bluuebunny/crossref_metadata_2025 huggingface dataset:
The Bacteriophage T4 Inhibitor and Coactivator AsiA InhibitsEscherichia coliRNA Polymerase More Rapidly in the Absence of σ70Region 1.1: Evidence that Region 1.1 Stabilizes the Interaction between σ70and Core
Authors: Hinton Deborah M. , Vuthoori Srilatha , Mulamba Rebecca
Published: February 2006

The N-terminal region (region 1.1) of σ70, the primary σ subunit ofEscherichia coliRNA polymerase, is a negatively charged domain that affects the DNA binding properties of σ70regions 2 and 4. Region 1.1 prevents the interaction of free σ70with DNA and modulates the formation of stable (open) polymerase/promoter complexes at certain promoters. The bacteriophage T4 AsiA protein is an inhibitor of σ70-dependent transcription from promoters that require an interaction between σ70region 4 and the −35 DNA element and is the coactivator of transcription at T4 MotA-dependent promoters. Like AsiA, the T4 activator MotA also interacts with σ70region 4. We have investigated the effect of region 1.1 on AsiA inhibition and MotA/AsiA activation. We show that σ70region 1.1 is not required for MotA/AsiA activation at the T4 middle promoter PuvsX. However, the rate of AsiA inhibition and of MotA/AsiA activation of polymerase is significantly increased when region 1.1 is missing. We also find that RNA polymerase reconstituted with σ70that lacks region 1.1 is less stable than polymerase with full-length σ70. Our previous work has demonstrated that the AsiA-inhibited polymerase is formed when AsiA binds to region 4 of free σ70and then the AsiA/σ70complex binds to core. Our results suggest that in the absence of region 1.1, there is a shift in the dynamic equilibrium between polymerase holoenzyme and free σ70plus core, yielding more free σ70at any given time. Thus, the rate of AsiA inhibition and AsiA/MotA activation increases when RNA polymerase lacks region 1.1 because of the increased availability of free σ70. Previous work has argued both for and against a direct interaction between regions 1.1 and 4. Using anE. colitwo-hybrid assay, we do not detect an interaction between these regions. This result supports the idea that the ability of region 1.1 to prevent DNA binding by free σ70arises through an indirect effect.
Sll1717 Affects the Redox State of the Plastoquinone Pool by Modulating Quinol Oxidase Activity in Thylakoids
Authors: Kufryk Galyna I. , Vermaas Wim F. J.
Published: February 2006

ASynechocystissp. strain PCC 6803 mutant lacking CtaI, a main subunit of cytochromecoxidase, is not capable of growing at light intensities below 5 μmol photons m−2s−1, presumably due to an overreduced plastoquinone pool in the thylakoid membrane. Upon selection for growth at light intensities below 5 μmol photons m−2s−1, a secondary mutant was generated that retained the CtaI deletion and had fully assembled photosystem II complexes; in this secondary mutant (pseudorevertant), oxygen evolution and respiratory activities were similar to those in the wild type. Functional complementation of the original CtaI-less strain to low-light tolerance by transformation with restriction fragments of genomic DNA of the pseudorevertant and subsequent mapping of the pseudoreversion site showed that the point mutation led to a Ser186Cys substitution in Sll1717, a protein of as-yet-unknown function and with a predicted ATP/GTP-binding domain. This mutation caused a decrease in the plastoquinone pool reduction level of thylakoids compared to that observed for the wild type. Based on a variety of experimental evidence, the most plausible mechanism to cause this effect is an activation of plastoquinol oxidation in thylakoids by the quinol oxidase CydAB that occurs without upregulation of the corresponding gene and that may be caused by an increased CydAB activity in thylakoids, conceivably due to altered CydAB sorting between cytoplasmic and thylakoid membranes. Sll1717 appears to be unique toSynechocystissp. strain PCC 6803 and has a close homologue encoded in the genome of this organism. The transcript level of sll1717 is low, which suggests that the corresponding protein is regulatory rather than structural.
The
Brucella abortus xthA-1
Gene Product Participates in Base Excision Repair and Resistance to Oxidative Killing but Is Not Required for Wild-Type Virulence in the Mouse Model
Authors: Hornback Michael L. , Roop R. Martin
Published: February 2006

Exonuclease III, encoded by the
xthA
gene, plays a central role in the base excision pathway of DNA repair in bacteria. Studies with
Escherichia coli xthA
mutants have also shown that exonuclease III participates in the repair of oxidative damage to DNA. An isogenic
xthA-1
mutant (designated CAM220) derived from virulent
Brucella abortus
2308 exhibited increased sensitivity to the alkylating agent methyl methanesulfonate (MMS) compared to the parent strain. In contrast, 2308 and the isogenic
xthA-1
mutant displayed similar levels of resistance to the DNA cross-linker mitomycin C. These phenotypic properties are those that would be predicted for a strain defective in base excision repair. The
B. abortus xthA-1
mutant also displayed reduced resistance to killing by H
2
O
2
and the ONOO
−
-generating compound 3-morpholinosydnonimine (SIN-1) compared to strain 2308, indicating that the
xthA-1
gene product participates in protecting
B. abortus
2308 from oxidative damage. Introducing a plasmid-borne copy of the parental
xthA-1
gene into CAM220 restored wild-type resistance of this mutant to MMS, H
2
O
2
, and SIN-1. Although the
B. abortus xthA-1
mutant exhibited increased sensitivity to oxidative killing compared to the parental strain in laboratory assays, CAM220 and 2308 displayed equivalent spleen colonization profiles in BALB/c mice through 8 weeks postinfection and equivalent intracellular survival and replication profiles in cultured murine macrophages. Thus, although the
xthA-1
gene product participates in base excision repair and resistance to oxidative killing in
B. abortus
2308, XthA-1 is not required for wild-type virulence of this strain in the mouse model.
The Innate Immune Modulators Staphylococcal Complement Inhibitor and Chemotaxis Inhibitory Protein ofStaphylococcus aureusAre Located on β-Hemolysin-Converting Bacteriophages
Authors: van Wamel Willem J. B. , Rooijakkers Suzan H. M. , Ruyken Maartje , van Kessel Kok P. M. , van Strijp Jos A. G.
Published: February 2006

Two newly discovered immune modulators, chemotaxis inhibitory protein ofStaphylococcus aureus(CHIPS) and staphylococcal complement inhibitor (SCIN), cluster on the conserved 3′ end of β-hemolysin (hlb)-converting bacteriophages (βC-φs). Since these βC-φs also carry the genes for the immune evasion molecules staphylokinase (sak) and enterotoxin A (sea), this 8-kb region at the 3′ end of βC-φ represents an innate immune evasion cluster (IEC). By PCR and Southern analyses of 85 clinicalStaphylococcus aureusstrains and 5 classical laboratory strains, we show that 90% ofS. aureusstrains carry a βC-φ with an IEC. Seven IEC variants were discovered, carrying different combinations ofchp,sak, orsea(orsep), always in the same 5′-to-3′ orientation and on the 3′ end of a βC-φ. From most IEC variants we could isolate active bacteriophages by mitomycin C treatment, of which lysogens were generated inS. aureusR5 (broad phage host). All IEC-carrying bacteriophages integrated intohlb, as was measured by Southern blotting of R5 lysogens. Large quantities of the different bacteriophages were obtained by mitomycin C treatment of the lysogens, and bacteriophages were collected and used to reinfect all lysogenic R5 strains. In total, five lytic families were found. Furthermore, phage DNA was isolated and digested with EcoR1, revealing that one IEC variant can be found on different βI-φs. In conclusion, the four human-specific innate immune modulators SCIN, CHIPS, SAK, and SEA form an IEC that is easily transferred amongS. aureusstrains by a diverse group of β-hemolysin-converting bacteriophages.
The Transcriptional Antiterminator RfaH Represses Biofilm Formation inEscherichia coli
Authors: Beloin Christophe , Michaelis Kai , Lindner Karin , Landini Paolo , Hacker Jörg , Ghigo Jean-Marc , Dobrindt Ulrich
Published: February 2006

We investigated the influence of regulatory and pathogenicity island-associated factors (Hha, RpoS, LuxS, EvgA, RfaH, and tRNA5Leu) on biofilm formation by uropathogenicEscherichia coli(UPEC) strain 536. Only inactivation ofrfaH, which encodes a transcriptional antiterminator, resulted in increased initial adhesion and biofilm formation byE. coli536.rfaHinactivation in nonpathogenicE. coliK-12 isolate MG1655 resulted in the same phenotype. Transcriptome analysis of wild-type strain 536 and anrfaHmutant of this strain revealed that deletion ofrfaHcorrelated with increased expression offluorthologs.fluencodes antigen 43 (Ag43), which mediates autoaggregation and biofilm formation. We confirmed that deletion ofrfaHleads to increased levels offluandflu-like transcripts inE. coliK-12 and UPEC. Supporting the hypothesis that RfaH represses biofilm formation through reduction of the Ag43 level, the increased-biofilm phenotype ofE. coliMG1655rfaHwas reversed upon inactivation offlu. Deletion of the twofluorthologs, however, did not modify the behavior of mutant 536rfaH. Our results demonstrate that the strong initial adhesion and biofilm formation capacities of strain MG1655rfaHare mediated by both increased steady-state production of Ag43 and likely increased Ag43 presentation due to nullrfaH-dependent lipopolysaccharide depletion. Although the roles ofrfaHin the biofilm phenotype are different in UPEC strain 536 and K-12 strain MG1655, this study shows that RfaH, in addition to affecting the expression of bacterial virulence factors, also negatively controls expression and surface presentation of Ag43 and possibly another Ag43-independent factor(s) that mediates cell-cell interactions and biofilm formation.
Characterization of the Twin-Arginine Translocase Secretion System of
Mycobacterium smegmatis
Authors: Posey James E. , Shinnick Thomas M. , Quinn Frederick D.
Published: February 2006

The twin-arginine translocation (TAT) system secretes fully folded proteins that contain a twin-arginine motif within their signal sequence across the cytoplasmic membrane in bacteria. Using a green fluorescent protein fused with a TAT signal sequence, we demonstrated that
Mycobacterium smegmatis
contains a TAT system. By inactivating individual genes, we showed that three genes (
tatA
,
tatB
, and
tatC
) are required for a functional TAT system in
M. smegmatis
. The
tat
mutants exhibited a decreased growth rate and altered colony morphology compared to the parent strain. Comparison of the secreted proteins of the Δ
tatC
and parent strain by two-dimensional polyacrylamide gel electrophoresis revealed an alteration in the secretion of at least five proteins, and one of the major TAT-dependent secreted proteins was identified as β-lactamase (BlaS). The genome of
M. smegmatis
was analyzed with the TATFIND program, and 49 putative TAT substrates were identified, including the succinate transporter DctP. Because disruption of the TAT secretion system has a direct effect on the physiology of
M. smegmatis
and homologs of the TAT proteins are also present in the genome of
Mycobacterium tuberculosis
, the TAT secretion system or its substrates may be good candidates for drug or vaccine development.
Pyruvate:Quinone Oxidoreductase in
Corynebacterium glutamicum
: Molecular Analysis of the
pqo
Gene, Significance of the Enzyme, and Phylogenetic Aspects
Authors: Schreiner Mark E. , Riedel Christian , Holátko Jiři , Pátek Miroslav , Eikmanns Bernhard J.
Published: February 2006

Corynebacterium glutamicum
recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO
2
with a quinone as the electron acceptor. Here, we analyze the expression of the
C. glutamicum pqo
gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of
pqo
is initiated 45 bp upstream of the translational start site and that it is organized in an operon together with genes encoding a putative metal-activated pyridoxal enzyme and a putative activator protein. Inactivation of the chromosomal
pqo
gene led to the absence of PQO activity; however, growth and amino acid production were not affected under either condition tested. Introduction of plasmid-bound
pqo
into a pyruvate dehydrogenase complex-negative
C. glutamicum
strain partially relieved the growth phenotype of this mutant, indicating that high PQO activity can compensate for the function of the pyruvate dehydrogenase complex. To investigate the distribution of PQO enzymes in prokaryotes and to clarify the relationship between PQO, pyruvate oxidase (POX), and acetohydroxy acid synthase enzymes, we compiled and analyzed the phylogeny of respective proteins deposited in public databases. The analyses revealed a wide distribution of PQOs among prokaryotes, corroborated the hypothesis of a common ancestry of the three enzymes, and led us to propose that the POX enzymes of
Lactobacillales
were derived from a PQO.
lbtAandlbtBAre Required for Production of theLegionella pneumophilaSiderophore Legiobactin
Authors: Allard Kimberly A. , Viswanathan V. K. , Cianciotto Nicholas P.
Published: February 2006

Under iron stress,Legionella pneumophilasecretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes,lbtAandlbtB, which are involved in legiobactin production.lbtAappears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein ofL. pneumophila. lbtBencodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lackinglbtAorlbtBwere defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2′,2′-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. ThelbtAmutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of thelbtmutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. ThelbtAmutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However,L. pneumophilaexpresseslbtAin the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery oflbtABrepresents the first identification of genes required forL. pneumophilasiderophore expression.
Disruption of the Operon Encoding Ehb Hydrogenase Limits Anabolic CO
2
Assimilation in the Archaeon
Methanococcus maripaludis
Authors: Porat Iris , Kim Wonduck , Hendrickson Erik L. , Xia Qiangwei , Zhang Yi , Wang Tiansong , Taub Fred , Moore Brian C. , Anderson Iain J. , Hackett Murray , Leigh John A. , Whitman William B.
Published: February 2006

Methanococcus maripaludis
is a mesophilic archaeon that reduces CO
2
to methane with H
2
or formate as an energy source. It contains two membrane-bound energy-conserving hydrogenases, Eha and Ehb. To determine the role of Ehb, a deletion in the
ehb
operon was constructed to yield the mutant, strain S40. Growth of S40 was severely impaired in minimal medium. Both acetate and yeast extract were necessary to restore growth to nearly wild-type levels, suggesting that Ehb was involved in multiple steps in carbon assimilation. However, no differences in the total hydrogenase specific activities were found between the wild type and mutant in either cell extracts or membrane-purified fractions. Methanogenesis by resting cells with pyruvate as the electron donor was also reduced by 30% in S40, suggesting a defect in pyruvate oxidation. CO dehydrogenase/acetyl coenzyme A (CoA) synthase and pyruvate oxidoreductase had higher specific activities in the mutant, and genes encoding these enzymes, as well as AMP-forming acetyl-CoA synthetase, were expressed at increased levels. These observations support a role for Ehb in anabolic CO
2
assimilation in methanococci.
Characterization of Late Acyltransferase Genes ofYersinia pestisand Their Role in Temperature-Dependent Lipid A Variation
Authors: Rebeil Roberto , Ernst Robert K. , Jarrett Clayton O. , Adams Kristin N. , Miller Samuel I. , Hinnebusch B. Joseph
Published: February 2006

Yersinia pestisis an important human pathogen that is maintained in flea-rodent enzootic cycles in many parts of the world. During its life cycle,Y. pestissenses host-specific environmental cues such as temperature and regulates gene expression appropriately to adapt to the insect or mammalian host. For example,Y. pestissynthesizes different forms of lipid A when grown at temperatures corresponding to the in vivo environments of the mammalian host and the flea vector. At 37°C, tetra-acylated lipid A is the major form; but at 26°C or below, hexa-acylated lipid A predominates. In this study, we show that theY. pestis msbB(lpxM) andlpxPhomologs encode the acyltransferases that add C12and C16:1groups, respectively, to lipid IVAto generate the hexa-acylated form, and that their expression is upregulated at 21°C in vitro and in the flea midgut. AY. pestis ΔmsbB ΔlpxPdouble mutant that did not produce hexa-acylated lipid A was more sensitive to cecropin A, but not to polymyxin B. This mutant was able to infect and block fleas as well as the parental wild-type strain, indicating that the low-temperature-dependent change to hexa-acylated lipid A synthesis is not required for survival in the flea gut.
Inhibition of Cell Division Suppresses Heterocyst Development in
Anabaena
sp. Strain PCC 7120
Authors: Sakr Samer , Jeanjean Robert , Zhang Cheng-Cai , Arcondeguy Tania
Published: February 2006

When the filamentous cyanobacterium
Anabaena
PCC 7120 is exposed to combined nitrogen starvation, 5 to 10% of the cells along each filament at semiregular intervals differentiate into heterocysts specialized in nitrogen fixation. Heterocysts are terminally differentiated cells in which the major cell division protein FtsZ is undetectable. In this report, we provide molecular evidence indicating that cell division is necessary for heterocyst development. FtsZ, which is translationally fused to the green fluorescent protein (GFP) as a reporter, is found to form a ring structure at the mid-cell position. SulA from
Escherichia coli
inhibits the GTPase activity of FtsZ in vitro and prevents the formation of FtsZ rings when expressed in
Anabaena
PCC 7120. The expression of
sulA
arrests cell division and suppresses heterocyst differentiation completely. The antibiotic aztreonam, which is targeted to the FtsI protein necessary for septum formation, has similar effects on both cell division and heterocyst differentiation, although in this case, the FtsZ ring is still formed. Therefore, heterocyst differentiation is coupled to cell division but independent of the formation of the FtsZ ring. Consistently, once the inhibitory pressure of cell division is removed, cell division should take place first before heterocyst differentiation resumes at a normal frequency. The arrest of cell division does not affect the accumulation of 2-oxoglutarate, which triggers heterocyst differentiation. Consistently, a nonmetabolizable analogue of 2-oxoglutarate does not rescue the failure of heterocyst differentiation when cell division is blocked. These results suggest that the control of heterocyst differentiation by cell division is independent of the 2-oxoglutarate signal.
Spo0A-Dependent Activation of an Extended −10 Region Promoter in
Bacillus subtilis
Authors: Chen Guangnan , Kumar Amrita , Wyman Travis H. , Moran Charles P.
Published: February 2006

At the onset of endospore formation in
Bacillus subtilis
the DNA-binding protein Spo0A directly activates transcription from promoters of about 40 genes. One of these promoters, P
skf
, controls expression of an operon encoding a killing factor that acts on sibling cells. AbrB-mediated repression of P
skf
provides one level of security ensuring that this promoter is not activated prematurely. However, Spo0A also appears to activate the promoter directly, since Spo0A is required for P
skf
activity in a Δ
abrB
strain. Here we investigate the mechanism of P
skf
activation. DNase I footprinting was used to determine the locations at which Spo0A bound to the promoter, and mutations in these sites were found to significantly reduce promoter activity. The sequence near the −10 region of the promoter was found to be similar to those of extended −10 region promoters, which contain a TRTGn motif. Mutational analysis showed that this extended −10 region, as well as other base pairs in the −10 region, is required for Spo0A-dependent activation of the promoter. We found that a substitution of the consensus base pair for the nonconsensus base pair at position −9 of P
skf
produced a promoter that was active constitutively in both Δ
abrB
and Δ
spo0A
Δ
abrB
strains. Therefore, the base pair at position −9 of P
skf
makes its activity dependent on Spo0A binding, and the extended −10 region motif of the promoter contributes to its high level of activity.
The Genome of the Novel Phage Rtp, with a Rosette-Like Tail Tip, IsHomologous to the Genome of Phage T1
Authors: Wietzorrek Andreas , Schwarz Heinz , Herrmann Christina , Braun Volkmar
Published: February 2006

AnewEscherichia coliphage, named Rtp, was isolated and shown to be closely related to phage T1. Electron microscopy revealed that phage Rtp has a morphologically unique tail tip consisting of four leaf-like structures arranged in a rosette, whereas phage T1 has thinner, flexible leaves that thicken toward the ends. In contrast to T1, Rtp did not require FhuA and TonB for infection. The 46.2-kb genome of phage Rtp encodes 75 open reading frames, 47 of which are homologous to phage T1 genes. Like phage T1, phage Rtp encodes a large number of small genes at the genome termini that exhibit no sequence similarity to known genes. Six predicted genes larger than 300 nucleotides in the highly homologous region of Rtp are not found in T1. Two predicted HNH endonucleases are encoded at positions different from those in phage T1. The sequence similarity ofrtp37, -38, -39, -41, -42, and -43to equally arranged genes of lambdoid phages suggests a common tail assembly initiation complex. Protein Rtp43 is homologous to the λ J protein, which determines λ host specificity. Since the two proteins differ most in the C-proximal area, where the binding site to the LamB receptor resides in the J protein, we propose that Rtp43 contributes to Rtp host specificity. Lipoproteins similar to the predicted lipoprotein Rtp45 are found in a number of phages (encoded bycorgenes) in which they prevent superinfection by inactivating the receptors. We propose that, similar to the proposed function of the phage T5 lipoprotein, Rtp45 prevents inactivation of Rtp by adsorption to its receptor during cells lysis. Rtp52 is a putative transcriptional regulator, for which 10 conserved inverted repeats were identified upstream of genes in the Rtp genome. In contrast, the much largerE. coligenome has only one such repeat sequence.
Lethal Mutations in the Isoprenoid Pathway of
Salmonella enterica
Authors: Cornish Rita M. , Roth John R. , Poulter C. Dale
Published: February 2006

Essential isoprenoid compounds are synthesized using the 2-
C
-methyl-
d
-erythritol 4-phosphate (MEP) pathway in many gram-negative bacteria, some gram-positive bacteria, some apicomplexan parasites, and plant chloroplasts. The alternative mevalonate pathway is found in archaea and eukaryotes, including cytosolic biosynthesis in plants. The existence of orthogonal essential pathways in eukaryotes and bacteria makes the MEP pathway an attractive target for the development of antimicrobial agents. A system is described for identifying mutations in the MEP pathway of
Salmonella enterica
serovar Typhimurium. Using this system, point mutations induced by diethyl sulfate were found in the all genes of the essential MEP pathway and also in genes involved in uptake of methylerythritol. Curiously, none of the MEP pathway genes could be identified in the same parent strain by transposon mutagenesis, despite extensive searches. The results complement the biochemical and bioinformatic approaches to the elucidation of the genes involved in the MEP pathway and also identify key residues for activity in the enzymes of the pathway.
Quorum Sensing inYersinia enterocoliticaControls Swimming and Swarming Motility
Authors: Atkinson Steve , Chang Chien-Yi , Sockett R. Elizabeth , Cámara Miguel , Williams Paul
Published: February 2006

TheYersinia enterocoliticaLuxI homologue YenI directs the synthesis ofN-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) andN-hexanoylhomoserine lactone (C6-HSL). In aY. enterocolitica yenImutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent andyenImutant. Electrophoresis revealed that in contrast to the parent strain, theyenImutant grown for 17 h at 26°C lacked the 45-kDa flagellin protein FleB. Reverse transcription-PCR indicated that while mutation ofyenIhad no effect onyenR,flhDC(the motility master regulator) orfliA(the flagellar sigma factor) expression,fleB(the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in theyenImutant, we reexamined theN-acylhomoserine lactone (AHL) profile ofY. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI:N-(3-oxodecanoyl)homoserine lactone,N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), andN-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in theyenImutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into theyenImutant we demonstrate that the inability of exogenous AHLs to restore motility to theyenImutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of theyenImutant with a plasmid-borne copy ofyenI.
Genetic Diversity inMycobacterium ulceransIsolates from Ghana Revealed by a Newly Identified Locus Containing a Variable Number of Tandem Repeats
Authors: Hilty Markus , Yeboah-Manu Dorothy , Boakye Daniel , Mensah-Quainoo Ernestina , Rondini Simona , Schelling Esther , Ofori-Adjei David , Portaels Françoise , Zinsstag Jakob , Pluschke Gerd
Published: February 2006

The molecular typing methods used so far forMycobacterium ulceransisolates have not been able to identify genetic differences among isolates from Africa. This apparent lack of genetic diversity amongM. ulceransisolates is indicative of a clonal population structure. We analyzed the genetic diversity of 72 African isolates, including 57 strains from Ghana, by variable number of tandem repeat (VNTR) typing based on a newly identified polymorphic locus designated ST1 and the previously described locus MIRU 1. Three different genotypes were found in Ghana, demonstrating for the first time the genetic diversity ofM. ulceransin an African country. While the ST1/MIRU 1 allele combination BD/BAA seems to dominate in Africa, it was only rarely found in isolates from Ghana, where the combination BD/B was dominant and observed in all districts studied. A third variant genotype (C/BAA) was found only in the Amansie-West district. The results indicate that new genetic variants ofM. ulceransemerged and spread within Ghana and support the potential of VNTR-based typing for genotyping ofM. ulcerans.
Roles of Charged Residues of Rotor and Stator in Flagellar Rotation: Comparative Study using H
+
-Driven and Na
+
-Driven Motors in
Escherichia coli
Authors: Yakushi Toshiharu , Yang Junghoon , Fukuoka Hajime , Homma Michio , Blair David F.
Published: February 2006

In
Escherichia coli
, rotation of the flagellar motor has been shown to depend upon electrostatic interactions between charged residues of the stator protein MotA and the rotor protein FliG. These charged residues are conserved in the Na
+
-driven polar flagellum of
Vibrio alginolyticus
, but mutational studies in
V. alginolyticus
suggested that they are relatively unimportant for motor rotation. The electrostatic interactions detected in
E. coli
therefore might not be a general feature of flagellar motors, or, alternatively, the
V. alginolyticus
motor might rely on similar interactions but incorporate additional features that make it more robust against mutation. Here, we have carried out a comparative study of chimeric motors that were resident in
E. coli
but engineered to use
V. alginolyticus
stator components, rotor components, or both. Charged residues in the
V. alginolyticus
rotor and stator proteins were found to be essential for motor rotation when the proteins functioned in the setting of the
E. coli
motor. Patterns of synergism and suppression in rotor/stator double mutants indicate that the
V. alginolyticus
proteins interact in essentially the same way as their counterparts in
E. coli
. The robustness of the rotor-stator interface in
V. alginolyticus
is in part due to the presence of additional charged residues in PomA but appears mainly due to other factors, because an
E. coli
motor using both rotor and stator components from
V. alginolyticus
remained sensitive to mutation. Motor function in
V. alginolyticus
may be enhanced by the proteins MotX and MotY.
The Genome Sequence of the Obligately Chemolithoautotrophic, Facultatively Anaerobic Bacterium
Thiobacillus denitrificans
Authors: Beller Harry R. , Chain Patrick S. G. , Letain Tracy E. , Chakicherla Anu , Larimer Frank W. , Richardson Paul M. , Coleman Matthew A. , Wood Ann P. , Kelly Donovan P.
Published: February 2006

The complete genome sequence of
Thiobacillus denitrificans
ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, β-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding
c
-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for
T. denitrificans
and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfur-compound oxidation (including
sox
genes,
dsr
genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of
T. denitrificans
will enable elucidation of the mechanisms of aerobic and anaerobic sulfur-compound oxidation by β-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.
ParABS Systems of the Four Replicons ofBurkholderia cenocepacia: New Chromosome Centromeres Confer Partition Specificity
Authors: Dubarry Nelly , Pasta Franck , Lane David
Published: February 2006

Most bacterial chromosomes carry an analogue of theparABSsystems that govern plasmid partition, but their role in chromosome partition is ambiguous.parABSsystems might be particularly important for orderly segregation of multipartite genomes, where their role may thus be easier to evaluate. We have characterizedparABSsystems inBurkholderia cenocepacia, whose genome comprises three chromosomes and one low-copy-number plasmid. A singleparABlocus and a set of ParB-binding (parS) centromere sites are located near the origin of each replicon. ParA and ParB of the longest chromosome are phylogenetically similar to analogues in other multichromosome and monochromosome bacteria but are distinct from those of smaller chromosomes. The latter form subgroups that correspond to the taxa of their hosts, indicating evolution from plasmids. TheparSsites on the smaller chromosomes and the plasmid are similar to the “universal”parSof the main chromosome but with a sequence specific to their replicon. In anEscherichia coliplasmid stabilization test, eachparABexhibits partition activity only with theparSof its own replicon. Hence,parABSfunction is based on the independent partition of individual chromosomes rather than on a single communal system or network of interacting systems. Stabilization by the smaller chromosome and plasmid systems was enhanced by mutation ofparSsites and a promoter internal to theirparABoperons, suggesting autoregulatory mechanisms. The small chromosome ParBs were found to silence transcription, a property relevant to autoregulation.
The Bifunctional FtsK Protein Mediates Chromosome Partitioning and Cell Division in
Caulobacter
Authors: Wang Sherry C. E. , West Lisandra , Shapiro Lucy
Published: February 2006

Bacterial chromosome partitioning and cell division are tightly connected cellular processes. We show here that the
Caulobacter crescentus
FtsK protein localizes to the division plane, where it mediates multiple functions involved in chromosome segregation and cytokinesis. The first 258 amino acids of the N terminus are necessary and sufficient for targeting the protein to the division plane. Furthermore, the FtsK N terminus is required to either assemble or maintain FtsZ rings at the division plane. The FtsK C terminus is essential in
Caulobacter
and is involved in maintaining accurate chromosome partitioning. In addition, the C-terminal region of FtsK is required for the localization of the topoisomerase IV ParC subunit to the replisome to facilitate chromosomal decatenation prior to cell division. These results suggest that the interdependence between chromosome partitioning and cell division in
Caulobacte
r is mediated, in part, by the FtsK protein.
Solution Structure of a Low-Molecular-Weight Protein Tyrosine Phosphatase from
Bacillus subtilis
Authors: Xu Huimin , Xia Bin , Jin Changwen
Published: February 2006

The low-molecular-weight (LMW) protein tyrosine phosphatases (PTPs) exist ubiquitously in prokaryotes and eukaryotes and play important roles in cellular processes. We report here the solution structure of YwlE, an LMW PTP identified from the gram-positive bacteria
Bacillus subtilis.
YwlE consists of a twisted central four-stranded parallel β-sheet with seven α-helices packing on both sides. Similar to LMW PTPs from other organisms, the conformation of the YwlE active site is favorable for phosphotyrosine binding, indicating that it may share a common catalytic mechanism in the hydrolysis of phosphate on tyrosine residue in proteins. Though the overall structure resembles that of the eukaryotic LMW PTPs, significant differences were observed around the active site. Residue Asp115 is likely interacting with residue Arg13 through electrostatic interaction or hydrogen bond interaction to stabilize the conformation of the active cavity, which may be a unique character of bacterial LMW PTPs. Residues in the loop region from Phe40 to Thr48 forming a wall of the active cavity are more flexible than those in other regions. Ala41 and Gly45 are located near the active cavity and form a noncharged surface around it. These unique properties demonstrate that this loop may be involved in interaction with specific substrates. In addition, the results from spin relaxation experiments elucidate further insights into the mobility of the active site. The solution structure in combination with the backbone dynamics provides insights into the mechanism of substrate specificity of bacterial LMW PTPs.
A Mobile Quorum-Sensing System inSerratia marcescens
Authors: Wei Jun-Rong , Tsai Yu-Huan , Horng Yu-Tze , Soo Po-Chi , Hsieh Shang-Chen , Hsueh Po-Ren , Horng Jim-Tong , Williams Paul , Lai Hsin-Chih
Published: February 2006

Quorum-sensing systems that have been widely identified in bacteria play important roles in the regulation of bacterial multicellular behavior by which bacteria sense population density to control various biological functions, including virulence. One characteristic of theluxIRquorum-sensing genes is their diverse and discontinuous distribution among proteobacteria. Here we report that thespnIRquorum-sensing system identified in the enterobacteriumSerratia marcescensstrain SS-1 is carried in a transposon, TnTIR, which has common characteristics of Tn3family transposons and is mobile between chromosomes and plasmids of different enterobacterial hosts. SpnIR functions in the new host and was shown to negatively regulate the TnTIRtransposition frequency. This finding may help reveal the horizontal transfer and evolutionary mechanism of quorum-sensing genes and alter the way that we perceive regulation of bacterial multicellular behavior.
Assembly of Distinct Pilus Structures on the Surface of
Corynebacterium diphtheriae
Authors: Gaspar Andrew H. , Ton-That Hung
Published: February 2006

Different surface organelles contribute to specific interactions of a pathogen with host tissues or infectious partners. Multiple pilus gene clusters potentially encoding different surface structures have been identified in several gram-positive bacterial genomes sequenced to date, including actinomycetales, clostridia, corynebacteria, and streptococci.
Corynebacterium diphtheriae
has been shown to assemble a pilus structure, with sortase SrtA essential for the assembly of a major subunit SpaA and two minor proteins, SpaB and SpaC. We report here the characterization of a second pilus consisting of SpaD, SpaE, and SpaF, of which SpaD and SpaE form the pilus shaft and SpaF may be located at the pilus tip. The structure of the SpaDEF pilus contains no SpaABC pilins as detected by immunoelectron microscopy. Neither deletion of
spaA
nor sortase
srtA
abolishes SpaDEF pilus formation. The assembly of the SpaDEF pilus requires specific sortases located within the SpaDEF pilus gene cluster. Although either sortase SrtB or SrtC is sufficient to polymerize SpaDF, the incorporation of SpaE into the SpaD pili requires sortase SrtB. In addition, an alanine in place of the lysine of the SpaD pilin motif abrogates pilus polymerization. Thus, SpaD, SpaE, and SpaF constitute a different pilus structure that is independently assembled and morphologically distinct from the SpaABC pili and possibly other pili of
C. diphtheriae
.
In the
Bacillus stearothermophilus
DnaB-DnaG Complex, the Activities of the Two Proteins Are Modulated by Distinct but Overlapping Networks of Residues
Authors: Thirlway Jenny , Soultanas Panos
Published: February 2006

We demonstrate the primase activity of
Bacillus stearothermophilus
DnaG and show that it initiates at 3′-ATC-5′ and 3′-ATT-5′ sites synthesizing primers that are 22 or 23 nucleotides long. In the presence of the helicase DnaB the size distribution of primers is different, and a range of additional smaller primers are also synthesized. Nine residues from the N- and C-terminal domains of DnaB, as well as its linker region, have been reported previously to affect this interaction. In
Bacillus stearothermophilus
only three residues from the linker region (I119 and I125) and the N-terminal domain (Y88) of DnaB have been shown previously to have direct structural importance, and I119 and I125 mediate DnaG-induced effects on DnaB activity. The functions of the other residues (L138, T191, E192, R195, and M196) are still a mystery. Here we show that the E15A, Y88A, and E15A Y88A mutants bind DnaG but are not able to modulate primer size, whereas the R195A M196A mutant inhibited the primase activity. Therefore, four of these residues, E15 and Y88 (N-terminal domain) and R195 and M196 (C-terminal domain), mediate DnaB-induced effects on DnaG activity. Overall, the data suggest that the effects of DnaB on DnaG activity and vice versa are mediated by distinct but overlapping networks of residues.
Influence of CrgA on Assembly of the Cell Division Protein FtsZ during Development of
Streptomyces coelicolor
Authors: Del Sol Ricardo , Mullins Jonathan G. L. , Grantcharova Nina , Flärdh Klas , Dyson Paul
Published: February 2006

The product of the
crgA
gene of
Streptomyces coelicolor
represents a novel family of small proteins. A single orthologous gene is located close to the origin of replication of all fully sequenced actinomycete genomes and borders a conserved gene cluster implicated in cell growth and division. In
S. coelicolor
, CrgA is important for coordinating growth and cell division in sporogenic hyphae. In this study, we demonstrate that CrgA is an integral membrane protein whose peak expression is coordinated with the onset of development of aerial hyphae. The protein localizes to discrete foci away from growing hyphal tips. Upon overexpression, CrgA localizes to apical syncytial cells of aerial hyphae and inhibits the formation of productive cytokinetic rings of the bacterial tubulin homolog FtsZ, leading to proteolytic turnover of this major cell division determinant. In the absence of known prokaryotic cell division inhibitors in actinomycetes, CrgA may have an important conserved function influencing Z-ring formation in these bacteria.
Genetic and Transcriptional Analysis of the Siderophore Malleobactin Biosynthesis and Transport Genes in the Human Pathogen
Burkholderia pseudomallei
K96243
Authors: Alice Alejandro F. , López Claudia S. , Lowe Carolyn A. , Ledesma Maria A. , Crosa Jorge H.
Published: February 2006

Burkholderia pseudomallei
is a gram-negative facultative intracellular pathogen that causes melioidosis, an invasive disease of humans and animals. To address the response of this bacterium to iron-limiting conditions, we first performed a global transcriptional analysis of RNA extracted from bacteria grown under iron-limiting and iron-rich conditions by microarrays. We focused our study on those open reading frames (ORFs) induced under iron limitation, which encoded predicted proteins that could be involved in the biosynthesis and uptake of the siderophore malleobactin. We purified this siderophore and determined that it consisted of at least three compounds with different molecular weights. We demonstrated that ORFs BPSL1776 and BPSL1774, designated
mbaA
and
mbaF
, respectively, are involved in the biosynthesis of malleobactin, while BPSL1775, named
fmtA
, is involved in its transport. These genes are in an operon with two other ORFs (
mbaJ
and
mbaI
) whose transcription is under the control of MbaS, a protein that belongs to the extracytoplasmic function sigma factors. Interestingly, the transcription of the
mbaA
,
fmtA
, and
mbaS
genes is not controlled by the availability of the siderophore malleobactin.
Tetrapyrrole Biosynthesis in
Rhodobacter capsulatus
Is Transcriptionally Regulated by the Heme-Binding Regulatory Protein, HbrL
Authors: Smart James L. , Bauer Carl E.
Published: February 2006

We demonstrate that the expression of
hem
genes in
Rhodobacter capsulatus
is transcriptionally repressed in response to the exogenous addition of heme. A high-copy suppressor screen for regulators of
hem
gene expression resulted in the identification of an LysR-type transcriptional regulator, called HbrL, that regulates
hem
promoters in response to the availability of heme. HbrL is shown to activate the expression of
hemA
and
hemZ
in the absence of exogenous hemin and repress
hemB
expression in the presence of exogenous hemin. Heterologously expressed HbrL apoprotein binds heme
b
and is purified with bound heme
b
when expressed in the presence of 5-aminolevulinic acid. Electrophoretic gel shift analysis demonstrated that HbrL binds the promoter region of
hemA
,
hemB
, and
hemZ
as well as its own promoter and that the presence of heme increases the binding affinity of HbrL to
hemB.
A Na
+
:H
+
Antiporter and a Molybdate Transporter Are Essential for Arsenite Oxidation in
Agrobacterium tumefaciens
Authors: Kashyap Des R. , Botero Lina M. , Lehr Corinne , Hassett Daniel J. , McDermott Timothy R.
Published: February 2006

Transposon Tn
5
-B22 mutagenesis was used to identify genetic determinants required for arsenite [As(III)] oxidation in an
Agrobacterium tumefaciens
soil isolate, strain 5A. In one mutant, the transposon interrupted
modB
, which codes for the permease component of a high-affinity molybdate transporter. In a second mutant, the transposon insertion occurred in
mrpB
, which is part of a seven-gene operon encoding an Mrp-type Na
+
:H
+
antiporter complex. Complementation experiments with
mod
and
mrp
operons PCR cloned from the genome-sequenced
A. tumefaciens
strain C58 resulted in complementation back to an As(III)-oxidizing phenotype, confirming that these genes encode activities essential for As(III) oxidation in this strain of
A. tumefaciens
. As expected, the
mrp
mutant was extremely sensitive to NaCl and LiCl, indicating that the Mrp complex in
A. tumefaciens
is involved in Na
+
circulation across the membrane. Gene expression studies (
lacZ
reporter and reverse transcriptase PCR experiments) failed to show evidence of transcriptional regulation of the
mrp
operon in response to As(III) exposure, whereas expression of the
mod
operon was found to be up-regulated by As(III) exposure. In each mutant, the loss of As(III)-oxidizing capacity resulted in conversion to an arsenate [As(V)]-reducing phenotype. Neither mutant was more sensitive to As(III) than the parental strain.