bluuebunny/crossref_metadata_2025_split huggingface dataset:
Multiple Replication Origins ofHalobacteriumsp. Strain NRC-1: Properties of the Conservedorc7-DependentoriC1
Authors: Coker James A. , DasSarma Priya , Capes Melinda , Wallace Tammitia , McGarrity Karen , Gessler Rachael , Liu Jingfang , Xiang Hua , Tatusov Roman , Berquist Brian R. , DasSarma Shiladitya 
Published: June 2009

ABSTRACTThe eukaryote-like DNA replication system of the model haloarchaeonHalobacteriumNRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2andorc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence,oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal toorc7(oriC1) andorc10(oriC2), and the largest peaks on the extrachromosomal elements were nearorc9(oriP1) in both pNRC100 and -200 and nearorc4(oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomalorcgenes showed that replication initiation atoriC1requiresorc7but notorc6andorc8. The initiation sites atoriC1were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. TheoriC1region, Orc binding sites, andorc7gene orthologs were conserved in all sequenced haloarchaea. Serial deletion oforcgenes resulted in the construction of a minimal strain containing not onlyorc2andorc10but alsoorc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.
An Alternative Succinate (2-Oxoglutarate) Transport System in
Rhizobium tropici
Is Induced in Nodules of
Phaseolus vulgaris
Authors: Batista Silvia , Patriarca Eduardo J. , Tatè Rosarita , Martínez-Drets Gloria , Gill Paul R. 
Published: June 2009

ABSTRACT

The rhizobial DctA permease is essential for the development of effective nitrogen-fixing bacteroids, which was correlated with its requirement for growth on C
4
-dicarboxylates. A previously described
dctA
mutant of
Rhizobium tropici
CIAT899, strain GA1 (
dctA
), however, was unexpectedly still able to grow on succinate as a sole carbon source but less efficiently than CIAT899. Like other rhizobial
dctA
mutants, GA1 was unable to grow on fumarate or malate as a carbon source and induced the formation of ineffective nodules. We report an alternative succinate uptake system identified by Tn
5
mutagenesis of strain GA1 that was required for the remaining ability to transport and utilize succinate. The alternative uptake system required a three-gene cluster that is highly characteristic of a
dctABD
locus. The predicted permease-encoding gene had high sequence similarity with open reading frames encoding putative 2-oxoglutarate permeases (KgtP) of
Ralstonia solanacearum
and
Agrobacterium tumefaciens
. This analysis was in agreement with the requirement for this gene for optimal growth on and induction by 2-oxoglutarate. The permease-encoding gene of the alternative system was also designated
kgtP
in
R. tropici
. The
dctBD
-like genes in this cluster were found to be required for
kgtP
expression and were designated
kgtSR
. Analysis of a
kgtP
::
lacZ
transcriptional fusion indicated that a
kgtSR
-dependent promoter of
kgtP
was specifically induced by 2-oxoglutarate. The expression of
kgtPp
was found in bacteroids of nodules formed with either CIAT899 or GA1 on roots of
Phaseolus vulgaris
. Results suggested that 2-oxoglutarate might be transported or conceivably exported in nodules induced by
R. tropici
on roots of
P. vulgaris
.
Structural Basis for the Localization of the Chemotaxis Phosphatase CheZ by CheA
S
Authors: Hao Shufeng , Hamel Damon , Zhou Hongjun , Dahlquist Frederick W. 
Published: June 2009

ABSTRACT
CheA-short interacts with CheZ to localize CheZ to cell poles. The fifth helical region (residues 112 to 133) from the phosphotransfer domain of CheA interacts with CheZ and becomes ordered and helical, although it lacks a stable fold in the CheA fragment comprising residues 98 to 150 alone. One CheA molecule binds to one CheZ dimer.
The
Staphylococcus aureus
LytSR Two-Component Regulatory System Affects Biofilm Formation
Authors: Sharma-Kuinkel Batu K. , Mann Ethan E. , Ahn Jong-Sam , Kuechenmeister Lisa J. , Dunman Paul M. , Bayles Kenneth W. 
Published: June 2009

ABSTRACT

Studies of the
Staphylococcus aureus
LytSR two-component regulatory system have led to the identification of the
cid
and
lrg
operons, which affect murein hydrolase activity, stationary-phase survival, antibiotic tolerance, and biofilm formation. The
cid
gene products enhance murein hydrolase activity and antibiotic tolerance whereas the
lrg
gene products inhibit these processes in a manner believed to be analogous to bacteriophage-encoded holins and antiholins, respectively. Importantly, these operons have been shown to play significant roles in biofilm development by controlling the release of genomic DNA, which then becomes an important structural component of the biofilm matrix. To determine the role of LytSR in biofilm development, a
lytS
knockout mutant was generated from a clinical
S. aureus
isolate (UAMS-1) and the effects on gene expression and biofilm formation were examined. As observed in laboratory isolates, LytSR was found to be required for
lrgAB
expression. Furthermore, the
lytS
mutant formed a more adherent biofilm than the wild-type and complemented strains. Consistent with previous findings, the increased adherence of the mutant was attributed to the increased prevalence of matrix-associated eDNA. Transcription profiling studies indicated that the
lrgAB
operon is the primary target of LytSR-mediated regulation but that this regulatory system also impacts expression of a wide variety of genes involved in basic metabolism. Overall, the results of these studies demonstrate that the LytSR two-component regulatory system plays an important role in
S. aureus
biofilm development, likely as a result of its direct influence on
lrgAB
expression.
Functional and Expression Analyses of the
cop
Operon, Required for Copper Resistance in
Agrobacterium tumefaciens
Authors: Nawapan Sirikan , Charoenlap Nisanart , Charoenwuttitam Anchalee , Saenkham Panatda , Mongkolsuk Skorn , Vattanaviboon Paiboon 
Published: June 2009

ABSTRACT

The copper resistance determinant
copARZ
, which encodes a CPx-type copper ATPase efflux protein, a transcriptional regulator, and a putative intracellular copper chaperone, was functionally characterized for the phytopathogenic bacterium
Agrobacterium tumefaciens
. These genes are transcribed as an operon, and their expression is induced in response to increasing copper and silver ion concentrations in a
copR
-dependent fashion. Analysis of the
copARZ
promoter revealed a putative CopR binding box located within the spacer of the −35 and −10 promoter motifs. In vitro, purified CopR could specifically bind to the box. The inactivation of the
copARZ
operon or
copZ
reduces the level of resistance to copper but not to other metal ions. Also, the
copARZ
operon mutant shows increased sensitivity to the superoxide generators menadione and plumbagin. In addition, the loss of functional
copZ
does not affect the ability of copper ions to induce the
copARZ
promoter, indicating that CopZ is not involved in the copper-sensing mechanism of CopR. Altogether, the results demonstrate a crucial role for the
copARZ
operon as a component of the copper resistance machinery in
A. tumefaciens.
Feedback Regulation of Lac Repressor Expression inEscherichia coli
Authors: Oehler Stefan 
Published: June 2009

ABSTRACTNegative feedback regulation, mediated through repressor binding siteO3, which overlaps thelacIgene, could explain the robustness of the weak expression of Lac repressor. Significant autorepression of Lac repressor has never been ruled out. In the work presented here, the degree of autoregulation of Lac repressor was determined. It is negligible.
Replication Initiator DnaA ofEscherichia coliChanges Its Assembly Form on the Replication Origin during the Cell Cycle
Authors: Nozaki Shingo , Niki Hironori , Ogawa Tohru 
Published: June 2009

ABSTRACTDnaA is a replication initiator protein that is conserved among bacteria. It plays a central role in the initiation of DNA replication. In order to monitor its behavior in livingEscherichia colicells, a nonessential portion of the protein was replaced by a fluorescent protein. Such a strain grew normally, and flow cytometry data suggested that the chimeric protein has no substantial loss of the initiator activity. The initiator was distributed all over the nucleoid. Furthermore, a majority of the cells exhibited certain distinct foci that emitted bright fluorescence. These foci colocalized with the replication origin (oriC) region and were brightest during the period spanning the initiation event. In cells that had undergone the initiation, the foci were enriched in less intense ones. In addition, a significant portion of theoriCregions at this cell cycle stage had no colocalized DnaA-enhanced yellow fluorescent protein (EYFP) focus point. It was difficult to distinguish the initiator titration locus (datA) from theoriCregion. However, involvement ofdatAin the initiation control was suggested from the observation that, inΔdatAcells, DnaA-EYFP maximally colocalized with theoriCregion earlier in the cell cycle than it did in wild-type cells andoriCconcentration was increased.
Sxy Induces a CRP-S Regulon in
Escherichia coli
Authors: Sinha Sunita , Cameron Andrew D. S. , Redfield Rosemary J. 
Published: June 2009

ABSTRACT

Escherichia coli
is not considered naturally competent, yet it has homologues of the genes that most competent bacteria use for DNA uptake and processing. In
Haemophilus influenzae
and
Vibrio cholerae
, these genes are regulated by the Sxy and cyclic AMP receptor (CRP) proteins. We used microarrays to find out whether similar regulation occurs in
E. coli
. Expression of
sxy
strongly induced 63 transcriptional units, 34 of which required CRP for transcriptional activation and had promoter sites resembling the Sxy- and CRP-dependent CRP-S motif previously characterized in
H. influenzae
. As previously reported,
sxy
expression also induced the sigma-H regulon. Flagellar operons were downregulated by
sxy
expression, although motility remained unaffected. The CRP-S regulon included all of
E. coli
's known competence gene homologues, so we investigated Sxy's effect on competence-associated phenotypes. A
sxy
knockout reduced both “natural” plasmid transformation and competitive fitness in long-term culture. In addition, expression of plasmid-borne
sxy
led to production of type IV pilin, the main subunit of the DNA uptake machinery of most bacteria. Although
H. influenzae
Sxy only weakly activated the
E. coli
Sxy regulon, induction was dramatically improved when it was coexpressed with its cognate CRP, suggesting that intimate interactions between Sxy and CRP are required for transcriptional activation at CRP-S sites.
Directed Evolution of Ionizing Radiation Resistance in
Escherichia coli
Authors: Harris Dennis R. , Pollock Steve V. , Wood Elizabeth A. , Goiffon Reece J. , Klingele Audrey J. , Cabot Eric L. , Schackwitz Wendy , Martin Joel , Eggington Julie , Durfee Timothy J. , Middle Christina M. , Norton Jason E. , Popelars Michael C. , Li Hao , Klugman Sarit A. , Hamilton Lindsay L. , Bane Lukas B. , Pennacchio Len A. , Albert Thomas J. , Perna Nicole T. , Cox Michael M. , Battista John R. 
Published: June 2009

ABSTRACT

We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species,
Escherichia coli
, by directed evolution. Four populations of
Escherichia coli
K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D
37
values for strains isolated from two of the populations approached that exhibited by
Deinococcus radiodurans
. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the
recA
gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.
An Excretory Function for theEscherichia coliOuter Membrane Pore TolC: Upregulation ofmarAandsoxSTranscription and Rob Activity Due to Metabolites Accumulated intolCMutants
Authors: Rosner Judah L. , Martin Robert G. 
Published: June 2009

ABSTRACTEfflux pumps function to rid bacteria of xenobiotics, including antibiotics, bile salts, and organic solvents. TolC, which forms an outer membrane channel, is an essential component of several efflux pumps inEscherichia coli. We asked whether TolC has a role during growth in the absence of xenobiotics. BecausetolCtranscription is activated by three paralogous activators, MarA, SoxS, and Rob, we examined the regulation of these activators intolCmutants. Using transcriptional fusions, we detected significant upregulation ofmarRABandsoxStranscription and Rob protein activity intolCmutants. Three mechanisms could be distinguished: (i) activation ofmarRABtranscription was independent ofmarRAB,soxR, androbfunctions; (ii) activation ofsoxStranscription required SoxR, a sensor of oxidants; and (iii) Rob protein was activated posttranscriptionally. This mechanism is similar to the mechanisms of upregulation ofmarRAB,soxS, and Rob by treatment with certain phenolics, superoxides, and bile salts, respectively. The transcription of othermarA/soxS/robregulon promoters, includingtolCitself, was also elevated intolCmutants. We propose that TolC is involved in the efflux of certain cellular metabolites, not only xenobiotics. As these metabolites accumulate during growth, they trigger the upregulation of MarA, SoxS, and Rob, which in turn upregulatetolCand help rid the bacteria of these metabolites, thereby restoring homeostasis.
Shiga Toxin as a Bacterial Defense against a Eukaryotic Predator,
Tetrahymena thermophila
Authors: Lainhart William , Stolfa Gino , Koudelka Gerald B. 
Published: June 2009

ABSTRACT

Bacterially derived exotoxins kill eukaryotic cells by inactivating factors and/or pathways that are universally conserved among eukaryotic organisms. The genes that encode these exotoxins are commonly found in bacterial viruses (bacteriophages). In the context of mammals, these toxins cause diseases ranging from cholera to diphtheria to enterohemorrhagic diarrhea. Phage-carried exotoxin genes are widespread in the environment and are found with unexpectedly high frequency in regions lacking the presumed mammalian “targets,” suggesting that mammals are not the primary targets of these exotoxins. We suggest that such exotoxins may have evolved for the purpose of bacterial antipredator defense. We show here that
Tetrahymena thermophila
, a bacterivorous predator, is killed when cocultured with bacteria bearing a Shiga toxin (Stx)-encoding temperate bacteriophage. In cocultures with
Tetrahymena
, the Stx-encoding bacteria display a growth advantage over those that do not produce Stx.
Tetrahymena
is also killed by purified Stx. Disruption of the gene encoding the StxB subunit or addition of an excess of the nontoxic StxB subunit substantially reduced Stx holotoxin toxicity, suggesting that this subunit mediates intake and/or trafficking of Stx by
Tetrahymena
. Bacterially mediated
Tetrahymena
killing was blocked by mutations that prevented the bacterial SOS response (
recA
mutations) or by enzymes that breakdown H
2
O
2
(catalase), suggesting that the production of H
2
O
2
by
Tetrahymena
signals its presence to the bacteria, leading to bacteriophage induction and production of Stx.
A Critical Process Controlled by MalT and OmpR Is Revealed through Synthetic Lethality
Authors: Reimann Sylvia A. , Wolfe Alan J. 
Published: June 2009

ABSTRACT
The death of cells harboring defects in two distinct pathways implicates these pathways in the control of an essential process. Here we report that cells lacking OmpR and harboring constitutively active MalT undergo premature death that involves increased expression of the outer membrane porin LamB.
Specific Interactions between Four Molybdenum-Binding Proteins Contribute to Mo-Dependent Gene Regulation in
Rhodobacter capsulatus
Authors: Wiethaus Jessica , Müller Alexandra , Neumann Meina , Neumann Sandra , Leimkühler Silke , Narberhaus Franz , Masepohl Bernd 
Published: June 2009

ABSTRACT

The phototrophic purple bacterium
Rhodobacter capsulatus
encodes two transcriptional regulators, MopA and MopB, with partially overlapping and specific functions in molybdate-dependent gene regulation. Both MopA and MopB consist of an N-terminal DNA-binding helix-turn-helix domain and a C-terminal molybdate-binding di-MOP domain. They formed homodimers as apo-proteins and in the molybdate-bound state as shown by yeast two-hybrid (Y2H) studies, glutaraldehyde cross-linking, gel filtration chromatography, and copurification experiments. Y2H studies suggested that both the DNA-binding and the molybdate-binding domains contribute to dimer formation. Analysis of molybdate binding to MopA and MopB revealed a binding stoichiometry of four molybdate oxyanions per homodimer. Specific interaction partners of MopA and MopB were the molybdate transporter ATPase ModC and the molbindin-like Mop protein, respectively. Like other molbindins, the
R. capsulatus
Mop protein formed hexamers, which were stabilized by binding of six molybdate oxyanions per hexamer. Heteromer formation of MopA and MopB was shown by Y2H studies and copurification experiments. Reporter gene activity of a strictly MopA-dependent
mop
-
lacZ
fusion in mutant strains defective for either
mopA
,
mopB
, or both suggested that MopB negatively modulates expression of the
mop
promoter. We propose that depletion of the active MopA homodimer pool by formation of MopA-MopB heteromers might represent a fine-tuning mechanism controlling
mop
gene expression.
Comparative Genomic Analysis of Ten
Streptococcus pneumoniae
Temperate Bacteriophages
Authors: Romero Patricia , Croucher Nicholas J. , Hiller N. Luisa , Hu Fen Z. , Ehrlich Garth D. , Bentley Stephen D. , García Ernesto , Mitchell Tim J. 
Published: June 2009

ABSTRACT

Streptococcus pneumoniae
is an important human pathogen that often carries temperate bacteriophages. As part of a program to characterize the genetic makeup of prophages associated with clinical strains and to assess the potential roles that they play in the biology and pathogenesis in their host, we performed comparative genomic analysis of 10 temperate pneumococcal phages. All of the genomes are organized into five major gene clusters: lysogeny, replication, packaging, morphogenesis, and lysis clusters. All of the phage particles observed showed a
Siphoviridae
morphology. The only genes that are well conserved in all the genomes studied are those involved in the integration and the lysis of the host in addition to two genes, of unknown function, within the replication module. We observed that a high percentage of the open reading frames contained no similarities to any sequences catalogued in public databases; however, genes that were homologous to known phage virulence genes, including the
pblB
gene of
Streptococcus mitis
and the
vapE
gene of
Dichelobacter nodosus
, were also identified. Interestingly, bioinformatic tools showed the presence of a toxin-antitoxin system in the phage φSpn_6, and this represents the first time that an addition system in a pneumophage has been identified. Collectively, the temperate pneumophages contain a diverse set of genes with various levels of similarity among them.
Implementation of the Thin Layer Agar Method for Diagnosis of Smear-Negative Pulmonary Tuberculosis in a Setting with a High Prevalence of Human Immunodeficiency Virus Infection in Homa Bay, Kenya
Authors: Martin Anandi , Munga Waweru Peter , Babu Okatch Fred , Amondi Ouma Naureen , Bonte Laurence , Varaine Francis , Portaels Françoise 
Published: June 2009

ABSTRACT
The objective of this study was to evaluate the performance of a low-cost method, the thin layer agar (TLA) method, for the diagnosis of smear-negative patients. This prospective study was performed in Homa Bay District Hospital in Kenya. Out of 1,584 smear-negative sputum samples, 212 (13.5%) were positive by culture in Löwenstein-Jensen medium (LJ) and 220 (14%) were positive by the TLA method. The sensitivities of LJ and TLA were 71% and 74%, respectively. TLA could become an affordable method for the diagnosis of smear-negative tuberculosis in resource-limited settings, with results available within 2 weeks.
Human Papillomavirus Genotype Specificity of Hybrid Capture 2 Low-Risk Probe Cocktail
Authors: Poljak Mario , Kocjan Boštjan J. , Kovanda Anja , Lunar Maja M. , Lepej Snježana Židovec , Planinić Ana , Seme Katja , Vince Adriana 
Published: June 2009

ABSTRACT
A genotyping study of 285 Hybrid Capture 2 low-risk probe cocktail-positive specimens showed cross-reactivity with several untargeted human papillomavirus genotypes. Cross-reactivity was often clinically beneficial due to the detection of untargeted low-risk genotypes. A total of 8.4% of positive results, usually weak, were due to cross-reactivity with high-risk genotypes. Establishment of a gray zone is recommended.
First Case of Bloodstream Infection Caused by
Rhodococcus erythropolis
Authors: Baba Hisashi , Nada Toshi , Ohkusu Kiyofumi , Ezaki Takayuki , Hasegawa Yoshinori , Paterson David L. 
Published: June 2009

ABSTRACT

We describe the first case of bloodstream infection caused by
Rhodococcus erythropolis
. The identification was performed using 16S rRNA sequencing. This case illustrates that non-
equi Rhodococcus
infections may be underdiagnosed due to difficulties in identification in the routine clinical microbiology laboratory.