bluuebunny/crossref_metadata_2025_split huggingface dataset:
Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation
Authors: Che Yong-Suk , Nakamura Shuichi , Kojima Seiji , Kami-ike Nobunori , Namba Keiichi , Minamino Tohru 
Published: August 2008

ABSTRACT

MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of
Salmonella enterica
serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile
motB
(
D33E
) mutant and its pseudorevertants. We first confirmed that the poor motility of the
motB
(
D33E
) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the
motB
(
D33E
) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.
Reexamination of the Role of the Amino Terminus of SecA in Promoting Its Dimerization and Functional State
Authors: Das Sanchaita , Stivison Elizabeth , Folta-Stogniew Ewa , Oliver Donald 
Published: August 2008

ABSTRACT

The SecA nanomotor promotes protein translocation in eubacteria by binding both protein cargo and the protein-conducting channel and by undergoing ATP-driven conformation cycles that drive this process. There are conflicting reports about whether SecA functions as a monomer or dimer during this dynamic process. Here we reexamined the roles of the amino and carboxyl termini of SecA in promoting its dimerization and functional state by examining three
secA
mutants and the corresponding proteins: SecAΔ8 lacking residues 2 to 8, SecAΔ11 lacking residues 2 to 11, and SecAΔ11/N95 lacking both residues 2 to 11 and the carboxyl-terminal 70 residues. We demonstrated that whether SecAΔ11 or SecAΔ11/N95 was functional for promoting cell growth depended solely on the vivo level of the protein, which appeared to govern residual dimerization. All three SecA mutant proteins were defective for promoting cell growth unless they were highly overproduced. Cell fractionation revealed that SecAΔ11 and SecAΔ11/N95 were proficient in membrane association, although the formation of integral membrane SecA was reduced. The presence of a modestly higher level of SecAΔ11/N95 in the membrane and the ability of this protein to form dimers, as detected by chemical cross-linking, were consistent with the higher level of
secA
expression and better growth of the SecAΔ11/N95 mutant than of the SecAΔ11 mutant. Biochemical studies showed that SecAΔ11 and SecAΔ11/N95 had identical dimerization defects, while SecAΔ8 was intermediate between these proteins and wild-type SecA in terms of dimer formation. Furthermore, both SecAΔ11 and SecAΔ11/N95 were equally defective in translocation ATPase specific activity. Our studies showed that the nonessential carboxyl-terminal 70 residues of SecA play no role in its dimerization, while increasing the truncation of the amino-terminal region of SecA from 8 to 11 residues results in increased defects in SecA dimerization and poor in vivo function unless the protein is highly overexpressed. They also clarified a number of conflicting previous reports and support the essential nature of the SecA dimer.
NorB, an Efflux Pump in
Staphylococcus aureus
Strain MW2, Contributes to Bacterial Fitness in Abscesses
Authors: Ding Yanpeng , Onodera Yoshikuni , Lee Jean C. , Hooper David C. 
Published: August 2008

ABSTRACT

While remaining a major problem in hospitals,
Staphylococcus aureus
is now spreading in communities. Strain MW2 (USA400 lineage) and other community methicillin-resistant
S. aureus
strains most commonly cause skin infections with abscess formation. Multidrug resistance (MDR) efflux pumps contribute to antimicrobial resistance but may also contribute to bacterial survival by removal of environmental toxins. In
S. aureus
, NorA, NorB, NorC, and Tet38 are chromosomally encoded efflux pumps whose overexpression can confer MDR to quinolones and other compounds (Nor pumps) or tetracyclines alone (Tet38), but the natural substrates of these pumps are not known. To determine the role of these efflux pumps in a natural environment in the absence of antibiotics, we used strain MW2 in a mouse subcutaneous abscess model and compared pump gene expression as determined by reverse transcription-PCR in the abscesses and in vitro.
norB
and
tet38
were selectively upregulated in vivo more than 171- and 24-fold, respectively, whereas
norA
and
norC
were downregulated. These changes were associated with an increase in expression of
mgrA
, which encodes a transcriptional regulator known to affect pump gene expression. In competition experiments using equal inocula of a
norB
or
tet38
mutant and parent strain MW2, each mutant exhibited growth defects of about two- to threefold in vivo. In complementation experiments, a single-copy insertion of
norB
(but not a single-copy insertion of
tet38
) in the
attB
site within
geh
restored the growth fitness of the
norB
mutant in vivo. Our findings indicate that some MDR pumps, like NorB, can facilitate bacterial survival when they are overexpressed in a staphylococcal abscess and may contribute to the relative resistance of abscesses to antimicrobial therapy, thus linking bacterial fitness and resistance in vivo.
Purification and Characterization of the Bacterial UDP-GlcNAc:Undecaprenyl-Phosphate GlcNAc-1-Phosphate Transferase WecA
Authors: Al-Dabbagh Bayan , Mengin-Lecreulx Dominique , Bouhss Ahmed 
Published: August 2008

ABSTRACT

To date, the structural and functional characterization of proteins belonging to the polyprenyl-phosphate
N
-acetylhexosamine-1-phosphate transferase superfamily has been relentlessly held back by problems encountered with their overexpression and purification. In the present work and for the first time, the integral membrane protein WecA that catalyzes the transfer of the GlcNAc-1-phosphate moiety from UDP-GlcNAc onto the carrier lipid undecaprenyl phosphate, yielding undecaprenyl-pyrophosphoryl-GlcNAc, the lipid intermediate involved in the synthesis of various bacterial cell envelope components, was overproduced and purified to near homogeneity in milligram quantities. An enzymatic assay was developed, and the kinetic parameters of WecA as well as the effects of pH, salts, cations, detergents, and temperature on the enzyme activity were determined. A minimal length of 35 carbons was required for the lipid substrate, and tunicamycin was shown to inhibit the enzyme at submicromolar concentrations.
SpoT Regulates DnaA Stability and Initiation of DNA Replication in Carbon-StarvedCaulobacter crescentus
Authors: Lesley Joseph A. , Shapiro Lucy 
Published: August 2008

ABSTRACTCell cycle progression and polar differentiation are temporally coordinated inCaulobacter crescentus. This oligotrophic bacterium divides asymmetrically to produce a motile swarmer cell that represses DNA replication and a sessile stalked cell that replicates its DNA. The initiation of DNA replication coincides with the proteolysis of the CtrA replication inhibitor and the accumulation of DnaA, the replication initiator, upon differentiation of the swarmer cell into a stalked cell. We analyzed the adaptive response ofC. crescentusswarmer cells to carbon starvation and found that there was a block in both the swarmer-to-stalked cell polar differentiation program and the initiation of DNA replication. SpoT is a bifunctional synthase/hydrolase that controls the steady-state level of the stress-signaling nucleotide (p)ppGpp, and carbon starvation caused a SpoT-dependent increase in (p)ppGpp concentration. Carbon starvation activates DnaA proteolysis (B. Gorbatyuk and G. T. Marczynski, Mol. Microbiol.55:1233-1245, 2005). We observed that SpoT is required for this phenomenon in swarmer cells, and in the absence of SpoT, carbon-starved swarmer cells inappropriately initiated DNA replication. Since SpoT controls (p)ppGpp abundance, we propose that this nucleotide relays carbon starvation signals to the cellular factors responsible for activating DnaA proteolysis, thereby inhibiting the initiation of DNA replication. SpoT, however, was not required for the carbon starvation block of the swarmer-to-stalked cell polar differentiation program. Thus, swarmer cells utilize at least two independent signaling pathways to relay carbon starvation signals: a SpoT-dependent pathway mediating the inhibition of DNA replication initiation, and a SpoT-independent pathway(s) that blocks morphological differentiation.
Genome Sequence of the Fish Pathogen
Renibacterium salmoninarum
Suggests Reductive Evolution away from an Environmental
Arthrobacter
Ancestor
Authors: Wiens Gregory D. , Rockey Daniel D. , Wu Zaining , Chang Jean , Levy Ruth , Crane Samuel , Chen Donald S. , Capri Gina R. , Burnett Jeffrey R. , Sudheesh Ponnerassery S. , Schipma Matthew J. , Burd Henry , Bhattacharyya Anamitra , Rhodes Linda D. , Kaul Rajinder , Strom Mark S. 
Published: August 2008

ABSTRACT

Renibacterium salmoninarum
is the causative agent of bacterial kidney disease and a significant threat to healthy and sustainable production of salmonid fish worldwide. This pathogen is difficult to culture in vitro, genetic manipulation is challenging, and current therapies and preventative strategies are only marginally effective in preventing disease. The complete genome of
R. salmoninarum
ATCC 33209 was sequenced and shown to be a 3,155,250-bp circular chromosome that is predicted to contain 3,507 open-reading frames (ORFs). A total of 80 copies of three different insertion sequence elements are interspersed throughout the genome. Approximately 21% of the predicted ORFs have been inactivated via frameshifts, point mutations, insertion sequences, and putative deletions. The
R. salmoninarum
genome has extended regions of synteny to the
Arthrobacter
sp. strain FB24 and
Arthrobacter aurescens
TC1 genomes, but it is approximately 1.9 Mb smaller than both
Arthrobacter
genomes and has a lower G+C content, suggesting that significant genome reduction has occurred since divergence from the last common ancestor. A limited set of putative virulence factors appear to have been acquired via horizontal transmission after divergence of the species; these factors include capsular polysaccharides, heme sequestration molecules, and the major secreted cell surface antigen p57 (also known as major soluble antigen). Examination of the genome revealed a number of ORFs homologous to antibiotic resistance genes, including genes encoding β-lactamases, efflux proteins, macrolide glycosyltransferases, and rRNA methyltransferases. The genome sequence provides new insights into
R. salmoninarum
evolution and may facilitate identification of chemotherapeutic targets and vaccine candidates that can be used for prevention and treatment of infections in cultured salmonids.
The Origins of 168, W23, and Other
Bacillus subtilis
Legacy Strains
Authors: Zeigler Daniel R. , Prágai Zoltán , Rodriguez Sabrina , Chevreux Bastien , Muffler Andrea , Albert Thomas , Bai Renyuan , Wyss Markus , Perkins John B. 
Published: August 2008

ABSTRACT

Bacillus subtilis
is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of
B. subtilis
genetics. For three—NCIB 3610
T
, PY79, and SMY—we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more “W23 islands” in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its
trp
and
sac
loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these
B. subtilis
legacy strains.
Stygiolobus
Rod-Shaped Virus and the Interplay of Crenarchaeal Rudiviruses with the CRISPR Antiviral System
Authors: Vestergaard Gisle , Shah Shiraz A. , Bize Ariane , Reitberger Werner , Reuter Monika , Phan Hien , Briegel Ariane , Rachel Reinhard , Garrett Roger A. , Prangishvili David 
Published: August 2008

ABSTRACT

A newly characterized archaeal rudivirus
Stygiolobus
rod-shaped virus (SRV), which infects a hyperthermophilic
Stygiolobus
species, was isolated from a hot spring in the Azores, Portugal. Its virions are rod-shaped, 702 (± 50) by 22 (± 3) nm in size, and nonenveloped and carry three tail fibers at each terminus. The linear double-stranded DNA genome contains 28,096 bp and an inverted terminal repeat of 1,030 bp. The SRV shows morphological and genomic similarities to the other characterized rudiviruses
Sulfolobus
rod-shaped virus 1 (SIRV1), SIRV2, and
Acidianus
rod-shaped virus 1, isolated from hot acidic springs of Iceland and Italy. The single major rudiviral structural protein is shown to generate long tubular structures in vitro of similar dimensions to those of the virion, and we estimate that the virion constitutes a single, superhelical, double-stranded DNA embedded into such a protein structure. Three additional minor conserved structural proteins are also identified. Ubiquitous rudiviral proteins with assigned functions include glycosyl transferases and a
S
-adenosylmethionine-dependent methyltransferase, as well as a Holliday junction resolvase, a transcriptionally coupled helicase and nuclease implicated in DNA replication. Analysis of matches between known crenarchaeal chromosomal CRISPR spacer sequences, implicated in a viral defense system, and rudiviral genomes revealed that about 10% of the 3,042 unique acidothermophile spacers yield significant matches to rudiviral genomes, with a bias to highly conserved protein genes, consistent with the widespread presence of rudiviruses in hot acidophilic environments. We propose that the 12-bp indels which are commonly found in conserved rudiviral protein genes may be generated as a reaction to the presence of the host CRISPR defense system.
The Carboxyl-Terminal Domain of TraR, a
Streptomyces
HutC Family Repressor, Functions in Oligomerization
Authors: Kataoka Masakazu , Tanaka Takeshi , Kohno Toshiyuki , Kajiyama Yusuke 
Published: August 2008

ABSTRACT

Efficient conjugative transfer of the
Streptomyces
plasmid pSN22 is accomplished by regulated expression of the
tra
operon genes,
traA, traB
, and
spdB
. The TraR protein is the central transcriptional repressor regulating the expression of the
tra
operon and itself and is classified as a member of the HutC subfamily in the helix-turn-helix (HTH) GntR protein family. Sequence information predicts that the N-terminal domain (NTD) of TraR, containing an HTH motif, functions in binding of DNA to the
cis
element; however, the function of the C-terminal region remains obscure, like that for many other GntR family proteins. Here we demonstrate the domain structure of the TraR protein and explain the role of the C-terminal domain (CTD). The TraR protein can be divided into two structural domains, the NTD of M1 to R95 and the CTD of Y96 to E246, revealed by limited proteolysis. Domain expression experiments revealed that both domains retained their function. An in vitro pull-down assay using recombinant TraR proteins revealed that TraR oligomerization depended on the CTD. A bacterial two-hybrid system interaction assay revealed that the minimum region necessary for this binding is R95 to P151. A mutant TraR protein in which Leu121 was replaced by His exhibited a loss of both oligomerization ability and repressor function. An in vitro cross-linking assay revealed preferential tetramer formation by TraR and the minimum CTD. These results indicate that the C-terminal R95-to-P151 region of TraR functions to form an oligomer, preferentially a tetramer, that is essential for the repressor function of TraR.
Use of CDP-Glycerol as an Alternate Acceptor for the Teichoic Acid Polymerase Reveals that Membrane Association Regulates Polymer Length
Authors: Schertzer Jeffrey W. , Brown Eric D. 
Published: August 2008

ABSTRACT

The study of bacterial extracellular polysaccharide biosynthesis is hampered by the fact that these molecules are synthesized on membrane-resident carrier lipids. To get around this problem, a practical solution has been to synthesize soluble lipid analogs and study the biosynthetic enzymes using a soluble system. This has been done for the
Bacillus subtilis
teichoic acid polymerase, TagF, although several aspects of catalysis were inconsistent with the results obtained with reconstituted membrane systems or physiological observations. In this work we explored the acceptor substrate promiscuity and polymer length disregulation that appear to be characteristic of TagF activity away from biological membranes. Using isotope labeling, steady-state kinetics, and chemical lability studies, we demonstrated that the enzyme can synthesize poly(glycerol phosphate) teichoic acid using the elongation substrate CDP-glycerol as an acceptor. This suggests that substrate specificity is relaxed in the region distal to the glycerol phosphate moiety in the acceptor molecule under these conditions. Polymer synthesis proceeded at a rate (27 min
−1
) comparable to that in the reconstituted membrane system after a distinct lag period which likely represented slower initiation on the unnatural CDP-glycerol acceptor. We confirmed that polymer length became disregulated in the soluble system as the polymers synthesized on CDP-glycerol acceptors were much larger than the polymers synthesized on the membrane or previously found attached to bacterial cell walls. Finally, polymer synthesis on protease-treated membranes suggested that proper length regulation is retained in the absence of accessory proteins and provided evidence that such regulation is conferred through proper association of the polymerase with the membrane.
Functional Definition and Global Regulation of Zur, a Zinc Uptake Regulator in a
Streptococcus suis
Serotype 2 Strain Causing Streptococcal Toxic Shock Syndrome
Authors: Feng Youjun , Li Ming , Zhang Huimin , Zheng Beiwen , Han Huiming , Wang Changjun , Yan Jinghua , Tang Jiaqi , Gao George F. 
Published: August 2008

ABSTRACT

Zinc is an essential trace element for all living organisms and plays pivotal roles in various cellular processes. However, an excess of zinc is extremely deleterious to cells. Bacteria have evolved complex machineries (such as efflux/influx systems) to control the concentration at levels appropriate for the maintenance of zinc homeostasis in cells and adaptation to the environment. The Zur (zinc uptake regulator) protein is one of these functional members involved in the precise control of zinc homeostasis. Here we identified a
zur
homologue designated 310 from
Streptococcus suis
serotype 2, strain 05ZYH33, a highly invasive isolate causing streptococcal toxic shock syndrome. Biochemical analysis revealed that the protein product of gene 310 exists as a dimer form and carries zinc ions. An isogenic gene replacement mutant of gene 310, the Δ310 mutant, was obtained by homologous recombination. Physiological tests demonstrated that the Δ310 mutant is specifically sensitive to Zn
2+
, while functional complementation of the Δ310 mutant can restore its duration capability, suggesting that 310 is a functional member of the Zur family. Two-dimensional electrophoresis indicated that nine proteins in the Δ310 mutant are overexpressed in comparison with those in the wild type. DNA microarray analyses suggested that 121 genes in the Δ310 mutant are affected, of which 72 genes are upregulated and 49 are downregulated. The transcriptome of
S. suis
serotype 2 with high Zn
2+
concentrations also showed 117 differentially expressed genes, with 71 upregulated and 46 downregulated. Surprisingly, more than 70% of the genes differentially expressed in the Δ310 mutant were the same as those in
S. suis
serotype 2 that were differentially expressed in response to high Zn
2+
concentration, consistent with the notion that 310 is involved in zinc homeostasis. We thus report for the first time a novel zinc-responsive regulator, Zur, from
Streptococcus suis
serotype 2.
TheACTN3R577X polymorphism in Russian endurance athletes
Authors: Ahmetov I I , Druzhevskaya A M , Astratenkova I V , Popov D V , Vinogradova O L , Rogozkin V A 
Published: August 2008

ObjectiveThe functional 577R allele of the α-actinin-3 (ACTN3) gene has been reported to be associated with elite power athlete status, while the nonfunctional 577XX genotype (predicts an α-actinin-3 deficient phenotype) has been hypothesised as providing some sort of advantage for endurance athletes. In the present study, the distribution ofACTN3genotypes and alleles in Russian endurance-oriented athletes were examined and association betweenACTN3genotypes and the competition results of rowers were sought.Methods456 Russian endurance-oriented athletes of regional or national competitive standard were involved in the study.ACTN3genotype and allele frequencies were compared with 1211 controls. The data from the Russian Cup Rowing Tournament were used to search for possible association between theACTN3genotype and the long-distance (∼6 km) rowing results of 54 athletes. DNA was extracted from mouthwash samples. Genotyping for the R577X variant was performed by PCR and restriction enzyme digestion.ResultsThe frequencies of theACTN3577XX genotype (5.7% vs 14.5%; p<0.0001) and 577X allele (33.2% vs 39.0%; p = 0.0025) were significantly lower in endurance-oriented athletes compared with the controls, and none of the highly elite athletes had the 577XX genotype. Furthermore, male rowers withACTN3577RR genotype showed better results (1339 (11) s) in long-distance rowing than carriers of 577RX (1386 (12) s) or 577XX (1402 (10) s) genotypes (p = 0.016).ConclusionOur data show that theACTN3577X allele is under-represented in Russian endurance athletes and is associated with the rowers’ competition results.