bluuebunny/crossref_metadata_2025 huggingface dataset:
Methanosarcina acetivorans
Flap Endonuclease 1 Activity Is Inhibited by a Cognate Single-Stranded-DNA-Binding Protein
Authors: Lin Yuyen , Guzman Claudia E. , McKinney Mary C. , Nair Satish K. , Ha Taekjip , Cann Isaac K. O.
Published: August 2006

The oligonucleotide/oligosaccharide-binding (OB) fold is central to the architecture of single-stranded- DNA-binding proteins, which are polypeptides essential for diverse cellular processes, including DNA replication, repair, and recombination. In archaea, single-stranded DNA-binding proteins composed of multiple OB folds and a zinc finger domain, in a single polypeptide, have been described. The OB folds of these proteins were more similar to their eukaryotic counterparts than to their bacterial ones. Thus, the archaeal protein is called replication protein A (RPA), as in eukaryotes. Unlike most organisms,
Methanosarcina acetivorans
harbors multiple functional RPA proteins, and it was our interest to determine whether the different proteins play different roles in DNA transactions. Of particular interest was lagging-strand DNA synthesis, where recently RPA has been shown to regulate the size of the 5′ region cleaved during Okazaki fragment processing. We report here that
M. acetivorans
RPA1 (MacRPA1), a protein composed of four OB folds in a single polypeptide, inhibits cleavage of a long flap (20 nucleotides) by
M. acetivorans
flap endonuclease 1 (MacFEN1). To gain a further insight into the requirement of the different regions of MacRPA1 on its inhibition of MacFEN1 endonuclease activity, N-terminal and C-terminal truncated derivatives of the protein were made and were biochemically and biophysically analyzed. Our results suggested that MacRPA1 derivatives with at least three OB folds maintained the properties required for inhibition of MacFEN1 endonuclease activity. Despite these interesting observations, further biochemical and genetic analyses are required to gain a deeper understanding of the physiological implications of our findings.
Regulation of
ppk
Expression and In Vivo Function of Ppk in
Streptomyces lividans
TK24
Authors: Ghorbel Sofiane , Smirnov Aleksey , Chouayekh Hichem , Sperandio Brice , Esnault Catherine , Kormanec Jan , Virolle Marie-Joelle
Published: August 2006

The
ppk
gene of
Streptomyces lividans
encodes an enzyme catalyzing, in vitro, the reversible polymerization of the γ phosphate of ATP into polyphosphate and was previously shown to play a negative role in the control of antibiotic biosynthesis (H. Chouayekh and M. J. Virolle, Mol. Microbiol.
43:
919-930, 2002). In the present work, some regulatory features of the expression of
ppk
were established and the polyphosphate content of
S. lividans
TK24 and the
ppk
mutant was determined. In P
i
sufficiency, the expression of
ppk
was shown to be low but detectable. DNA gel shift experiments suggested that
ppk
expression might be controlled by a repressor using ATP as a corepressor. Under these conditions, short acid-soluble polyphosphates accumulated upon entry into the stationary phase in the wild-type strain but not in the
ppk
mutant strain. The expression of
ppk
under P
i
-limiting conditions was shown to be much higher than that under P
i
-sufficient conditions and was under positive control of the two-component system PhoR/PhoP. Under these conditions, the polyphosphate content of the cell was low and polyphosphates were reproducibly found to be longer and more abundant in the
ppk
mutant strain than in the wild-type strain, suggesting that Ppk might act as a nucleoside diphosphate kinase. In light of our results, a novel view of the role of this enzyme in the regulation of antibiotic biosynthesis in
S. lividans
TK24 is proposed.
Probing the Substrate Specificity of Aminopyrrolnitrin Oxygenase (PrnD) by Mutational Analysis
Authors: Lee Jung-Kul , Ang Ee-Lui , Zhao Huimin
Published: August 2006

Molecular modeling and mutational analysis (site-directed mutagenesis and saturation mutagenesis) were used to probe the molecular determinants of the substrate specificity of aminopyrrolnitrin oxygenase (PrnD) from
Pseudomonas fluorescens
Pf-5. There are 17 putative substrate-contacting residues, and mutations at two of the positions, positions 312 and 277, could modulate the enzyme substrate specificity separately or in combination. Interestingly, several of the mutants obtained exhibited higher catalytic efficiency (approximately two- to sevenfold higher) with the physiological substrate aminopyrrolnitrin than the wild-type enzyme exhibited.
Superoxide Dismutase B Gene (sodB)-Deficient Mutants ofFrancisella tularensisDemonstrate Hypersensitivity to Oxidative Stress and Attenuated Virulence
Authors: Bakshi Chandra Shekhar , Malik Meenakshi , Regan Kevin , Melendez J. Andres , Metzger Dennis W. , Pavlov Vitaly M. , Sellati Timothy J.
Published: August 2006

AFrancisella tularensislive vaccine strain mutant (sodBFt) with reduced Fe-superoxide dismutase gene expression was generated and found to exhibit decreasedsodBactivity and increased sensitivity to redox cycling compounds compared to wild-type bacteria. ThesodBFtmutant also was significantly attenuated for virulence in mice. Thus, this study has identifiedsodBas an importantF. tularensisvirulence factor.
PBP5 Complementation of a PBP3 Deficiency inEnterococcus hirae
Authors: Leimanis S. , Hoyez N. , Hubert S. , Laschet M. , Sauvage Eric , Brasseur R. , Coyette J.
Published: August 2006

The low susceptibility of enterococci to β-lactams is due to the activity of the low-affinity penicillin-binding protein 5 (PBP5). One important feature of PBP5 is its ability to substitute for most, if not all, penicillin-binding proteins when they are inhibited. That substitution activity was analyzed inEnterococcus hiraeSL2, a mutant whosepbp5gene was interrupted by thenisRKgenes and whose PBP3 synthesis was submitted to nisin induction. Noninduced SL2 cells were unable to divide except when plasmid-bornepbp5genes were present, provided that the PBP5 active site was functional. Potential protein-protein interaction sites of the PBP5 N-terminal module were mutagenized by site-directed mutagenesis. The T167-L184region (designated site D) appeared to be an essential intramolecular site needed for the stability of the protein. Mutations made in the two globular domains present in the N-terminal module indicated that they were needed for the suppletive activity. The P197-N209segment (site E) in one of these domains seemed to be particularly important, as single and double mutations reduced or almost completely abolished, respectively, the action of PBP5.
The Structure of MbtI from
Mycobacterium tuberculosis
, the First Enzyme in the Biosynthesis of the Siderophore Mycobactin, Reveals It To Be a Salicylate Synthase
Authors: Harrison Anthony J. , Yu Minmin , Gårdenborg Therés , Middleditch Martin , Ramsay Rochelle J. , Baker Edward N. , Lott J. Shaun
Published: August 2006

The ability to acquire iron from the extracellular environment is a key determinant of pathogenicity in mycobacteria.
Mycobacterium tuberculosis
acquires iron exclusively via the siderophore mycobactin T, the biosynthesis of which depends on the production of salicylate from chorismate. Salicylate production in other bacteria is either a two-step process involving an isochorismate synthase (chorismate isomerase) and a pyruvate lyase, as observed for
Pseudomonas aeruginosa
, or a single-step conversion catalyzed by a salicylate synthase, as with
Yersinia enterocolitica
. Here we present the structure of the enzyme MbtI (Rv2386c) from
M. tuberculosis
, solved by multiwavelength anomalous diffraction at a resolution of 1.8 Å, and biochemical evidence that it is the salicylate synthase necessary for mycobactin biosynthesis. The enzyme is critically dependent on Mg
2+
for activity and produces salicylate via an isochorismate intermediate. MbtI is structurally similar to salicylate synthase (Irp9) from
Y. enterocolitica
and the large subunit of anthranilate synthase (TrpE) and shares the overall architecture of other chorismate-utilizing enzymes, such as the related aminodeoxychorismate synthase PabB. Like Irp9, but unlike TrpE or PabB, MbtI is neither regulated by nor structurally stabilized by bound tryptophan. The structure of MbtI is the starting point for the design of inhibitors of siderophore biosynthesis, which may make useful lead compounds for the production of new antituberculosis drugs, given the strong dependence of pathogenesis on iron acquisition in
M. tuberculosis
.
Exo-Oligosaccharides of
Rhizobium
sp. Strain NGR234 Are Required for Symbiosis with Various Legumes
Authors: Staehelin Christian , Forsberg Lennart S. , D'Haeze Wim , Gao Mu-Yun , Carlson Russell W. , Xie Zhi-Ping , Pellock Brett J. , Jones Kathryn M. , Walker Graham C. , Streit Wolfgang R. , Broughton William J.
Published: August 2006

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from
Rhizobium
sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with β-1,3, β-1,4, β-1,6, α-1,3, and α-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the
exoF
,
exoL
,
exoP
,
exoQ
, and
exoY
genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in
Escherichia coli
. Mutation of
exoK
(NGRΩ
exoK
), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGRΩ
exoK
is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)
5
(GlcA)
2
PvGal. When used as inoculants, both the
exo
-deficient mutants and NGRΩ
exoK
were unable to form nitrogen-fixing nodules on some hosts (e.g.,
Albizia lebbeck
and
Leucaena leucocephala
), but they were able to form nitrogen-fixing nodules on other hosts (e.g.,
Vigna unguiculata
). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, ∼50 μg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-β-1,4-glycanase encoded by
exoK
(glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.
Study of the Five
Rickettsia prowazekii
Proteins Annotated as ATP/ADP Translocases (Tlc): Only Tlc1 Transports ATP/ADP, While Tlc4 and Tlc5 Transport Other Ribonucleotides
Authors: Audia Jonathon P. , Winkler Herbert H.
Published: August 2006

The obligate intracytoplasmic pathogen
Rickettsia prowazekii
relies on the transport of many essential compounds from the cytoplasm of the eukaryotic host cell in lieu of de novo synthesis, an evolutionary outcome undoubtedly linked to obligatory growth in this metabolite-replete niche. The paradigm for the study of rickettsial transport systems is the ATP/ADP translocase Tlc1, which exchanges bacterial ADP for host cell ATP as a source of energy, rather than as a source of adenylate. Interestingly, the
R. prowazekii
genome encodes four open reading frames that are highly homologous to the well-characterized ATP/ADP translocase Tlc1. Therefore, by annotation, the
R. prowazekii
genome encodes a total of five ATP/ADP translocases: Tlc1, Tlc2, Tlc3, Tlc4, and Tlc5. We have confirmed by quantitative reverse transcriptase PCR that mRNAs corresponding to all five
tlc
homologues are expressed in
R. prowazekii
growing in L-929 cells and have shown their heterologous protein expression in
Escherichia coli
, suggesting that none of the
tlc
genes are pseudogenes in the process of evolutionary meltdown. However, we demonstrate by heterologous expression in
E. coli
that only Tlc1 functions as an ATP/ADP transporter. A survey of nucleotides and nucleosides has determined that Tlc4 transports CTP, UTP, and GDP. Intriguingly, although GTP was not transported by Tlc4, it was an inhibitor of CTP and UTP uptake and demonstrated a

K
i

similar to that of GDP. In addition, we demonstrate that Tlc5 transports GTP and GDP. We postulate that Tlc4 and Tlc5 serve the primary function of maintaining intracellular pools of nucleotides for rickettsial nucleic acid biosynthesis and do not provide the cell with nucleoside triphosphates as an energy source, as is the case for Tlc1. Although heterologous expression of Tlc2 and Tlc3 was observed in
E. coli
, we were unable to identify substrates for these proteins.
Regulation of Uptake Hydrogenase and Effects of Hydrogen Utilization on Gene Expression in
Rhodopseudomonas palustris
Authors: Rey Federico E. , Oda Yasuhiro , Harwood Caroline S.
Published: August 2006

Rhodopseudomonas palustris
is a purple, facultatively phototrophic bacterium that uses hydrogen gas as an electron donor for carbon dioxide fixation during photoautotrophic growth or for ammonia synthesis during nitrogen fixation. It also uses hydrogen as an electron supplement to enable the complete assimilation of oxidized carbon compounds, such as malate, into cell material during photoheterotrophic growth. The
R. palustris
genome predicts a membrane-bound nickel-iron uptake hydrogenase and several regulatory proteins to control hydrogenase synthesis. There is also a novel sensor kinase gene (RPA0981) directly adjacent to the hydrogenase gene cluster. Here we show that the
R. palustris
regulatory sensor hydrogenase HupUV acts in conjunction with the sensor kinase-response regulator protein pair HoxJ-HoxA to activate hydrogenase expression in response to hydrogen gas. Transcriptome analysis indicated that the HupUV-HoxJA regulatory system also controls the expression of genes encoding a predicted dicarboxylic acid transport system, a putative formate transporter, and a glutamine synthetase. RPA0981 had a small effect in repressing hydrogenase synthesis. We also determined that the two-component system RegS-RegR repressed expression of the uptake hydrogenase, probably in response to changes in intracellular redox status. Transcriptome analysis indicated that about 30 genes were differentially expressed in
R. palustris
cells that utilized hydrogen when growing photoheterotrophically on malate under nitrogen-fixing conditions compared to a mutant strain that lacked uptake hydrogenase. From this it appears that the recycling of reductant in the form of hydrogen does not have extensive nonspecific effects on gene expression in
R. palustris
.
A Mycothiol Synthase Mutant of
Mycobacterium tuberculosis
Has an Altered Thiol-Disulfide Content and Limited Tolerance to Stress
Authors: Buchmeier Nancy A. , Newton Gerald L. , Fahey Robert C.
Published: August 2006

Mycothiol (MSH) (acetyl-Cys-GlcN-Ins) is the major low-molecular-mass thiol in
Mycobacterium tuberculosis
. MSH has antioxidant activity, can detoxify a variety of toxic compounds, and helps to maintain the reducing environment of the cell. The production of MSH provides a potential novel target for tuberculosis treatment. Biosynthesis of MSH requires at least four genes. To determine which of these genes is essential in
M. tuberculosis
, we have been constructing targeted gene disruptions. Disruption in the
mshC
gene is lethal to
M. tuberculosis
, while disruption in the
mshB
gene results in MSH levels 20 to 100% of those of the wild type. For this study, we have constructed a targeted gene disruption in the
mshD
gene that encodes mycothiol synthase, the final enzyme in MSH biosynthesis. The
mshD
mutant produced ∼1% of normal MSH levels but high levels of the MshD substrate Cys-GlcN-Ins and the novel thiol
N
-formyl-Cys-GlcN-Ins. Although
N
-formyl-Cys-GlcN-Ins was maintained in a highly reduced state, Cys-GlcN-Ins was substantially oxidized. In both the wild type and the
mshD
mutant, cysteine was predominantly oxidized. The
M. tuberculosis mshD
mutant grew poorly on agar plates lacking catalase and oleic acid and in low-pH media and had heightened sensitivity to hydrogen peroxide. The inability of the
mshD
mutant to survive and grow in macrophages may be associated with its altered thiol-disulfide status. It appears that
N
-formyl-Cys-GlcN-Ins serves as a weak surrogate for MSH but is not sufficient to support normal growth of
M. tuberculosis
under stress conditions such as those found within the macrophage.
Rubredoxin:Oxygen Oxidoreductase Enhances Survival of
Desulfovibrio vulgaris
Hildenborough under Microaerophilic Conditions
Authors: Wildschut Janine D. , Lang R. Michael , Voordouw Johanna K. , Voordouw Gerrit
Published: August 2006

Genes for superoxide reductase (Sor), rubredoxin (Rub), and rubredoxin:oxygen oxidoreductase (Roo) are located in close proximity in the chromosome of
Desulfovibrio vulgaris
Hildenborough. Protein blots confirmed the absence of Roo from
roo
mutant and
sor-rub-roo
(
srr
) mutant cells and its presence in
sor
mutant and wild-type cells grown under anaerobic conditions. Oxygen reduction rates of the
roo
and
srr
mutants were 20 to 40% lower than those of the wild type and the
sor
mutant, indicating that Roo functions as an O
2
reductase in vivo. Survival of single cells incubated for 5 days on agar plates under microaerophilic conditions (1% air) was 85% for the
sor
, 4% for the
roo
, and 0.7% for the
srr
mutant relative to that of the wild type (100%). The similar survival rates of
sor
mutant and wild-type cells suggest that O
2
reduction by Roo prevents the formation of reactive oxygen species (ROS) under these conditions; i.e., the ROS-reducing enzyme Sor is only needed for survival when Roo is missing. In contrast, the
sor
mutant was inactivated much more rapidly than the
roo
mutant when liquid cultures were incubated in 100% air, indicating that O
2
reduction by Roo and other terminal oxidases did not prevent ROS formation under these conditions. Competition of Sor and Roo for limited reduced Rub was suggested by the observation that the
roo
mutant survived better than the wild type under fully aerobic conditions. The
roo
mutant was more strongly inhibited than the wild type by the nitric oxide (NO)-generating compound
S
-nitrosoglutathione, indicating that Roo may also serve as an NO reductase in vivo.
Staphylococcus aureus
Mutants with Increased Lysostaphin Resistance
Authors: Gründling Angelika , Missiakas Dominique M. , Schneewind Olaf
Published: August 2006

Staphylococcus simulans
secretes lysostaphin, a bacteriolytic enzyme that specifically binds to the cell wall envelope of
Staphylococcus aureus
and cleaves the pentaglycine cross bridges of peptidoglycan, thereby killing staphylococci. The study of
S. aureus
mutants with resistance to lysostaphin-mediated killing has revealed biosynthetic pathways for cell wall assembly. To identify additional genes involved in cell wall envelope biosynthesis, we have screened a collection of
S. aureus
strain Newman transposon mutants for lysostaphin resistance.
Bursa aurealis
insertion in SAV2335, encoding a polytopic membrane protein with predicted protease domain, caused a high degree of lysostaphin resistance, similar to the case for a previously described
femAB
promoter mutant. In contrast to the case for this
femAB
mutant, transposon insertion in SAV2335, herein named
lyrA
(
ly
sostaphin
r
esistance
A
), did not cause gross alterations of cell wall cross bridges such as truncations of pentaglycine to tri- or monoglycine. Also, inactivation of LyrA in a methicillin-resistant
S. aureus
strain did not precipitate a decrease in β-lactam resistance as observed for
fem
(
f
actor
e
ssential for
m
ethicillin resistance) mutants. Lysostaphin bound to the cell wall envelopes of
lyrA
mutants in a manner similar to that for wild-type staphylococci. Lysostaphin resistance of
lyrA
mutants is attributable to altered cell wall envelope properties and may in part be due to increased abundance of altered cross bridges. Other
lyr
mutants with intermediate lysostaphin resistance carried
bursa aurealis
insertions in genes specifying GTP pyrophosphokinase or enzymes of the purine biosynthetic pathway.
Comparative Genome Hybridization Reveals Substantial Variation among Clinical Isolates ofBorrelia burgdorferiSensu Stricto with Different Pathogenic Properties
Authors: Terekhova Darya , Iyer Radha , Wormser Gary P. , Schwartz Ira
Published: August 2006

Clinical and murine studies suggest that there is a differential pathogenicity of different genotypes ofBorrelia burgdorferi, the spirochetal agent of Lyme disease. Comparative genome hybridization was used to explore the relationship between different genotypes. The chromosomes of all studied isolates were highly conserved (>93%) with respect to both sequence and gene order. Plasmid sequences were substantially more diverse. Plasmids lp54, cp26, and cp32 were present in all tested isolates, and their sequences and gene order were conserved. The majority of linear plasmids showed variation both in terms of presence among different isolates and in terms of sequence and gene order. The data strongly imply that allB. burgdorfericlinical isolates contain linear plasmids related to each other, but the structure of these replicons may vary substantially from isolate to isolate. These alterations include deletions and presumed rearrangements that are likely to result in unique plasmid elements in many isolates. There is a strong correlation between complete genome hybridization profiles and other typing methods, which, in turn, also correlate to differences in pathogenicity. Because there is substantially less variation in the chromosomal and circular plasmid portions of the genome, the major differences in open reading frame content and genomic diversity among isolates are linear plasmid driven.
General Mutagenesis of F Plasmid TraI Reveals Its Role in Conjugative Regulation
Authors: Haft Rembrandt J. F. , Palacios Gilberto , Nguyen Tran , Mally Manuela , Gachelet Eliora G. , Zechner Ellen L. , Traxler Beth
Published: August 2006

Bacteria commonly exchange genetic information by the horizontal transfer of conjugative plasmids. In gram-negative conjugation, a relaxase enzyme is absolutely required to prepare plasmid DNA for transit into the recipient via a type IV secretion system. Here we report a mutagenesis of the F plasmid relaxase gene
traI
using in-frame, 31-codon insertions. Phenotypic analysis of our mutant library revealed that several mutant proteins are functional in conjugation, highlighting regions of TraI that can tolerate insertions of a moderate size. We also demonstrate that wild-type TraI, when overexpressed, plays a dominant-negative regulatory role in conjugation, repressing plasmid transfer frequencies ∼100-fold. Mutant TraI proteins with insertions in a region of approximately 400 residues between the consensus relaxase and helicase sequences did not cause conjugative repression. These unrestrictive TraI variants have normal relaxase activity in vivo, and several have wild-type conjugative functions when expressed at normal levels. We postulate that TraI negatively regulates conjugation by interacting with and sequestering some component of the conjugative apparatus. Our data indicate that the domain responsible for conjugative repression resides in the central region of TraI between the protein's catalytic domains.
Separate Pathways for O Acetylation of Polymeric and Monomeric Sialic Acids and Identification of Sialyl
O
-Acetyl Esterase in
Escherichia coli
K1
Authors: Steenbergen Susan M. , Lee Young-Choon , Vann Willie F. , Vionnet Justine , Wright Lori F. , Vimr Eric R.
Published: August 2006

O acetylation at carbon positions 7 or 9 of the sialic acid residues in the polysialic acid capsule of
Escherichia coli
K1 is catalyzed by a phase-variable contingency locus,
neuO
, carried by the K1-specific prophage, CUS-3. Here we describe a novel method for analyzing polymeric sialic acid O acetylation that involves the release of surface sialic acids by endo-
N
-acetylneuraminidase digestion, followed by fluorescent labeling and detection of quinoxalinone derivatives by chromatography. The results indicated that NeuO is responsible for the majority of capsule modification that takes place in vivo. However, a minor
neuO
-independent O acetylation pathway was detected that is dependent on the bifunctional polypeptide encoded by
neuD
. This pathway involves O acetylation of monomeric sialic acid and is regulated by another bifunctional enzyme, NeuA, which includes N-terminal synthetase and C-terminal sialyl
O
-esterase domains. A homologue of the NeuA C-terminal domain (Pm1710) in
Pasteurella multocida
was also shown to be an esterase, suggesting that it functions in the catabolism of acetylated environmental sialic acids. Our combined results indicate a previously unexpected complexity in the synthesis and catabolism of microbial sialic and polysialic acids. These findings are key to understanding the biological functions of modified sialic acids in
E. coli
K1 and other species and may provide new targets for drug or vaccine development.
Positive Selection in the ComC-ComD System of Streptococcal Species
Authors: Ichihara Hisako , Kuma Kei-ichi , Toh Hiroyuki
Published: August 2006

Competence-stimulating peptide (CSP) and ComD of the streptococcal species are a pheromone and its receptor, respectively, involved in the regulation of competence for natural genetic transformation. We show here that these molecules have undergone positive selection. This study is the first report of positive selection due to competition among bacterial populations.
Genetic Analysis of the Requirement for
flp-2
,
tadV
, and
rcpB
in
Actinobacillus actinomycetemcomitans
Biofilm Formation
Authors: Perez B. A. , Planet P. J. , Kachlany S. C. , Tomich M. , Fine D. H. , Figurski D. H.
Published: August 2006

The
tad
locus of
Actinobacillus actinomycetemcomitans
encodes a molecular transport system required for tenacious, nonspecific adherence to surfaces and formation of extremely strong biofilms. This locus is dedicated to the biogenesis of Flp pili, which are required for colonization and virulence. We have previously shown that 11 of the 14
tad
locus genes are required for adherence and Flp pilus production. Here, we present genetic and phylogenetic analyses of
flp-2
,
tadV
, and
rcpB
genes in biofilm formation. We show that
tadV
, predicted to encode prepilin peptidase, is required for adherence. In contrast, targeted insertional inactivation of
flp
-2, a gene closely related to the prepillin gene
flp
-1, did not abrogate biofilm formation. Expression studies did not detect Flp2-T7 protein under standard laboratory conditions. We present phylogenetic data showing that there is no significant evidence for natural selection in the available
flp-2
sequences from
A. actinomycetemcomitans
, suggesting that
flp-2
does not play a significant role in the biology of this organism. Mutants with insertions at the 3′ end of
rcpB
formed biofilms equivalent to wild-type
A. actinomycetemcomitans
. Surprisingly, 5′ end chromosomal insertion mutants in
rcpB
were obtained only when a wild-type copy of the
rcpB
gene was provided in
trans
or when the Tad secretion system was inactivated. Together, our results strongly suggest that
A. actinomycetemcomitans rcpB
is essential in the context of a functional
tad
locus. These data show three different phenotypes for the three genes.
Cluster of DnaA Boxes Involved in Regulation of
Streptomyces
Chromosome Replication: from In Silico to In Vivo Studies
Authors: Smulczyk-Krawczyszyn Aleksandra , Jakimowicz Dagmara , Ruban-Ośmiałowska Beata , Zawilak-Pawlik Anna , Majka Jerzy , Chater Keith , Zakrzewska-Czerwińska Jolanta
Published: August 2006

In
Streptomyces coelicolor
, replication is initiated by the DnaA protein in the centrally located
oriC
region and proceeds bidirectionally until the replication forks reach the ends of the linear chromosome. We identified three clusters of DnaA boxes (H69, H24, and D78) which are in a relatively short segment of the chromosome centered on the
oriC
region. Of the clusters analyzed, D78 exhibited the highest affinity for the DnaA protein; the affinity of DnaA for the D78 cluster was about eightfold higher than the affinity for
oriC
. The high-affinity DnaA boxes appear to be involved in the control of chromosome replication. Deletion of D78 resulted in more frequent chromosome replication (an elevated ratio of origins to chromosome ends was observed) and activated aerial mycelium formation, leading to earlier colony maturation. In contrast, extra copies of D78 (delivered on a plasmid) caused slow colony growth, presumably because of a reduction in the frequency of initiation of chromosome replication. This suggests that the number of high-affinity DnaA boxes is relatively constant in hyphal compartments and that deletion of D78 therefore permits an increased copy number of either the chromosomal origin region or a plasmid harboring the D78 cluster. This system conceivably influences the timing of decisions to initiate aerial mycelial formation and sporulation.
Detection and Quantification of Superoxide Formed within the Periplasm ofEscherichia coli
Authors: Korshunov Sergei , Imlay James A.
Published: August 2006

Many gram-negative bacteria harbor a copper/zinc-containing superoxide dismutase (CuZnSOD) in their periplasms. In pathogenic bacteria, one role of this enzyme may be to protect periplasmic biomolecules from superoxide that is released by host phagocytic cells. However, the enzyme is also present in many nonpathogens and/or free-living bacteria, includingEscherichia coli. In this study we were able to detect superoxide being released into the medium from growing cultures ofE. coli. Exponential-phase cells do not normally synthesize CuZnSOD, which is specifically induced in stationary phase. However, the engineered expression of CuZnSOD in growing cells eliminated superoxide release, confirming that this superoxide was formed within the periplasm. The rate of periplasmic superoxide production was surprisingly high and approximated the estimated rate of cytoplasmic superoxide formation when both were normalized to the volume of the compartment. The rate increased in proportion to oxygen concentration, suggesting that the superoxide is generated by the adventitious oxidation of an electron carrier. Mutations that eliminated menaquinone synthesis eradicated the superoxide formation, while mutations in genes encoding respiratory complexes affected it only insofar as they are likely to affect the redox state of menaquinone. We infer that the adventitious autoxidation of dihydromenaquinone in the cytoplasmic membrane releases a steady flux of superoxide into the periplasm ofE. coli. This endogenous superoxide may create oxidative stress in that compartment and be a primary substrate of CuZnSOD.
Essential Role of Flavohemoglobin in Long-Term Anaerobic Survival of
Bacillus subtilis
Authors: Nakano Michiko M.
Published: August 2006

A
Bacillus subtilis
culture incubated anaerobically in nitrate-containing medium lost viability during the first 3 days but recovered thereafter. A flavohemoglobin mutant showed very poor survival under these conditions unless the cells were prevented from carrying out nitrate respiration.
Independent Control of Replication Initiation of the Two
Vibrio cholerae
Chromosomes by DnaA and RctB
Authors: Duigou Stéphane , Knudsen Kristine G. , Skovgaard Ole , Egan Elizabeth S. , Løbner-Olesen Anders , Waldor Matthew K.
Published: August 2006

Although the two
Vibrio cholerae
chromosomes initiate replication in a coordinated fashion, we show here that each chromosome appears to have a specific replication initiator. DnaA overproduction promoted overinitiation of chromosome I and not chromosome II. In contrast, overproduction of RctB, a protein that binds to the origin of replication of chromosome II, promoted overinitiation of chromosome II and not chromosome I.
Proline Betaine Uptake in
Sinorhizobium meliloti
: Characterization of Prb, an Opp-Like ABC Transporter Regulated by both Proline Betaine and Salinity Stress
Authors: Alloing Geneviève , Travers Isabelle , Sagot Brice , Le Rudulier Daniel , Dupont Laurence
Published: August 2006

Sinorhizobium meliloti
uses proline betaine (PB) as an osmoprotectant when osmotically stressed and as an energy source in low-osmolarity environments. To fulfill this dual function, two separate PB transporters, BetS and Hut, that contribute to PB uptake at high and low osmolarity, respectively, have been previously identified. Here, we characterized a novel transport system that mediates the uptake of PB at both high and low osmolarities. Sequence analysis of Tn
5-luxAB
chromosomal insertions from several PB-inducible mutants has revealed the presence of a four-gene locus encoding the components of an ABC transporter, Prb, which belongs to the oligopeptide permease (Opp) family. Surprisingly,
prb
mutants were impaired in their ability to transport PB, and oligopeptides were not shown to be competitors for PB uptake. Further analysis of Prb specificity has shown its ability to take up other quaternary ammonium compounds such as choline and, to a lesser extent, glycine betaine. Interestingly, salt stress and PB were found to control
prb
expression in a positive and synergistic way and to increase Prb transport activity. At low osmolarity, Prb is largely implicated in PB uptake by stationary-phase cells, likely to provide PB as a source of carbon and nitrogen. Furthermore, at high osmolarity, the analysis of
prb
and
betS
single and double mutants demonstrated that Prb, together with BetS, is a key system for protection by PB.
The Highly Efficient Translation Initiation Region from the
Escherichia coli rpsA
Gene Lacks a Shine-Dalgarno Element
Authors: Skorski Patricia , Leroy Prune , Fayet Olivier , Dreyfus Marc , Hermann-Le Denmat Sylvie
Published: August 2006

The translational initiation region (TIR) of the
Escherichia coli rpsA
gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position −91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the “specialized ribosome” approach. Depending upon the constructs used, translation initiation was decreased three- to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins.
Type III Pilus of Corynebacteria: Pilus Length Is Determined by the Level of Its Major Pilin Subunit
Authors: Swierczynski Arlene , Ton-That Hung
Published: August 2006

Multiple pilus gene clusters have been identified in several gram-positive bacterial genomes sequenced to date, including the
Actinomycetales
, clostridia, streptococci, and corynebacteria. The genome of
Corynebacterium diphtheriae
contains three pilus gene clusters, two of which have been previously characterized. Here, we report the characterization of the third pilus encoded by the
spaHIG
cluster. By using electron microscopy and biochemical analysis, we demonstrate that SpaH forms the pilus shaft, while SpaI decorates the structure and SpaG is largely located at the pilus tip. The assembly of the SpaHIG pilus requires a specific sortase located within the
spaHIG
pilus gene cluster. Deletion of genes specific for the synthesis and polymerization of the other two pilus types does not affect the SpaHIG pilus. Moreover, SpaH but not SpaI or SpaG is essential for the formation of the filament. When expressed under the control of an inducible promoter, the amount of the SpaH pilin regulates pilus length; no pili are assembled from an SpaH precursor that has an alanine in place of the conserved lysine of the SpaH pilin motif. Thus, the
spaHIG
pilus gene cluster encodes a pilus structure that is independently assembled and antigenically distinct from other pili of
C. diphtheriae
. We incorporate these findings in a model of sortase-mediated pilus assembly that may be applicable to many gram-positive pathogens.
Genetic Evidence for an Interaction of the UbiG
O
-Methyltransferase with UbiX in
Escherichia coli
Coenzyme Q Biosynthesis
Authors: Gulmezian Melissa , Zhang Haitao , Javor George T. , Clarke Catherine F.
Published: August 2006

IS16 is a thiol-sensitive, Q-deficient mutant strain of
Escherichia coli.
Here, we show that IS16 harbors a mutation in the
ubiG
gene encoding a methyltransferase required for two O-methylation steps of Q biosynthesis. Complementation of IS16 with either
ubiG
or
ubiX
K-12
reverses this phenotype, suggesting that UbiX may interact with UbiG.
The Blue-Light Receptor YtvA Acts in the Environmental Stress Signaling Pathway of
Bacillus subtilis
Authors: Gaidenko Tatiana A. , Kim Tae-Jong , Weigel Andrea L. , Brody Margaret S. , Price Chester W.
Published: August 2006

The general stress response of the bacterium
Bacillus subtilis
is regulated by a partner-switching mechanism in which serine and threonine phosphorylation controls protein interactions in the stress-signaling pathway. The environmental branch of this pathway contains a family of five paralogous proteins that function as negative regulators. Here we present genetic evidence that a sixth paralog, YtvA, acts as a positive regulator in the same environmental signaling branch. We also present biochemical evidence that YtvA and at least three of the negative regulators can be isolated from cell extracts in a large environmental signaling complex. YtvA differs from these associated negative regulators by its flavin mononucleotide (FMN)-containing light-oxygen-voltage domain. Others have shown that this domain has the photochemistry expected for a blue-light sensor, with the covalent linkage of the FMN chromophore to cysteine 62 composing a critical part of the photocycle. Consistent with the view that light intensity modifies the output of the environmental signaling pathway, we found that cysteine 62 is required for YtvA to exert its positive regulatory role in the absence of other stress. Transcriptional analysis of the
ytvA
structural gene indicated that it provides the entry point for at least one additional environmental input, mediated by the Spx global regulator of disulfide stress. These results support a model in which the large signaling complex serves to integrate multiple environmental signals in order to modulate the general stress response.
Blue Light Activates the σ
B
-Dependent Stress Response of
Bacillus subtilis
via YtvA
Authors: Ávila-Pérez Marcela , Hellingwerf Klaas J. , Kort Remco
Published: August 2006

Here we present evidence for a physiologically relevant light response mediated by the LOV domain-containing protein YtvA in the soil bacterium
Bacillus subtilis
. The loss and overproduction of YtvA abolish and enhance, respectively, the increase in σ
B
-controlled
ctc
promoter activity at moderate light intensities. These effects were absent in the dark and in red light but present under blue-light illumination. Thus, activation of the general stress response in
B. subtilis
is modulated by blue light.
Pseudomonas syringae
HrpJ Is a Type III Secreted Protein That Is Required for Plant Pathogenesis, Injection of Effectors, and Secretion of the HrpZ1 Harpin
Authors: Fu Zheng Qing , Guo Ming , Alfano James R.
Published: August 2006

The bacterial plant pathogen
Pseudomonas syringae
requires a type III protein secretion system (TTSS) to cause disease. The
P. syringae
TTSS is encoded by the
hrp-hrc
gene cluster. One of the genes within this cluster,
hrpJ
, encodes a protein with weak similarity to YopN, a type III secreted protein from the animal pathogenic
Yersinia
species. Here, we show that HrpJ is secreted in culture and translocated into plant cells by the
P. syringae
pv. tomato DC3000 TTSS. A DC3000
hrpJ
mutant, UNL140, was greatly reduced in its ability to cause disease symptoms and multiply in
Arabidopsis thaliana
. UNL140 exhibited a reduced ability to elicit a hypersensitive response (HR) in nonhost tobacco plants. UNL140 was unable to elicit an AvrRpt2- or AvrB1-dependent HR in
A. thaliana
but maintained its ability to secrete AvrB1 in culture via the TTSS. Additionally, UNL140 was defective in its ability to translocate the effectors AvrPto1, HopB1, and AvrPtoB. Type III secretion assays showed that UNL140 secreted HrpA1 and AvrPto1 but was unable to secrete HrpZ1, a protein that is normally secreted in culture in relatively large amounts, into culture supernatants. Taken together, our data indicate that HrpJ is a type III secreted protein that is important for pathogenicity and the translocation of effectors into plant cells. Based on the failure of UNL140 to secrete HrpZ1, HrpJ may play a role in controlling type III secretion, and in its absence, specific accessory proteins, like HrpZ1, may not be extracellularly localized, resulting in disabled translocation of effectors into plant cells.
Transcriptional Profiling of the
Bacillus anthracis
Life Cycle In Vitro and an Implied Model for Regulation of Spore Formation
Authors: Bergman Nicholas H. , Anderson Erica C. , Swenson Ellen E. , Niemeyer Matthew M. , Miyoshi Amy D. , Hanna Philip C.
Published: August 2006

The life cycle of
Bacillus anthracis
includes both vegetative and endospore morphologies which alternate based on nutrient availability, and there is considerable evidence indicating that the ability of this organism to cause anthrax depends on its ability to progress through this life cycle in a regulated manner. Here we report the use of a custom
B. anthracis
GeneChip in defining the gene expression patterns that occur throughout the entire life cycle in vitro. Nearly 5,000 genes were expressed in five distinct waves of transcription as the bacteria progressed from germination through sporulation, and we identified a specific set of functions represented within each wave. We also used these data to define the temporal expression of the spore proteome, and in doing so we have demonstrated that much of the spore's protein content is not synthesized de novo during sporulation but rather is packaged from preexisting stocks. We explored several potential mechanisms by which the cell could control which proteins are packaged into the developing spore, and our analyses were most consistent with a model in which
B. anthracis
regulates the composition of the spore proteome based on protein stability. This study is by far the most comprehensive survey yet of the
B. anthracis
life cycle and serves as a useful resource in defining the growth-phase-dependent expression patterns of each gene. Additionally, the data and accompanying bioinformatics analyses suggest a model for sporulation that has broad implications for
B. anthracis
biology and offer new possibilities for microbial forensics and detection.
The RgpB C-Terminal Domain Has a Role in Attachment of RgpB to the Outer Membrane and Belongs to a Novel C-Terminal-Domain Family Found in
Porphyromonas gingivalis
Authors: Seers Christine A. , Slakeski Nada , Veith Paul D. , Nikolof Todd , Chen Yu-Yen , Dashper Stuart G. , Reynolds Eric C.
Published: August 2006

Porphyromonas gingivalis
produces outer membrane-attached proteins that include the virulence-associated proteinases RgpA and RgpB (Arg-gingipains) and Kgp (Lys-gingipain). We analyzed the
P. gingivalis
outer membrane proteome and identified numerous proteins with C-terminal domains similar in sequence to those of RgpB, RgpA, and Kgp, indicating that these domains may have a common function. Using RgpB as a model to investigate the role of the C-terminal domain, we expressed RgpB as a full-length zymogen (recombinant RgpB [rRgpB]), with a catalytic Cys244Ala mutation [rRgpB(C244A)], or with the C-terminal 72 amino acids deleted (rRgpB435) in an Arg-gingipain
P. gingivalis
mutant (YH522AB) and an Arg- and Lys-gingipain mutant (YH522KAB). rRgpB was catalytically active and located predominantly attached to the outer membrane of both background strains. rRgpB(C244A) was inactive and outer membrane attached, with a typical attachment profile for both background strains according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but in YH522KAB, the prodomain was not removed. Thus, in vivo, RgpB export and membrane attachment are independent of the proteolytic activity of RgpA, RgpB, or Kgp. However, for maturation involving proteolytic processing of RgpB, the proteolytic activity of RgpB, RgpA, or Kgp is required. The C-terminally-truncated rRgpB435 was not attached to the outer membrane and was located as largely inactive, discrete 71-kDa and 48-kDa isoforms in the culture supernatant and the periplasm. These results suggest that the C-terminal domain is essential for outer membrane attachment and may be involved in a coordinated process of export and attachment to the cell surface.
Residues Involved in FecR Binding Are Localized on One Side of the FecA Signaling Domain in
Escherichia coli
Authors: Breidenstein Elena , Mahren Susanne , Braun Volkmar
Published: August 2006

Ferric citrate transport in
Escherichia coli
involves proteins encoded by the
fec
genes, including the transport and signaling protein FecA and the signal transducing protein FecR. Randomly isolated FecA point mutants showed a reduced interaction with FecR and a reduced transcription initiation of the ferric citrate transport genes. The mutations were localized on one side of the FecA signaling domain, which might form the interface to FecR. Some of the mutants showed strongly reduced iron transport rates, which suggests that the signaling domain affects the structure of the FecA transporter domain.
Two Genes Encoding New Carotenoid-Modifying Enzymes in the Green Sulfur Bacterium
Chlorobium tepidum
Authors: Maresca Julia A. , Bryant Donald A.
Published: August 2006

The green sulfur bacterium
Chlorobium tepidum
produces chlorobactene as its primary carotenoid. Small amounts of chlorobactene are hydroxylated by the enzyme CrtC and then glucosylated and acylated to produce chlorobactene glucoside laurate. The genes encoding the enzymes responsible for these modifications of chlorobactene, CT1987, and CT0967, have been identified by comparative genomics, and these genes were insertionally inactivated in
C. tepidum
to verify their predicted function. The gene encoding chlorobactene glucosyltransferase (CT1987) has been named
cruC
, and the gene encoding chlorobactene lauroyltransferase (CT0967) has been named
cruD
. Homologs of these genes are found in the genomes of all sequenced green sulfur bacteria and filamentous anoxygenic phototrophs as well as in the genomes of several nonphotosynthetic bacteria that produce similarly modified carotenoids. The other bacteria in which these genes are found are not closely related to green sulfur bacteria or to one another. This suggests that the ability to synthesize modified carotenoids has been a frequently transferred trait.