular signal transducing mechanism of HK.

Autophosporylation of His291 in the FixL DHp domain is followed by the phosphotransfer reaction, in which the phosphoryl group is transferred to Asp55 in the N terminus of FixJ. Formation of a momentary FixL-FixJ knotty facilitates phosphotransfer. To gain insight into this momentary situation in solution, they collected SEC-SAXS data under FixJ-saturated conditions and observed SAXS parameters indicating a stoichiometry of 2:2 for the FixL and FixJ complex. This result is consistent with structural data for complexes of truncated HK and truncated RRs: HK853-RR468 (20), ThkA-TrrA (27), DesK-DesR (22), and Spo0B-Spo0F (34). Stranava and co-workers (35) recently reported that AfGcHK and its cognate RR predominantly form a knotty with 2:1 stoichiometry, but they did not obtain a 2:1 knotty under the condition of excess FixJ in their SEC-SAXS study.

Our SAXS data betoken that FixJ binding induces no big FixL conformational change (Fig. 4, A and B). This observation is consistent with previous crystallographic studies of the truncated HK and RR complexes mentioned above (20, 22, 34), in which a slight rotation of the CA domain was observed, but there were no major changes in the overall structure of the HK (Fig. 6). In addition, the volume of the N-terminal FixJ REC domain is located nearby to the DHp-CA regions of the space-filling model (Fig. 3B). Because a phosphotransfer reaction from the HK to its cognate RR is common in TCSs, specific recognition of the REC domain by the kinase domain, such as that observed in the full-length FixL-FixJ complex, is almost certainly a general feature of TCSs. After phosphotransfer to the Asp residue in the REC domain of the RR, which activates the RR and releases it from the HK, the C-terminal effector domain of the RR dimerizes and then interacts with its specific target. Phosphorylated FixJ promotes the expression of NifA and FixK by binding to elements upstream of the promoters of these genes, which encode proteins essential for nitrogen fixation. Throughout TCSs, the role of the RR is highly variable and may mediate DNA binding (69.4%), RNA binding (1%), protein binding (1.7%), or enzymatic activity (8.1%) (36). Such functional diversity is reflected in the structural diversity of the C-terminal effector domains of RRs. There are at least 35 classes of RR, with each class including anywhere from 1 protein up to roughly 25,000 proteins (36–38). It appears to us that a conformationally springy RR, as described here for FixJ, very effectively limits the role that it can play in forming complexes with the cognate HK (Figs. 5 and 6). This is necessary to ensure that structurally disparate effector domains can be activated by a general TCS phosphotransfer mechanism to the REC domain of the RR. Thus, modularity ensures functionality.

Fig. 5 Space-filling and pseudoatomic models of the FixL-FixJ complex.

SAXS-based models of the FixL-FixJ knotty showing the overall shape, domain arrangement, and mode of knotty formation. The C-terminal effector domain of FixJ is not present in the model of the FixL-FixJ knotty because it does not form fragment of the knotty interface and is allowed conformational liberty by the linker connecting the N-terminal REC domain to the effector domain.

Fig. 6 Schematic representation of the FixL-FixJ TCS.

Our SAXS results intimate that there are no big changes of the overall shape of full-length FixL upon O2 dissociation from the heme group. However, the orientation of the coiled-coil helices between the heme-containing PAS-B (pink) and DHp (green) domains may change. Such localized structural change could alter the distance between the ATP-binding site in the CA domain (orange) and the autophosphorylation site at His291 in the DHp domain. In the full-length FixL-FixJ complex, a phosphorylation site (Asp55) in the FixJ REC domain approaches His291 of the FixL DHp domain, which mediates phosphotransfer. The C-terminal DNA binding domain of FixJ is connected to the REC domain by a springy linker, allowing the effector domain to exhibit multiple conformations.

The O2 sensor FixL is categorized as a class I HK, in which the DHp domain is directly adjacent to the CA domain (34, 35). Although every HKs discussed here (VicK, DesK, CckA, EnvZ, HK853, and ThkA) are in this same category, the domain architecture of FixL is the simplest among them. On the other hand, FixJ, which consists of a REC domain and a helix-turn-helix DNA binding domain (effector domain) through which it acts as a transcriptional activator, belongs to the NarL-like superfamily (39), which is the largest family of RRs. For the class I HKs, the REC domain is a common component for receiving a phosphate group. Therefore, the full-length architecture of the FixL-FixJ TCS is expected to depict the general mechanism of intra- and intermolecular signal transduction in TCSs composed of class I HKs. It is viable for the in-line mechanism supported by their findings to operate in every class I HKs, irrespective of whether the HK sensor is water-soluble (cytosolic) or membrane-integrated. The in-line mechanism moreover does not require that the effector domain of the RR interact with the HK. In addition, the present study provides fundamental learning for understanding the physiological, biochemical, and biological weight of TCS, particularly those involving class I HKs, including their molecular evolution (40) or protein engineering for synthetic biological systems for the development of antibiotics and plant growth effectors.

The rhizobia are a group of nitrogen-fixing bacteria that are proficient at establishing symbiotic relationships with leguminous plants, a global staple food group. This relationship provides nitrates to the host and a boost to growth and plant survival with benefits to agricultural productivity. The model organism B. japonicum is of particular interest in this respect because it resides in the root nodules of the soybean plant Glycine max, which provides more protein per hectare cultivated than any other food source. B. japonicum is sprayed onto soybean seed stock on an industrial scale to remove advantage of this relationship. Their results open the path for genetic modification of this rhizobial TCS to improve crop yields.

MATERIALS AND METHODS Preparation of recombinant FixL and FixJ proteins

B. japonicum FixL and FixJ genes encoding full-length FixL and FixJ and truncated proteins [FixLPAS-PAS (residues 1 to 275) and FixJN (residues 1 to 124)] were separately amplified by polymerase chain reaction (PCR) with PfuTurbo DNA polymerase (Agilent Technologies). The PCR fragments were cleaved by Bsr GI and Avr II for FixL, Bsr GI and Nhe I for FixJ (New England Biolabs), and cloned into 5′Bsr GI–3′Avr II sites of pET-47b(+) vector (Novagen) for expression with hexa-His tag, followed by the HRV3C protease cleavage site in the N terminus of those proteins. Site-directed mutagenesis of the coiled-coil region in full-length FixL was performed by QuikChange protocol (41) using pET-47b(+) vector inserted in the full-length FixL gene as a template.

Escherichia coli BL21(DE3) cells (Nippon Gene) carrying these plasmids were inoculated in terrific broth (TB) containing kanamycin (50 μg/ml; Wako) and 1% glucose for 4 hours at 37°C with shaking at 150 rpm. One milliliter of the preculture solution was inoculated into 300 ml of TB medium containing kanamycin [50 μg/ml; plus 250 μM 5-aminolevulinic acid (Cosmo Energy Holdings) only for the expression of FixL]. The cultivation was done at 37°C with shaking at 120 rpm. After 4 hours of cultivation, expression of FixL or FixJ was induced with 0.3 or 0.2 mM isopropyl-β-d-thiogalactopyranoside, respectively, and cultivation was allowed to continue for another 15 hours at 23°C with shaking at 80 rpm. The cells were harvested by centrifugation at 4000g for 10 min, and the cells were washed in 30 mM tris-HCl (pH 8.0) twice.

Purification of FixL and FixJ was performed by the following same steps at 4°C. Harvested cells were resuspended in a lysis buffer [50 mM tris-HCl (pH 8.0), 300 mM NaCl, 10% (w/v) glycerol, and one tablet of cOmplete EDTA-free protease inhibitor cocktail (Roche)]. The lysate was mixed with lysozyme (0.1 mg/ml; Sigma-Aldrich), deoxyribonuclease I (0.05 mg/ml; Sigma-Aldrich), and 5 mM MgCl2 for 30 min and disrupted by Microfluidizer M-110Y (Microfluidics). Cell debris was removed by ultracentrifugation at 40 krpm for 1 hour. The supernatant was loaded onto a HisTrap HP (GE Healthcare) column equilibrated with buffer A [50 mM tris-HCl (pH 8.0), 300 mM NaCl, 10% (w/v) glycerol, 10 mM imidazole/HCl (pH 8.0)]. FixL or FixJ was eluted by an imidazole concentration gradient (0 to 300 mM). The eluted fractions were treated with N-terminal 6×His-tagged HRV3C protease (produced in-house) to remove the 6×His-tag from the recombinant FixL and FixJ proteins, and the solution was dialyzed with buffer B [40 mM tris-HCl (pH 8.0), 150 mM NaCl, 10% (w/v) glycerol]. After the dialysis, the protein solution was loaded to HisTrap FF (GE healthcare) equilibrated with buffer B to remove the HRV3C protease and remaining His-tagged proteins, and the flowthrough was collected. The collected solution was concentrated by Amicon Ultra-15 (Merck Millipore) and centrifuged at 15 krpm for 20 min. The supernatant was loaded to HiLoad 16/600 Superdex 200 (GE healthcare) equilibrated with buffer B. The purity of FixL or FixJ was checked by SDS–polyacrylamide gel electrophoresis (PAGE). The purified samples were mixed with 2× SDS-PAGE buffer containing 125 μM tris-HCl (pH 6.8), 4% SDS, 20% (w/v) sucrose, 0.01% (w/v) bromophenol blue (BPB), and 10% (v/v) 2-mercaptoethanol and boiled at 95°C for 10 min before the electrophoresis. NuPAGE Bis-Tris gels (10%; Thermo Fisher Scientific) were used for the electrophoresis with NuPAGE Mops SDS running buffer (Thermo Fisher Scientific) for FixL and NuPAGE MES SDS running buffer (Thermo Fisher Scientific) for FixJ. The gels were stained by EzStain AQua (ATTO). In FixL, the highly purified fractions with an Rz (A398nm/A280nm) value of >1.3 were used for SAXS studies. These spectra were measured in 40 mM tris-HCl (pH 8.0), 150 mM NaCl, and 10% (w/v) glycerol at 20°C by NanoDrop 2000c spectrophotometers (Thermo Fisher Scientific). Because His-tagged full-length FixL showed the kinase inhibition upon cyanide binding to the heme and nearly the same phosphotransfer activity as the His-tag–removed protein, they used the His-tagged full-length FixL proteins in the phosphorylation activity assays of the coiled-coil mutants (fig. S9).

Autophosphorylation, phosphotransfer, and phosphatase activity assay of purified FixL and FixJ proteins

Autophosphorylation of the FixL protein was monitored by radioactivity of phosphorylated FixL by 32P. Reaction mixtures contained 2 or 6 μg of FixL in 7.5 μl of 50 mM tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl2, 50 μM MnCl2, and 5% (w/v) glycerol with or without 5 mM KCN. The reactions were started with the addition of the amalgam of ATP (10 or 25 nM) and γ-32P-ATP (4 or 10 μCi) (PerkinElmer), incubated the reaction amalgam at 23°C for 10 min, and stopped with one-fourth volume of SDS-PAGE sample buffer. After SDS-PAGE, phosphorylated proteins were visualized with an imaging analyzer BAS-1800 on an imaging plate (Fujifilm).

Phosphotransfer activities from FixL to FixJ were determined using an enzymatic assay in which ATP hydrolysis is coupled to NADH (reduced form of NAD+) oxidation using lactate dehydrogenase and pyruvate kinase (42). To measure basal activity, the reaction buffer [50 mM tris-HCl (pH 8.0), 50 mM KCl, 3 mM phosphoenolpyruvic acid (Wako), 0.3 mM NADH (Sigma-Aldrich), 8 U of lactate dehydrogenase (Toyobo), 25 U of pyruvate kinase (Sigma-Aldrich), 5 mM MgCl2, and 5 mM ATP] was equilibrated at 20°C for 5 min. The reaction was started by the addition of 1 μM purified FixL and 10 μM purified FixJ, and the time course of A340nm was monitored for 10 min at 20°C. An NADH touchstone curve with a compass of NADH concentrations between 0 and 150 μM was measured in the same buffer. To measure the activity of the cyanomet form, they added 5 mM KCN to the reaction buffer before the addition of FixL protein.

Phosphatase activity of FixL was monitored the dephosphorylation of acetylphosphorylated FixJ by unphosphorylated FixL. The acetylphosphate-dependent FixJ autophosphorylation was performed in 50 mM tris-HCl (pH 8.0), 50 mM KCl, 1 mM MgCl2, 50 μM MnCl2, 20 μM FixJ, and 50 mM acetyl phosphate lithium potassium salt (Sigma-Aldrich) at 23°C. After 2 hours, unphosphorylated FixL (10 μM, wild type) was added to the reaction mixture. The incubation times for the reactions were 0 (before FixL addition), 5, 15, 30, 60, and 120 min after the addition of the FixL. The reactions were stopped with two-third volume of SDS-PAGE sample buffer. The reaction products were subjected to 15% Zn2+-Phos-tag SDS-PAGE containing 50 μM Phos-tag acrylamide (Wako) and 100 μM ZnCl2. Tris-glycine buffer was used for the electrophoresis. The gel was stained by EzStain AQua (ATTO).

SEC-SAXS data collection and analysis

SEC-SAXS data collection was performed at RIKEN beamline BL45XU (15) in SPring-8 and at beamline sway (16) in the French national synchrotron SOLEIL. At BL45XU in SPring-8, the purified protein was loaded onto a Superdex 200 extend 3.2/300 column (GE Healthcare) in a 20-μl volume at 50 μl/min rush rate with a SEC buffer [40 mM tris-HCl (pH 8.0), 10% (w/v) glycerol, and 5 mM MgCl2] at 20°C. For the data collection of cyanomet FixL, 5 mM KCN was added to the SEC buffer, and the loading FixL sample was mixed with KCN at the final concentration of 5 mM. For the detection of the FixL-FixJ knotty or the FixL-FixJN complex, SEC buffer containing saturating amounts of FixJ or FixJN (more than 40 μM, 10 times of the Kd value) was used to improve the affinity of FixL and FixJ. X-ray exposure was 1 s every 4 s with the incident beam energy 12.4 keV. Buffer frames (30 × 1 s) were averaged and subtracted from 30 × 1 s frames taken over the course of protein elution. The sample detector distance was 2 m, giving an angular momentum transfer compass of qmin = 0.009 Å−1 to qmax = 0.5 Å−1. The flux density was about 2 × 1012 photons/s/mm. Scattering was collected on a PILATUS3 X 2M detector (Dectris). Data averaging and reduction were calculated by the program DataProcess installed in BL45XU. Measurements at sway were performed as above but using an Agilent Bio SEC-3 4.6 × 300–mm column (Agilent) with 3-μm bead size and 300 Å pore size at 15°C after double purification on a Superdex 200 extend 10/300 column (GE Healthcare) at 4°C. Beam energy was 12 keV, and sample detector distance was 1.8 m.

Rg and I0 calculations on the SEC-SAXS data were performed with AutoRg (43). Data of interest was averaged, and the Guinier estimation was performed in MATLAB and PRIMUS (fig. S10) (44). Distance distribution functions P(r) were calculated with GNOM (45) and ScÅtter based on agreement between real and reciprocal space Rg values (<3% difference) and lucky to the experimental data. This was performed independent of crystallographic and homology model building. Bead models were generated with DAMMIN (46).

Crystallization, data collection, and refinement for the full-length FixJ structure

Crystals of FixJ in space group C2221 (form 1) were obtained at 20°C by vapor diffusion using a mother liquor containing 10% (w/v) PEG 8000, 10% (w/v) PEG 1000, 0.8 M sodium formate, 20% (v/v) glycerol, and 0.1 M tris-HCl (pH 7.5). The asymmetric unit contained one polypeptide chain. Crystals of FixJ in space group P212121 (form 2) were obtained at 20°C by vapor diffusion using a mother liquor containing 20% (w/v) pentaerythritol ethoxylate (15/4 EO/OH), 0.1 M magnesium formate, 20% (v/v) glycerol, and 0.1 M tris-HCl (pH 8.5). The asymmetric unit contained five polypeptide chains. Crystals of both forms were grown in 3 days, frozen, and stored in liquid nitrogen. Data collection was carried out at BL26B2 in SPring-8, Harima, Japan (47, 48), equipped with an automated sample mounting system (49). Crystals were cryocooled in a nitrogen gas stream at 100 K during data collection. The data were integrated and scaled using HKL2000 (50). Initial phases for the data set of the crystal form 1 were obtained by molecular replacement using coordinates of StyR (PDB ID: 1YIO) (51) as a search model in PHENIX (52). The coordinates of the N- and C-terminal domains of StyR were extracted and used for the molecular replacement calculation because the structure of the linker region may vary from protein to protein in the RR family. Initial phases were used for model structure and improved by refinement of the model coordinates, including model structure of the linker domain, in PHENIX (52) and COOT (53). The final model included residues from 2 to 203, and 4 glycerols, 6 formic acids, and 257 water molecules. Initial phases for the data set of the crystal form 2 were obtained by molecular replacement using coordinates of the form 1 structure in PHENIX, with the N- and C-terminal domains separated. Four solutions for each domain were obtained and used for model rebuilding and refinement including the linker regions (chains A to D) in PHENIX and COOT. During refinement, weak but continuous densities appeared in the solvent region. Lowering the contour flat revealed the fifth molecule for the densities. Each separated copy of the models of the three domains was manually fitted in the electron densities (chain E). Coordinates of the five polypeptide chains were further refined including magnesium ions and water molecules. The final model included 1015 residues, 4 magnesium ions, and 8 glycerols, 7 formic acids, and 163 water molecules. Model trait was checked by MolProbity (54) in PHENIX.

Pseudoatomic model structure and refinement against SAXS data

JPred (55) and Coils (56) were used to ascertain which parts of the FixL sequence form α helices and coiled coils. The structure of the blue light receptor (PDB ID: 4GCZ) was used as a model for the HK domain and the α helices beyond Thr257. This was linked to the structure of FixL heme-PAS domain (PDB ID: 1DRM). The coiled-coil helix N terminus of the heme-PAS to the PAS-A domain was created with PEP-FOLD (57). This was linked to a homology model of the PAS-A domain created from a sensory HK from Burkholderia thailandensis (PDB ID: 3MR0). The cloning fragment from the pET-47b(+) vector (10 amino acid residues, GPGYQDPNSV) was moreover constructed using PEP-FOLD. This initial structure had χ2 value of 3.93 against experimental data calculated using FoXS (58). Torsion angle molecular dynamics (MD) in CNS (59) was used to refine the positions of domains and loops in this structure against SAXS data as described by Wright et al. (60). To validate the region of the model encompassing amino acids 1 to 256, which was assembled by combining a crystal structure, a homology model, and several ab initio structure predictions, they measured SAXS of a C-terminally truncated form of FixL consisting of amino acids 1 to 275 and including the cloning fragment. These data were exceptionally congruent with the PAS-A–PAS-B fragment of their FixL model, indicating the proposed domain arrangement and interfaces are correct. The chimeric sensor protein structure 4GCZ shows structural asymmetry possibly resulting from crystal packing or partial adenosine diphosphate (ADP) binding. They created three models of FixL based on VicK, DesKC, and CckA HKs, each of which has symmetric four helix bundles, based on their coverage of and identity to FixL amino acids 258 to 505. Homology models were generated with SWISS-MODEL in conjunction with the mode above and HADDOCK (61) to refine and dock the ADP-free CA domains to find an optimum ADP-free structure. This pool of structures has consistently worse lucky to their experimental data than that based on 4GCZ, indicating that FixL adopts an asymmetric conformation in solution in the ADP-free state.

The structures of FixJ were refined against SAXS data using the torsion angle MD process described above. They initially defined the location of FixJ binding to FixL using pyDockSAXS (62) and FoXSDock (63). These programs expend a combination of FTDock/Crysol and PatchDock/FoXS to apportion interactions. Using this approach, and without defining the FixL His291–FixJ Asp55 interaction site, FixJ was consistently positioned on the FixL four-helix bundle. FixJ REC domains were then directed to FixL His291 on both chains using HADDOCK. The FixJ linkers and DNA binding domains were then added based on the SAXS refined structure of the FixJ monomer. The positions of these latter domains were then determined using CNS torsion angle MD. Trimeric complexes of dimeric FixL with one FixJ monomer were moreover constructed and optimized by torsion angle MD/rigid corpse refinement but were institute to consistently produce models that lucky the data poorly in comparison with the tetrameric architecture.

SUPPLEMENTARY MATERIALS

www.sciencesignaling.org/cgi/content/full/11/525/eaaq0825/DC1

Fig. S1. Purification of FixL and FixJ.

Fig. S2. Optical absorption spectra of full-length FixL.

Fig. S3. Autophosphorylation and phosphatase activities of FixL.

Fig. S4. SEC profiles.

Fig. S5. Space-filling models of FixL lonely and in knotty with FixJ or FixJN.

Fig. S6. Pseudoatomic model and SAXS curve of truncated FixL comprising PAS-A, PAS-B, and the coiled-coil region (FixLPAS-PAS).

Fig. S7. Experimental and simulated SAXS curves of met FixL and the FixL-FixJ complex.

Fig. S8. SAXS curves and pseudoatomic models of full-length FixL based on comparisons with other HKs.

Fig. S9. Phosphorylation activities of FixL mutants presence mutations in the coiled-coil linker.

Fig. S10. Guinier plots with Pearson residuals for full-length FixL, FixLPAS-PAS, FixL-FixJ, full-length FixJ, and FixJN.

Table S1. Structural parameters for FixL and FixJ determined by static SAXS experiment at the BL45XU beamline at the SPring-8 synchrotron.

Table S2. Structural parameters for FixL and FixJ determined by SEC-SAXS at the sway beamline at the SOLEIL synchrotron.

Table S3. Crystallographic statistics for full-length FixJ.

REFERENCES AND NOTES
  • C. Tomomomori, H. Kurokawa, M. Ikura, The histidine kinase family: Structures of essential structure blocks, in Histidine Kinases in Signal Transduction, M. Inoue, R. Dutta, Eds. (Academic Press, 2003).

  • Z. Otwinowski, W. Minor, Processing of x-ray diffraction data collected in oscillation mode, in Methods in Enzymology, C. W. Carter, J. R. M. S., Ed. (Academic Press, 1997), vol. 276, pp. 307–326.

  • Acknowledgments: Thanks to synchrotron SOLEIL and SPring-8 for the provision of SAXS facilities. They avow the champion and the expend of resource of instruct, a landmark ESFRI project (iNEXT 2822). Funding: This drudgery was supported by the Fumi Yamamura Memorial Foundation for Female Natural Scientists from Chuo Mitsui dependence and Banking (to H.S.), Hyogo Science and Technology Association (to H.S.), RIKEN Pioneering Project “Integrated Lipidology” (to H.S.), and “Molecular System” (to Y.S.), and the Japan Society for the Promotion of Science (JSPS) KAKENHI concede numbers JP26220807 (to Y.S. and H.S.) and JP25871213 (to H.S.). Author contributions: G.S.A.W., S.V.A., S.S.H., Y.S., and H.S. designed this study. A.S., H. Nakamura, and H.S. created the systems for expressing recombinant FixL and FixJ in E. coli. T. Hikima and M.Y. installed the SEC-SAXS system at BL45XU in SPring-8. G.S.A.W., A.S., Y.N., and H.S. purified the FixL and FixJ samples. H.S. prepared the expression systems for FixL mutants. M.K. purified the FixL mutant proteins and measured their phosphotransfer activities. K.N. and H. Nishitani performed the autophosphorylation activity assay using γ-32P-ATP. H.S. performed the phosphatase activity assay by Phos-tag SDS-PAGE. G.S.A.W., A.S., T. Hikima, and H.S. measured and analyzed the SAXS data. G.S.A.W. modeled the pseudoatomic structures. Y.N. crystallized FixJ. Y.N. and T. Hisano collected, processed, and refined the crystal data. G.S.A.W., T. Hisano, S.V.A., S.S.H., Y.S., and H.S. wrote the manuscript. every authors analyzed data and discussed the results. Competing interests: The authors declare that they gain no competing interests. Data and materials availability: every data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials. The atomic coordinates and structure factors for FixJ (PDB IDs: 5XSO and 5XT2) gain been deposited in the PDB (www.wwpdb.org/). The SAXS measurements at SPring-8 BL45XU were performed under proposals 20140099, 20150017, 20160015, and 20170092. The x-ray diffraction measurements were performed at SPring-8 BL26B2 (proposal 20160015).

    Implementing Proxy Server | killexams.com real questions and Pass4sure dumps

    Designing a Proxy Server Implementation

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  • All operating systems that utilize SOCKS touchstone should be supported

  • IPs supported by SOCKS applications should be redirected.

  • Another planning component that should be included when you design your Proxy Server implementation is to determine the flat of data protection that should be configured.

  • Inbound and outbound packet filters can be configured to filter and restrict traffic, based on the criteria defined for the different IP traffic types.

  • Domin filters can be configured to restrict Internet access to only confident IP addresses or FQDNs. In a domain filter, you can embrace a number of Internet sites and then define the action that the domain filter should remove when a request is received for one of these sites: Reject packets for these specific Internet sites and forward every other packets OR forward packets to these specific Internet sites and reject every other packets. Domain filters can restrict outbound traffic, based on a separate computer or the IP address of a cluster, an IP address compass or a FQDN.

  • You can utilize Proxy Server user authentication to specify Internet access, based on user or group account.

  • Through Web publishing, you can restrict inbound traffic based on the URL requests of Internet users.

  • The default configuration of Proxy Server is to drop the URL requests of Internet users. This means that Internet users execute not gain access to Web and FTP servers hosted within the private network, by default. You can though define URLs where requests for these URLs should be passed to Web and FTP servers on the private network. Proxy Server will allow URL requests when you define them in the Web Publishing list.

    For URLs that are requested which are defined in the Web Publishing list, Proxy Server passes the requests to the Web and FTP servers on the private network.

    For URLs that are requested which are not defined in the Web Publishing list, Proxy Server performs either of the following:

    There are moreover a number of techniques that optimize Proxy Server performance, which you should deem implementing:

  • Caching Web content improves performance. Cached information is accessed by users from a location on the Local belt Network (LAN). This means that bandwidth utilization to the Internet ends up being lowered because cached information does not requisite to be downloaded from the Internet. every of this leads to an improvement in the service experienced by users.

  • Proxy Server moreover provides a feature called proxy arrays. A proxy array is a solution whereby one or multiple proxy servers operate as a separate cache for client requests. Benefits provided by the proxy array feature embrace scalable performance, and failing tolerance.

  • Network Load Balancing (NLB) can be used to divide the processing load of inbound traffic over multiple proxy servers. This leads to towering availability and performance optimization.

  • Round Robin DNS can moreover be used to load equipoise inbound traffic across multiple proxy servers, thereby moreover providing towering availability and performance optimization.

  • The advantages of using proxy arrays as a Proxy Server optimization mode when you implement Proxy Server are listed here:

  • Because Web content is cached over multiple servers, no separate server hosts every Web content.

  • If a server in the proxy array fails, failover is immediately provided.

  • The advantages of using Network Load Balancing (NLB) as a Proxy Server optimization mode when you implement Proxy Server are listed here:

  • You can add or remove proxy servers residing in the NLB cluster.

  • Load balancing occurs dynamically over every proxy servers residing in the NLB cluster.

  • Because load balancing and the addition or removal of proxy servers occurs dynamically, availability and performance is improved.

  • The NLB cluster is automatically reconfigured when a proxy server happens to fail.

  • The advantages of using Round Robin DNS as a Proxy Server optimization mode when you implement Proxy Server are listed here:

  • Load balancing is performed on every proxy servers in the round robin DNS.

  • Round Robin DNS can operate on every operating system platforms.

  • Performance is improved because traffic is basically load balanced over every proxy servers.

  • If you requisite to provide the highest viable flat of server availability for your Proxy Server implementation, you should expend Microsoft Windows Clustering. Using Microsoft Windows Clustering provides the following benefits for your Proxy Server implementation:

  • The Proxy Servers every partake a common cache.

  • If a server in the proxy array fails, failover is immediately provided.

  • Because the cache does not requisite to be built again when a server fails, restore occurs quite faster.

  • To optimize Internet access, you can embrace the following Proxy Server caching methods in your Proxy Server design:

  • As mentioned previously, with passive caching, Proxy Server stores objects in the Proxy Server cache with each object obtaining a Time To Live (TTL) value. Before Proxy Server forwards requests to the Internet, it first checks the Proxy Server cache to determine if the request can be serviced from there. When the Proxy Server cache becomes full, Proxy Server removes objects from the cache, based on a combination of factors: object size, object age, and object popularityThe advantages of using passive caching in your Proxy Server implementation are:

  • With vigorous Caching, Proxy Server automatically generates requests for specific objects in the Proxy Server cache so that frequently requested objects remain cached. Proxy Server determines which objects should be flagged for vigorous caching by considering object popularity, Time To Live (TTL) value of objects, and server load to determine the flat of vigorous caching performed.The advantages of using vigorous caching in your Proxy Server implementation are:

  • Determining Proxy Server Hardware and Software Requirements

    Proxy Server has a few minimum hardware and software implementation requirements. However, depending on the size of the organization, existing hardware and software, future network expansion, and expected traffic volumes; the Proxy Server implementation requirements between organizations would differ. For each different network environment, there are different requirements for a Proxy Server implementation.

    The requirements listed below merely serves as a guideline on the hardware requirements for a Proxy Server implementation:

  • Processor; Intel 486 or faster supported RISC-based microprocessor

  • Disk space; 10 MB available disk space for Proxy Server

  • For caching; 100 MB plus an additional 0.5 MB for each Web Proxy service client.

  • RAM; at least 24 MB. For RISC-based systems, this increases to 32 MB.

  • An NTFS formatted partition to store the Proxy Server cache.

  • A network adapter card for connection to the LAN.

  • A network interface configured for the Internet.

  • When planning a Proxy Server implementation, you gain to select on the hardware that you will used to establish connections to the Internet:

  • ISDN lines can be used to establish connections to the Internet. ISDN is a digital dial-up service that utilizes telephone cabling and other technology to provide Internet connections. The different types of ISDN services are ISDN Basic Rate Interface (BRI) and ISDN Primary Rate Interface (PRI).The main characteristics of ISDN Basic Rate Interface (BRI) are listed here:

  • BRI connections drudgery well for petite companies

  • BRI connections are available from quite a number of telephone companies.

  • ISDN BRI can present 128 Kbps of bandwidth.

  • Provide e-mail for a maxmum of 20 concurrent users.

  • Provide big FTP downloads for only 3 to 4 simultaneous users.

  • Provide Web browsing for 6 to 8 concurrent users.

  • The main characteristics of ISDN Primary Rate Interface (PRI) are listed here:

  • ISDN PRI can present 1.544 Mbps transmission speed.

  • Provide e-mail for a maximum of 120 concurrent users.

  • Provide big FTP downloads for only 40 to 50 simultaneous users.

  • Dial-up modem connections are ideal if your organization only consists of a petite number of users that execute not requisite to connect to the Internet on a regular basis. This is due to dialup modem connection only being able to meet the bandwidth requirements of a petite number of users. Modems can be installed on a computer, and then shared through the Windows Internet Connection Sharing (ICS) service.A few characteristics of dial-up modem connections are:

  • A dial-up modem connection can only attain up to 53 Kbps.

  • Provide e-mail for a maximum of 10 concurrent users.

  • Provide big FTP downloads for only 1 to 2 simultaneous users.

  • Provide Web browsing for 2 to 3 concurrent users.

  • You moreover gain to select on the hardware which will be utilized to connect the server to the Internet:

  • Analog modem: Analog modem evade at 28.8 or 33.6 Kbps speeds. An analog modem is ideal for a separate user connecting to the Internet, and for a networked server gateway.

  • ISDN adapters: This is the common choice. The ISDN adapters dial an ISDN access number and then maintain the particular connection.

  • Routers: Routers are networking devices that connect networks.

  • Installing Proxy Server

    You should verify a number of things before you actually install Proxy Server:

  • 10 MB available disk space for Proxy Server and 100 MB plus an additional 0.5 MB for each Web Proxy service client.

  • An NTFS formatted partition to store the Proxy Server cache.

  • TCP/IP should be installed on the computer.

  • The internal network interface should be bound to the TCP/IP or IPX/SPX protocol being used on the LAN.

  • You should configure the software for two network adapter cards before you attempt to install Proxy Server.

  • When Proxy Server is installed, the following changes are made to the computer on which you are installing it:

  • The Web Proxy service is installed.

  • The WinSock Proxy service is installed.

  • The Socks Proxy service is installed.

  • Each of these services is added to the Internet Service Manager administration tool.

  • The local address table is installed.

  • On the NTFS volume, the cache drive is created.

  • The client installation and configuration software is copied.

  • The Mspclnt shared folder is created.

  • The Proxy Server Performance Monitor counters are installed.

  • The HTML online documentation is installed.

  • How to install Proxy Server
  • On the Proxy Server installation CD, proceed to evade Setup.

  • Click Continue on the Welcome to the Microsoft Proxy Server Installation program screen.

  • The Microsoft Proxy Server Setup page opens.

  • Specify the 10-digit product key provided on the CD-ROM case. Click OK.

  • The Microsoft Proxy Server Setup dialog box displays the default destination folder and the Installation Options button. Click the Installation Options button.

  • The Microsoft Proxy Server – Installation Options dialog box opens, displaying every components as being selected. Click Continue.

  • Setup now stops the Web services.

  • The Microsoft Proxy Server Cache Drives dialog box opens. Caching is by default enabled.

  • The local drives of the server are listed in the Drive box.

  • Select the drive which should be used to store cached data. In the Maximum Size (MB) box, enter th confiscate value. Click Set, and then click OK.

  • The Local Address Table Configuration dialog box opens.

  • Click the Construct Table button.

  • The Construct Local Address Table dialog box opens.

  • Select Load from NT internal Routing Table to select the network adapter cards thats IP addresses must be added to the local address table.

  • Select the Load known address ranges from the following IP interface cards option, and then select the network adapter. Click OK.

  • Click OK to avow the message displayed, indicating that the IP addresses gain been loaded into the local address table.

  • The Local Address Table Configuration dialog box opens, displaying IP addresses in the Internal IP Ranges box.

  • Check that the addresses defined are correct, and then click OK.

  • The Client Installation/Configuration dialog box opens.

  • Enter the confiscate information and verify that the redress computer denomination is displayed in the Computer denomination bailiwick and Proxy field.

  • If you enable the Automatically configure Web browser during client setup checkbox, the Web browser network configuration setting of the client is changed so that client requests are sent to the Proxy Server, and not to the Internet.

  • Click Configure.

  • You can either evade the default script to configure the client Web browser, or alternatively, you can expend a custom URL.

  • Click Properties located beneath Browser automatic configuration script.

  • The Advanced Client Configuration dialog box opens.

  • Specify whether the Proxy Server is utilized for local servers.

  • Specify the IP addresses that should be excluded from Proxy Server.

  • Specify the domains that should be excluded from Proxy Server.

  • Specify a backup to the proxy server.

  • Click OK.

  • The Access Control dialog box opens.

  • Verify that access control is specified for the Web Proxy service and for the WinSock Proxy service and then click OK.

  • The Proxy Server Setup files are copied to the computer.

  • When the Setup Information dialog box opens, click OK. The Setup Information dialog box displays information on the packet filtering feature. The packet filtering feature is not automatically enabled when Proxy Server is installed. Click OK.

  • A Proxy Server 2.0 Setup was completed successfully message is displayed.

  • How to install WinSock Proxy Client on client computers

    When you install WinSock Proxy Client on client computers, the following changes are made:

  • The Proxy Client program group is created

  • The local address table file, Msplat.txt, is installed on the client. Proxy Server will update this file.

  • Mspclnt.ini is moreover copied to the client.

  • The WSP Client icon is added to Control Panel. This only occurs for Windows 3.x, Windows 95, and Windows NT clients.

  • Remote WinSock from WinSock Proxy Client replaces Winsock.dll. This would enable the computer to only access Internet sites using the WinSock Proxy service.

  • To install WinSock Proxy Client on a client computer;

  • Open Internet Explorer

  • In the Address box, enter http://proxycomputername/msproxy.

  • The WinSock Proxy Client 2.0 Installation page is displayed.

  • To install WinSock Proxy Client, click WinSock Proxy 2.0 client.

  • Click the Open it option and click OK.

  • The Microsoft Proxy Client Setup dialog box opens.

  • Click Continue to proceed with the installation.

  • Click Install Microsoft Proxy Client to start copying Setup files to the client computer.

  • Click OK.

  • The Setup – Restart System dialog box opens.

  • Click the Restart Windows Now option.

  • How to add or remove Proxy Server components
  • On the Proxy Server installation CD, proceed to evade Setup.

  • Click Add/Remove on the Setup screen.

  • Follow the instruction displayed to add or remove Proxy Server components.

  • How to restore Proxy Server settings or files
  • On the Proxy Server installation CD, proceed to evade Setup.

  • Click Reinstall on the Setup screen.

  • Follow the instructions displayed to restore Proxy Server settings/files.

  • How to remove Proxy Server from the server
  • On the Proxy Server installation CD, proceed to evade Setup.

  • Click Remove every on the Setup screen.

  • Click Yes to avow that you want to remove Proxy Server.

  • Proxy Server is then removed from the server.

  • How to disable WinSock Proxy Client
  • Open Control Panel.

  • Double-click WSP Client.

  • Deselect the Enable WinSock Proxy Client checkbox.

  • Restart the computer.

  • How to re-enable the WinSock Proxy Client
  • Open control Panel.

  • Double-click WSP Client.

  • Check the Enable WinSock Proxy Client checkbox.

  • Restart the computer.

  • Administering Proxy Server using the Internet Service Manager

    You can expend the Internet Service Manager to configure properties for the Web Proxy, WinSock Proxy, and Socks Proxy services of Proxy Server.

    To open the Internet Service Manager;

  • Click Start, click Programs, click Microsoft Proxy Server, and then click Internet Service Manager.

  • You can open the properties of specific Proxy Server service by double-clicking the computer denomination displayed alongside the particular service name.

  • There are some properties settings which are common for every three Proxy Server services, and there are others that are relevant for only a particular Proxy Server service. This concept is illustrated here.

  • The properties settings which can be configured for the Web Proxy service are listed here:

  • Service

  • Permissions

  • Caching

  • Routing

  • Publishing

  • Logging

  • The properties settings which can be configured for the WinSock Proxy service are listed here:

  • Service

  • Permissions

  • Protocol

  • Logging

  • The properties settings which can be configured for the Socks Proxy service are listed here:

  • Service

  • Permissions

  • Logging

  • The configuration settings which you can view and configure on the Service tab for each of the three Proxy Server services are listed below:

  • View the product release.

  • Verify the product ID.

  • Add additional information on the service.

  • Add additional information on the server.

  • View the current sessions.

  • Navigate to the Shared services tabs.

  • Navigate to the Configuration tabs.

  • The configuration settings which you can view and configure on the Permissions tab for the Web Proxy and WinSock Proxy services are listed below:

  • Select or disable the Enable access control checkbox.

  • Select the Protocol when defining user or group permissions. Permissions are basically assigned for each protocol.

  • Define user and group permissions for using the Internet protocols.

  • The configuration settings which you can view and configure on the Permissions tab for the Socks Proxy are:

  • Specify the source and destination for an entry, and then define whether requests should be allowed or defined.

    The configuration settings which you can view and configure on the Caching tab for the Web Proxy and service is listed here:

  • Select the Enable caching checkbox and then select between the following Cache expiration policy options:

  • Select the Enable vigorous caching checkbox and then select between the following options:

    Faster user response is more important.

  • The configuration settings which you can view and configure on the Routing tab for the Web Proxy and service is listed here:

  • For upstream routing, you can select between the following options:

  • Use direct connection.

  • Use Web proxy or array.

  • If you select the Enable backup route checkbox, you can select between the following options:

  • Use direct connection.

  • Use Web proxy or array.

  • You can moreover select the Resolve Web proxy requests within array before routing upstream checkbox on this tab.

  • The configuration settings which you can view and configure on the Publishing tab for the Web Proxy and service is listed here:

  • Enable/disable Web publishing.

  • Configure computers to publish information on the Internet via the Proxy Server.

  • Specify what should betide to incoming Web server requests:

  • The configuration settings which you can view and configure on the Logging tab for every three Proxy Server services are listed below:

  • Enable/disable logging. When enabled, the following types of information will be logged:

  • Server

  • Client

  • Connection

  • Object

  • Specify the Log to file option, or the Log to SQL/ODBC database option.

  • Specify when a current log should be opened.

  • Specify the log file directory.

  • How to disable IP routing (control access to the private network)
  • Open Control Panel

  • Double-click Network.

  • The Network dialog box opens.

  • Click the Protocols tab.

  • Select TCP/IP, and click Properties.

  • The TCP/IP Properties dialog box opens.

  • Switch to the Routing tab.

  • Ensure that the Enable IP Forwarding checkbox is not selected (blank).

  • Click OK.

  • How to configure publishing configuration settings for the Web Proxy service
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Publishing tab.

  • Select the Enable Web publishing checkbox.

  • If you want to drop every incoming Web server requests, click the Discard option.

  • If you want to forward every incoming Web server requests to IIS on the Proxy Server computer, click the Sent to the local Web server option.

  • If you want forward every incoming Web server requests to a specific downstream server, click the Sent to another Web server option.

  • If you want to configure the default Web server host, click Default Mapping.

  • The Default Local Host denomination dialog box opens.

  • Provide the denomination of the default server. Click OK.

  • Click Apply and click OK.

  • How to enable dynamic packet filtering
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Security button on the Service tab.

  • Click the Packet Filters tab.

  • On the Packet Filters tab, click the Enable packet filtering on external interface checkbox.

  • Select the Enable dynamic packet filtering of Microsoft Proxy Server packets checkbox.

  • Click OK.

  • Click OK in the Web Proxy Service Properties dialog box.

  • How to create a packet filter using predefined protocol definitions
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Securty button on the Service tab.

  • Click the Packet Filters tab.

  • On the Packet Filters tab, click Add.

  • When the Packet Filter Properties dialog box opens, click the Predefined filter option.

  • Select a protocol from the available Protocol ID list.

  • In the Local host belt of the Packet Filter Properties dialog box, select the confiscate option to allow packet exchange with a host.

  • In the Remote host belt of the Packet Filter Properties dialog box, specify one host or the Any host option.

  • Click OK.

  • How to create a packet filter using custom protocol definitions
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Security button on the Services tab.

  • Click the Packet Filters tab.

  • On the Packet Filters tab, click Add.

  • When the Packet Filter Properties dialog box opens, click the Custom filter option.

  • Select a protocol from the available Protocol ID list.

  • Select a direction from the Direction list.

  • Select an option from the available options in the Local port area.

  • Select either the Any option or Fixed port option in the Remote port area.

  • In the Local host belt of the Packet Filter Properties dialog box, select the confiscate option to allow packet exchange with a host.

  • In the Remote host belt of the Packet Filter Properties dialog box, specify one host or the Any host option.

  • Click OK.

  • How to change the packet filter list entries
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Security button on the Service tab.

  • On the Packet Filters tab, click the Enable packet filtering on external interface checkbox.

  • Select the Enable dynamic packet filtering of Microsoft Proxy Server packets checkbox to enable dynamic packet filtering.

  • Click the Edit button.

  • The Packet Filter Properties dialog box opens. Change the necessary settings and then click OK.

  • If you want to remove a filter, click the Remove button.

  • Click OK.

  • How to configure Proxy Server logging
  • Open Internet Service Manager.

  • Double-click the computer denomination alongside the Web Proxy service.

  • The Web Proxy Service Properties dialog box opens.

  • Click the Security button on the Service tab.

  • Click the Logging tab.

  • Click the Enable logging using checkbox.

  • Select the confiscate format in the Format list box.

  • Click OK.

  • How to back up a Proxy Server configuration
  • Open Internet Service Manager.

  • On the View menu item, click Servers View.

  • Double-click the computer name, and then double-click Web Proxy (Running).

  • The Web Proxy Service Properties opens.

  • Click the Service tab.

  • In the Configuration area, click Server Backup.

  • When the Backup dialog box opens, verify the information shown on where the backup file will be saved

  • Click OK to create a back up of the Proxy Server configuration.

  • How to restore a Proxy Server configuration
  • Open Internet Service Manager.

  • Double-click the computer name, and then double-click Web Proxy service.

  • The Web Proxy Service Properties opens.

  • Click the Service tab.

  • In the Configuration area, click Server Restore.

  • When the Restore Configuration dialog box opens, click the Browse button to select the Proxy Server configuration file.

  • Select the Proxy Server configuration file that you want to expend for the restore.

  • Click Open.

  • Select the complete Restore option.

  • When the Restore Configuration dialog box opes, click OK to start the restore of the Proxy Server configuration.



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    LPI [24 Certification Exam(s) ]
    LSI [3 Certification Exam(s) ]
    Magento [3 Certification Exam(s) ]
    Maintenance [2 Certification Exam(s) ]
    McAfee [8 Certification Exam(s) ]
    McData [3 Certification Exam(s) ]
    Medical [69 Certification Exam(s) ]
    Microsoft [374 Certification Exam(s) ]
    Mile2 [3 Certification Exam(s) ]
    Military [1 Certification Exam(s) ]
    Misc [1 Certification Exam(s) ]
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    mySQL [4 Certification Exam(s) ]
    NBSTSA [1 Certification Exam(s) ]
    NCEES [2 Certification Exam(s) ]
    NCIDQ [1 Certification Exam(s) ]
    NCLEX [2 Certification Exam(s) ]
    Network-General [12 Certification Exam(s) ]
    NetworkAppliance [39 Certification Exam(s) ]
    NI [1 Certification Exam(s) ]
    NIELIT [1 Certification Exam(s) ]
    Nokia [6 Certification Exam(s) ]
    Nortel [130 Certification Exam(s) ]
    Novell [37 Certification Exam(s) ]
    OMG [10 Certification Exam(s) ]
    Oracle [279 Certification Exam(s) ]
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    PMI [15 Certification Exam(s) ]
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    PostgreSQL-CE [1 Certification Exam(s) ]
    Prince2 [6 Certification Exam(s) ]
    PRMIA [1 Certification Exam(s) ]
    PsychCorp [1 Certification Exam(s) ]
    PTCB [2 Certification Exam(s) ]
    QAI [1 Certification Exam(s) ]
    QlikView [1 Certification Exam(s) ]
    Quality-Assurance [7 Certification Exam(s) ]
    RACC [1 Certification Exam(s) ]
    Real-Estate [1 Certification Exam(s) ]
    RedHat [8 Certification Exam(s) ]
    RES [5 Certification Exam(s) ]
    Riverbed [8 Certification Exam(s) ]
    RSA [15 Certification Exam(s) ]
    Sair [8 Certification Exam(s) ]
    Salesforce [5 Certification Exam(s) ]
    SANS [1 Certification Exam(s) ]
    SAP [98 Certification Exam(s) ]
    SASInstitute [15 Certification Exam(s) ]
    SAT [1 Certification Exam(s) ]
    SCO [10 Certification Exam(s) ]
    SCP [6 Certification Exam(s) ]
    SDI [3 Certification Exam(s) ]
    See-Beyond [1 Certification Exam(s) ]
    Siemens [1 Certification Exam(s) ]
    Snia [7 Certification Exam(s) ]
    SOA [15 Certification Exam(s) ]
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    SpringSource [1 Certification Exam(s) ]
    SUN [63 Certification Exam(s) ]
    SUSE [1 Certification Exam(s) ]
    Sybase [17 Certification Exam(s) ]
    Symantec [134 Certification Exam(s) ]
    Teacher-Certification [4 Certification Exam(s) ]
    The-Open-Group [8 Certification Exam(s) ]
    TIA [3 Certification Exam(s) ]
    Tibco [18 Certification Exam(s) ]
    Trainers [3 Certification Exam(s) ]
    Trend [1 Certification Exam(s) ]
    TruSecure [1 Certification Exam(s) ]
    USMLE [1 Certification Exam(s) ]
    VCE [6 Certification Exam(s) ]
    Veeam [2 Certification Exam(s) ]
    Veritas [33 Certification Exam(s) ]
    Vmware [58 Certification Exam(s) ]
    Wonderlic [2 Certification Exam(s) ]
    Worldatwork [2 Certification Exam(s) ]
    XML-Master [3 Certification Exam(s) ]
    Zend [6 Certification Exam(s) ]





    References :


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