bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTClinical 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT
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

ABSTRACT

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

ABSTRACT

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.