bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification ofsarV(SA2062), a New Transcriptional Regulator, Is Repressed by SarA and MgrA (SA0641) and Involved in the Regulation of Autolysis inStaphylococcus aureus
Authors: Manna Adhar C. , Ingavale Susham S. , Maloney MaryBeth , van Wamel Willem , Cheung Ambrose L. 
Published: August 2004

ABSTRACTThe expression of genes involved in the pathogenesis ofStaphylococcus aureusis known to be controlled by global regulatory loci, includingagr,sarA,sae,arlRS,lytSR, andsarA-like genes. Here we described a novel transcriptional regulator calledsarVof the SarA protein family. The transcription ofsarVis low or undetectable under in vitro conditions but is significantly augmented insarAandmgrA(norRorrat) (SA0641) mutants. ThesarAandmgrAgenes act as repressors ofsarVexpression, as confirmed by transcriptional fusion and Northern analysis data. Purified SarA and MgrA proteins bound specifically to separate regions of thesarVpromoter as determined by gel shift and DNase I footprinting assays. The expression of 19 potential target genes involved in autolysis and virulence, phenotypes affected bysarAandmgrA, was evaluated in an isogenicsarVmutant pair. Our data indicated that thesarVgene product played a role regulating some virulence genes and more genes involved in autolysis. ThesarVmutant was more resistant to Triton X-100 and penicillin-induced lysis compared to the wild type and thesarAmutant, whereas hyperexpression ofsarVin the parental strain or thesarVmutant rendered the resultant strain highly susceptible to lysis. Zymographic analysis of murein hydrolase activity revealed that inactivation of thesarVgene results in decreased extracellular murein hydrolase activity compared to that of wild-typeS. aureus. We propose thatsarVmay be part of the common pathway by whichmgrAandsarAgene products control autolysis inS. aureus.
Interaction of PomB with the Third Transmembrane Segment of PomA in the Na
+
-Driven Polar Flagellum of
Vibrio alginolyticus
Authors: Yakushi Toshiharu , Maki Shingo , Homma Michio 
Published: August 2004

ABSTRACT

The marine bacterium
Vibrio alginolyticus
has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na
+
-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na
+
than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na
+
conduction to flagellar rotation.
Biochemical Characterization of StyAB from
Pseudomonas
sp. Strain VLB120 as a Two-Component Flavin-Diffusible Monooxygenase
Authors: Otto Katja , Hofstetter Karin , Röthlisberger Martina , Witholt Bernard , Schmid Andreas 
Published: August 2004

ABSTRACT

Pseudomonas
sp. VLB120 uses styrene as a sole source of carbon and energy. The first step in this metabolic pathway is catalyzed by an oxygenase (StyA) and a NADH-flavin oxidoreductase (StyB). Both components have been isolated from wild-type
Pseudomonas
strain VLB120 as well as from recombinant
Escherichia coli
. StyA from both sources is a dimer, with a subunit size of 47 kDa, and catalyzes the enantioselective epoxidation of C═C double bonds. Styrene is exclusively converted to
S
-styrene oxide with a specific activity of 2.1 U mg
−1
(
k
cat
= 1.6 s
−1
) and

K
m

values for styrene of 0.45 ± 0.05 mM (wild type) and 0.38 ± 0.09 mM (recombinant). The epoxidation reaction depends on the presence of a NADH-flavin adenine dinucleotide (NADH-FAD) oxidoreductase for the supply of reduced FAD. StyB is a dimer with a molecular mass of 18 kDa and a NADH oxidation activity of 200 U mg
−1
(
k
cat
[NADH] = 60 s
−1
). Steady-state kinetics determined for StyB indicate a mechanism of sequential binding of NADH and flavin to StyB. This enzyme reduces FAD as well as flavin mononucleotide and riboflavin. The NADH oxidation activity does not depend on the presence of StyA. During the epoxidation reaction, no formation of a complex of StyA and StyB has been observed, suggesting that electron transport between reductase and oxygenase occurs via a diffusing flavin.
Defined Inactive FecA Derivatives Mutated in Functional Domains of the Outer Membrane Transport and Signaling Protein of
Escherichia coli
K-12
Authors: Sauter Annette , Braun Volkmar 
Published: August 2004

ABSTRACT

The FecA outer membrane protein of
Escherichia coli
functions as a transporter of ferric citrate and as a signal receiver and signal transmitter for transcription initiation of the
fec
transport genes. Three FecA regions for which functional roles have been predicted from the crystal structures were mutagenized: (i) loops 7 and 8, which move upon binding of ferric citrate and close the entrance to the ferric citrate binding site; (ii) the dinuclear ferric citrate binding site; and (iii) the interface between the globular domain and the β-barrel. Deletion of loops 7 and 8 abolished FecA transport and induction activities. Deletion of loops 3 and 11 also inactivated FecA, whereas deletion of loops 9 and 10 largely retained FecA activities. The replacement of arginine residue R365 or R380 and glutamine Q570, which are predicted to serve as binding sites for the negatively charged dinuclear ferric citrate, with alanine resulted in inactive FecA, whereas the binding site mutant R438A retained approximately 50% of the FecA induction and transport activities. Residues R150, E541, and E587, conserved among energy-coupled outer membrane transporters, are predicted to form salt bridges between the globular domain and the β-barrel and to contribute to the fixation of the globular domain inside the β-barrel. Mutations E541A and E541R affected FecA induction and transport activity slightly, whereas mutations E587A and E587R more strongly reduced FecA activity. The double mutations R150A E541R and R150A E587R nearly abolished FecA activity. Apparently, the salt bridges are less important than the individual functions these residues seem to have for FecA activity. Comparison of the properties of the FecA, FhuA, FepA, and BtuB transporters indicates that although they have very similar crystal structures, the details of their functional mechanisms differ.
Molecular Analysis of Cytolysin A (ClyA) in Pathogenic
Escherichia coli
Strains
Authors: Ludwig Albrecht , von Rhein Christine , Bauer Susanne , Hüttinger Christian , Goebel Werner 
Published: August 2004

ABSTRACT

Cytolysin A (ClyA) of
Escherichia coli
is a pore-forming hemolytic protein encoded by the
clyA
(
hlyE
,
sheA
) gene that was first identified in
E. coli
K-12. In this study we examined various clinical
E. coli
isolates with regard to the presence and integrity of
clyA
. PCR and DNA sequence analyses demonstrated that 19 of 23 tested Shiga toxin-producing
E. coli
(STEC) strains, all 7 tested enteroinvasive
E. coli
(EIEC) strains, 6 of 8 enteroaggregative
E. coli
(EAEC) strains, and 4 of 7 tested enterotoxigenic
E. coli
(ETEC) strains possess a complete
clyA
gene. The remaining STEC, EAEC, and ETEC strains and 9 of the 17 tested enteropathogenic
E. coli
(EPEC) strains were shown to harbor mutant
clyA
derivatives containing 1-bp frameshift mutations that cause premature termination of the coding sequence. The other eight EPEC strains and all tested uropathogenic and new-born meningitis-associated
E. coli
strains (
n
= 14 and 3, respectively) carried only nonfunctional
clyA
fragments due to the deletion of two sequences of 493 bp and 204 or 217 bp at the
clyA
locus. Expression of
clyA
from clinical
E. coli
isolates proved to be positively controlled by the transcriptional regulator SlyA. Several tested
E. coli
strains harboring a functional
clyA
gene produced basal amounts of ClyA when grown under standard laboratory conditions, but most of them showed a
clyA
-dependent hemolytic phenotype only when SlyA was overexpressed. The presented data indicate that cytolysin A can play a role only for some of the pathogenic
E. coli
strains.
The Usher N Terminus Is the Initial Targeting Site for Chaperone-Subunit Complexes and Participates in Subsequent Pilus Biogenesis Events
Authors: Ng Tony W. , Akman Leyla , Osisami Mary , Thanassi David G. 
Published: August 2004

ABSTRACT

Pilus biogenesis on the surface of uropathogenic
Escherichia coli
requires the chaperone/usher pathway, a terminal branch of the general secretory pathway. In this pathway, periplasmic chaperone-subunit complexes target an outer membrane (OM) usher for subunit assembly into pili and secretion to the cell surface. The molecular mechanisms of protein secretion across the OM are not well understood. Mutagenesis of the P pilus usher PapC and the type 1 pilus usher FimD was undertaken to elucidate the initial stages of pilus biogenesis at the OM. Deletion of residues 2 to 11 of the mature PapC N terminus abolished the targeting of the usher by chaperone-subunit complexes and rendered PapC nonfunctional for pilus biogenesis. Similarly, an intact FimD N terminus was required for chaperone-subunit binding and pilus biogenesis. Analysis of PapC-FimD chimeras and N-terminal fragments of PapC localized the chaperone-subunit targeting domain to the first 124 residues of PapC. Single alanine substitution mutations were made in this domain that blocked pilus biogenesis but did not affect targeting of chaperone-subunit complexes. Thus, the usher N terminus does not function simply as a static binding site for chaperone-subunit complexes but also participates in subsequent pilus assembly events.
Role of an Inducible Single-Domain Hemoglobin in Mediating Resistance to Nitric Oxide and Nitrosative Stress in
Campylobacter jejuni
and
Campylobacter coli
Authors: Elvers Karen T. , Wu Guanghui , Gilberthorpe Nicola J. , Poole Robert K. , Park Simon F. 
Published: August 2004

ABSTRACT

Campylobacter jejuni
expresses two hemoglobins, each of which exhibits a heme pocket and structural signatures in common with vertebrate and plant globins. One of these, designated Cgb, is homologous to Vgb from
Vitreoscilla stercoraria
and does not possess the reductase domain seen in the flavohemoglobins. A Cgb-deficient mutant of
C. jejuni
was hypersensitive to nitrosating agents (
S
-nitrosoglutathione [GSNO] or sodium nitroprusside) and a nitric oxide-releasing compound (spermine NONOate). The sensitivity of the Cgb-deficient mutant to methyl viologen, hydrogen peroxide, and organic peroxides, however, was the same as for the wild type. Consistent with the protective role of Cgb against NO-related stress,
cgb
expression was minimal in standard laboratory media but strongly and specifically induced after exposure to nitrosative stress. In contrast, the expression of Cgb was independent of aeration and the presence of superoxide. In the absence of preinduction by exposure to nitrosative stress, no difference was seen in the degree of respiratory inhibition by NO or the half-life of the NO signal when cells of the wild type and the
cgb
mutant were compared. However, cells expressing GSNO-upregulated levels of Cgb exhibited robust NO consumption and respiration that was relatively NO insensitive compared to the respiration of the
cgb
mutant. Based on similar studies in
Campylobacter coli
, we also propose an identical role for Cgb in this closely related species. We conclude that, unlike the archetypal single-domain globin Vgb, Cgb forms a specific and inducible defense against NO and nitrosating agents.
Penetration of Membrane-Containing Double-Stranded-DNA Bacteriophage PM2 intoPseudoalteromonasHosts
Authors: Kivelä Hanna M. , Daugelavičius Rimantas , Hankkio Riina H. , Bamford Jaana K. H. , Bamford Dennis H. 
Published: August 2004

ABSTRACTThe icosahedral bacteriophage PM2 has a circular double-stranded DNA (dsDNA) genome and an internal lipid membrane. It is the only representative of theCorticoviridaefamily. How the circular supercoiled genome residing inside the viral membrane is translocated into the gram-negative marinePseudoalteromonashost has been an intriguing question. Here we demonstrate that after binding of the virus to an abundant cell surface receptor, the protein coat is most probably dissociated. During the infection process, the host cell outer membrane becomes transiently permeable to lipophilic gramicidin D molecules proposing fusion with the viral membrane. One of the components of the internal viral lipid core particle is the integral membrane protein P7, with muralytic activity that apparently aids the process of peptidoglycan penetration. Entry of the virion also causes a limited depolarization of the cytoplasmic membrane. These phenomena differ considerably from those observed in the entry process of bacteriophage PRD1, a dsDNA virus, which uses its internal membrane to make a cell envelope-penetrating tubular structure.
Differential and Cross-Transcriptional Control of Duplicated Genes Encoding Alternative Sigma Factors inStreptomyces ambofaciens
Authors: Roth Virginie , Aigle Bertrand , Bunet Robert , Wenner Thomas , Fourrier Céline , Decaris Bernard , Leblond Pierre 
Published: August 2004

ABSTRACTThe duplicatedhasRandhasLgenes ofStreptomyces ambofaciensencode alternative sigma factors (named σBRand σBL) belonging to the σBgeneral stress response family inBacillus subtilis. The duplication appears to be the result of a recent event that occurred specifically inS. ambofaciens. The two genes are 98% identical, and their deduced protein products exhibit 97% identity at the amino acid level. In contrast with the coding sequences, their genetic environments and their transcriptional control are strongly divergent. WhilehasLis monocistronic,hasRis arranged in a polycistronic unit with two upstream open reading frames,arsRandprsR, that encode putative anti-anti-σ and anti-σ factors, respectively. Transcription of eachhasgene is initiated from two promoters. In each case, one promoter was shown to be developmentally controlled and to be similar to those recognized by theB. subtilisgeneral stress response sigma factor σB. Expression from this type of promoter for each of thehasgenes dramatically increases during the course of growth in liquid or on solid media and following oxidative and osmotic stresses. Reverse transcription-PCR measurements indicate thathasRis 100 times more strongly expressed thanhasLfrom the σB-like promoter. Transcription from the second promoter of each gene (located upstream ofarsRin the case of thehasRlocus) appears to be constitutive and weak. Quantitative transcriptional analysis in single and doublehasmutant strains revealed that σBRand σBLdirect their own transcription as well as that of their duplicates. Only a slight sensitivity in response to oxidative conditions could be assigned to either single or double mutants, revealing the probable redundancy of the σ factors implied in stress response inStreptomyces.
Role of the Pilot Protein YscW in the Biogenesis of the YscC Secretin in
Yersinia enterocolitica
Authors: Burghout Peter , Beckers Frank , de Wit Emmie , van Boxtel Ria , Cornelis Guy R. , Tommassen Jan , Koster Margot 
Published: August 2004

ABSTRACT

The YscC secretin is a major component of the type III protein secretion system of
Yersinia enterocolitica
and forms an oligomeric structure in the outer membrane. In a mutant lacking the outer membrane lipoprotein YscW, secretion is strongly reduced, and it has been proposed that YscW plays a role in the biogenesis of the secretin. To study the interaction between the secretin and this putative pilot protein, YscC and YscW were produced in
trans
in a
Y. enterocolitica
strain lacking all other components of the secretion machinery. YscW expression increased the yield of oligomeric YscC and was required for its outer membrane localization, confirming the function of YscW as a pilot protein. Whereas the pilot-binding site of other members of the secretin family has been identified in the C terminus, a truncated YscC derivative lacking the C-terminal 96 amino acid residues was functional and stabilized by YscW. Pulse-chase experiments revealed that ∼30 min were required before YscC oligomerization was completed. In the absence of YscW, oligomerization was delayed and the yield of YscC oligomers was strongly reduced. An unlipidated form of the YscW protein was not functional, although it still interacted with the secretin and caused mislocalization of YscC even in the presence of wild-type YscW. Hence, YscW interacts with the unassembled YscC protein and facilitates efficient oligomerization, likely at the outer membrane.
Bacillus subtilis
YhcR, a High-Molecular-Weight, Nonspecific Endonuclease with a Unique Domain Structure
Authors: Oussenko Irina A. , Sanchez Roberto , Bechhofer David H. 
Published: August 2004

ABSTRACT

In a continuing effort to identify ribonucleases that may be involved in mRNA decay in
Bacillus subtilis
, fractionation of a protein extract from a triple-mutant strain that was missing three previously characterized 3′-to-5′ exoribonucleases (polynucleotide phosphorylase [PNPase], RNase R, and YhaM) was undertaken. These experiments revealed the presence of a high-molecular-weight nuclease encoded by the
yhcR
gene that was active in the presence of Ca
2+
and Mn
2+
. YhcR is a sugar-nonspecific nuclease that cleaves endonucleolytically to yield nucleotide 3′-monophosphate products, similar to the well-characterized micrococcal nuclease of
Staphylococcus aureus
. YhcR appears to be located principally in the cell wall and is likely to be a substrate for a
B. subtilis
sortase. Zymogram analysis suggests that YhcR is the major Ca
2+
-activated nuclease of
B. subtilis
. In addition to having a unique overall domain structure, YhcR contains a hitherto unknown structural domain that we have named “NYD,” for “new YhcR domain.”
Indole-3-Acetic Acid Biosynthesis Is Deficient in
Gluconacetobacter diazotrophicus
Strains with Mutations in Cytochrome
c
Biogenesis Genes
Authors: Lee Sunhee , Flores-Encarnación M. , Contreras-Zentella M. , Garcia-Flores L. , Escamilla J. E. , Kennedy Christina 
Published: August 2004

ABSTRACT

Gluconacetobacter diazotrophicus
is an endophyte of sugarcane frequently found in plants grown in agricultural areas where nitrogen fertilizer input is low. Recent results from this laboratory, using mutant strains of
G. diazotrophicus
unable to fix nitrogen, suggested that there are two beneficial effects of
G. diazotrophicus
on sugarcane growth: one dependent and one not dependent on nitrogen fixation. A plant growth-promoting substance, such as indole-3-acetic acid (IAA), known to be produced by
G. diazotrophicus
, could be a nitrogen fixation-independent factor. One strain, MAd10, isolated by screening a library of Tn
5
mutants, released only ∼6% of the amount of IAA excreted by the parent strain in liquid culture. The mutation causing the IAA
−
phenotype was not linked to Tn
5
. A pLAFR3 cosmid clone that complemented the IAA deficiency was isolated. Sequence analysis of a complementing subclone indicated the presence of genes involved in cytochrome
c
biogenesis (
ccm
, for cytochrome
c
maturation). The
G. diazotrophicus ccm
operon was sequenced; the individual
ccm
gene products were 37 to 52% identical to
ccm
gene products of
Escherichia coli
and equivalent
cyc
genes of
Bradyrhizobium japonicum
. Although several
ccm
mutant phenotypes have been described in the literature, there are no reports of
ccm
gene products being involved in IAA production. Spectral analysis, heme-associated peroxidase activities, and respiratory activities of the cell membranes revealed that the
ccm
genes of
G. diazotrophicus
are involved in cytochrome
c
biogenesis.
Kinetic Analysis of tRNA-Directed Transcription Antitermination of the
Bacillus subtilis glyQS
Gene In Vitro
Authors: Grundy Frank J. , Henkin Tina M. 
Published: August 2004

ABSTRACT

Binding of uncharged tRNA to the nascent transcript promotes readthrough of a leader region transcription termination signal in genes regulated by the T box transcription antitermination mechanism. Each gene in the T box family responds independently to its cognate tRNA, with specificity determined by base pairing of the tRNA to the leader at the anticodon and acceptor ends of the tRNA. tRNA binding stabilizes an antiterminator element in the transcript that sequesters sequences that participate in formation of the terminator helix. tRNA
Gly
-dependent antitermination of the
Bacillus subtilis glyQS
leader was previously demonstrated in a purified in vitro assay system. This assay system was used to investigate the kinetics of transcription through the
glyQS
leader and the effect of tRNA and transcription elongation factors NusA and NusG on transcriptional pausing and antitermination. Several pause sites, including a major site in the loop of stem III of the leader, were identified, and the effect of modulation of pausing on antitermination efficiency was analyzed. We found that addition of tRNA
Gly
can promote antitermination as long as the tRNA is added before the majority of the transcription complexes reach the termination site, and variations in pausing affect the requirements for timing of tRNA addition.
Isoleucine Biosynthesis in
Leptospira interrogans
Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway
Authors: Xu Hai , Zhang Yuzhen , Guo Xiaokui , Ren Shuangxi , Staempfli Andreas A. , Chiao Juishen , Jiang Weihong , Zhao Guoping 
Published: August 2004

ABSTRACT

Three
leuA
-like protein-coding sequences were identified in
Leptospira interrogans
. One of these, the
cimA
gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded α-isopropylmalate synthase (EC 4.1.3.12). Expressed in
Escherichia coli
, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile
Methanococcus jannaschii
, the
L. interrogans
enzyme is temperature sensitive but exhibits a much lower

K
m

(0.04 mM) for pyruvate. The reaction product was characterized as (
R
)-citramalate, and the proposed β-methyl-
d
-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and β-isopropylmalate dehydrogenase (EC 1.1.1.85) in
E. coli
extracts bearing the corresponding clones and in vivo by complementation of
E. coli ilvA
,
leuC
/
D
, and
leuB
mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in
L. interrogans
under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the
cimA
and
leuA
genes were detected in
L. interrogans
grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes.