bluuebunny/crossref_metadata_2025_split huggingface dataset:
Horizontal Gene Transfer and the Evolution of Microvirid Coliphage Genomes
Authors: Rokyta D. R. , Burch C. L. , Caudle S. B. , Wichman H. A. 
Published: January 2006

ABSTRACT

Bacteriophage genomic evolution has been largely characterized by rampant, promiscuous horizontal gene transfer involving both homologous and nonhomologous source DNA. This pattern has emerged through study of the tailed double-stranded DNA (dsDNA) phages and is based upon a sparse sampling of the enormous diversity of these phages. The single-stranded DNA phages of the family
Microviridae
, including φX174, appear to evolve through qualitatively different mechanisms, possibly as result of their strictly lytic lifestyle and small genome size. However, this apparent difference could reflect merely a dearth of relevant data. We sought to characterize the forces that contributed to the molecular evolution of the
Microviridae
and to examine the genetic structure of this single family of bacteriophage by sequencing the genomes of microvirid phage isolated on a single bacterial host. Microvirids comprised 3.5% of the detectable phage in our environmental samples, and sequencing yielded 42 new microvirid genomes. Phylogenetic analysis of the genes contained in these and five previously described microvirid phages identified three distinct clades and revealed at least two horizontal transfer events between clades. All members of one clade have a block of five putative genes that are not present in any member of the other two clades. Our data indicate that horizontal transfer does contribute to the evolution of the microvirids but is both quantitatively and qualitatively different from what has been observed for the dsDNA phages.
Structural and Functional Studies Suggest a Catalytic Mechanism for the Phosphotransacetylase from
Methanosarcina thermophila
Authors: Lawrence Sarah H. , Luther Kelvin B. , Schindelin Hermann , Ferry James G. 
Published: January 2006

ABSTRACT

Phosphotransacetylase (EC 2.3.1.8) catalyzes reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA), forming acetyl-CoA and inorganic phosphate. Two crystal structures of phosphotransacetylase from the methanogenic archaeon
Methanosarcina thermophila
in complex with the substrate CoA revealed one CoA (CoA
1
) bound in the proposed active site cleft and an additional CoA (CoA
2
) bound at the periphery of the cleft. The results of isothermal titration calorimetry experiments are described, and they support the hypothesis that there are distinct high-affinity (equilibrium dissociation constant [

K
D

], 20 μM) and low-affinity (

K
D

, 2 mM) CoA binding sites. The crystal structures indicated that binding of CoA
1
is mediated by a series of hydrogen bonds and extensive van der Waals interactions with the enzyme and that there are fewer of these interactions between CoA
2
and the enzyme. Different conformations of the protein observed in the crystal structures suggest that domain movements which alter the geometry of the active site cleft may contribute to catalysis. Kinetic and calorimetric analyses of site-specific replacement variants indicated that there are catalytic roles for Ser
309
and Arg
310
, which are proximal to the reactive sulfhydryl of CoA
1
. The reaction is hypothesized to proceed through base-catalyzed abstraction of the thiol proton of CoA by the adjacent and invariant residue Asp
316
, followed by nucleophilic attack of the thiolate anion of CoA on the carbonyl carbon of acetyl phosphate. We propose that Arg
310
binds acetyl phosphate and orients it for optimal nucleophilic attack. The hypothesized mechanism proceeds through a negatively charged transition state stabilized by hydrogen bond donation from Ser
309
.
Suppression of Engulfment Defects inBacillus subtilisby Elevated Expression of the Motility Regulon
Authors: Perez Ana R. , Abanes-De Mello Angelica , Pogliano Kit 
Published: January 2006

ABSTRACTDuringBacillus subtilissporulation, the transient engulfment defect ofspoIIBstrains is enhanced byspoVGnull mutations and suppressed byspoVSnull mutations. These mutations have opposite effects on expression of the motility regulon, as thespoVGmutation reduces and thespoVSmutation increases σD-directed gene expression, cell separation, and autolysis. Elevating σDactivity by eliminating the anti-σ factor FlgM also suppressesspoIIB spoVG, and bothflgMandspoVSmutations cause continued expression of the σDregulon during sporulation. We propose that peptidoglycan hydrolases induced during motility can substitute for sporulation-specific hydrolases during engulfment. We find that sporulating cells are heterogeneous in their expression of the motility regulon, which could result in phenotypic variation between individual sporulating cells.
A
Bacteroides
Conjugative Transposon, CTnERL, Can Transfer a Portion of Itself by Conjugation without Excising from the Chromosome
Authors: Whittle Gabrielle , Hamburger Nathan , Shoemaker Nadja B. , Salyers Abigail A. 
Published: January 2006

ABSTRACT

CTnERL, a
Bacteroides
conjugative transposon, transferred DNA by an Hfr-type mechanism during conjugation when it was excision deficient due to an insertion in the integrase gene. Rescue of the conjugative transposon sequences required the recipient to be RecA proficient and to contain an integrated CTnERL. The transfer efficiency was only 10- to 30-fold lower than the normal element transfer efficiency, and the direction of transfer from the
oriT
gene showed that the integrase end was transferred first and that the transfer genes were transferred last.
Salmonella enterica
Strains Lacking the Frataxin Homolog CyaY Show Defects in Fe-S Cluster Metabolism In Vivo
Authors: Vivas E. , Skovran E. , Downs D. M. 
Published: January 2006

ABSTRACT

In
Salmonella enterica
, the
isc
operon contains genes necessary for the synthesis of Fe-S clusters and strains lacking this operon have severe defects in a variety of cellular processes. Other cellular loci that impact Fe-S cluster synthesis to a lesser extent have been described. The
cyaY
locus encodes a frataxin homolog, and it is shown here that lesions in this locus affect Fe-S cluster metabolism. When present in combination with other lesions, mutations in
cyaY
can result in a strain with more severe defects than those lacking the
isc
locus.
A
pmrA
Constitutive Mutant Sensitizes
Escherichia coli
to Deoxycholic Acid
Authors: Froelich Jamie M. , Tran Khoa , Wall Daniel 
Published: January 2006

ABSTRACT

An
Escherichia coli
mutant was isolated and shown to be polymyxin B resistant. Mapping and sequence analysis revealed a missense mutation at codon 53 within the
pmrA
(
basR
) gene that results in a G-to-V substitution. Fusions of promoters from the
pmrC
,
yibD
, and
pmrH
genes with the
lacZ
reporter showed that they were constitutively expressed in
pmrA53
cells. In
pmrA
+
strains, these promoters were induced by iron and zinc, while a Δ
pmrA
mutation blocked induction. The PmrA regulon regulates genes whose products remodel the composition and charge of lipid A and hence the barrier properties of the outer membrane. Along these lines, the
pmrA53
mutant was also found to be hypersensitive to the anionic bile detergent deoxycholic acid.
Topologically Fixed SecG Is Fully Functional
Authors: van der Sluis Eli O. , van der Vries Erhard , Berrelkamp Greetje , Nouwen Nico , Driessen Arnold J. M. 
Published: January 2006

ABSTRACT
It has been proposed that the bitopic membrane protein SecG undergoes topology inversion during translocation of (pre)proteins via SecYEG. Here we show that SecG covalently cross-linked to SecY cannot invert its topology while remaining fully functional in protein translocation. Our results strongly disfavor topology inversion of SecG during protein translocation.
Transcriptional Slippage in
mxiE
Controls Transcription and Translation of the Downstream
mxiD
Gene, Which Encodes a Component of the
Shigella flexneri
Type III Secretion Apparatus
Authors: Penno Christophe , Parsot Claude 
Published: January 2006

ABSTRACT

The
Shigella flexneri
transcription activator MxiE is produced by transcriptional slippage from two overlapping open reading frames. By using plasmids encoding a
mxiD-lacZ
translational fusion, we showed that transcriptional slippage in
mxiE
influences both transcription and translation of the downstream
mxiD
gene encoding an essential component of the type III secretion apparatus.
Transcriptome Analysis Applied to Survival of
Shewanella oneidensis
MR-1 Exposed to Ionizing Radiation
Authors: Qiu Xiaoyun , Daly Michael J. , Vasilenko Alexander , Omelchenko Marina V. , Gaidamakova Elena K. , Wu Liyou , Zhou Jizhong , Sundin George W. , Tiedje James M. 
Published: January 2006

ABSTRACT

The ionizing radiation (IR) dose that yields 20% survival (D
20
) of
Shewanella oneidensis
MR-1 is lower by factors of 20 and 200 than those for
Escherichia coli
and
Deinococcus radiodurans
, respectively. Transcriptome analysis was used to identify the genes of MR-1 responding to 40 Gy (D
20
). We observed the induction of 170 genes and repression of 87 genes in MR-1 during a 1-h recovery period after irradiation. The genomic response of MR-1 to IR is very similar to its response to UV radiation (254 nm), which included induction of systems involved in DNA repair and prophage synthesis and the absence of differential regulation of tricarboxylic acid cycle activity, which occurs in IR-irradiated
D. radiodurans
. Furthermore, strong induction of genes encoding antioxidant enzymes in MR-1 was observed. DNA damage may not be the principal cause of high sensitivity to IR, considering that MR-1 carries genes encoding a complex set of DNA repair systems and 40 Gy IR induces less than one double-strand break in its genome. Instead, a combination of oxidative stress, protein damage, and prophage-mediated cell lysis during irradiation and recovery might underlie this organism's great sensitivity to IR.
Analysis of the Lateral Flagellar Gene System ofAeromonas hydrophilaAH-3
Authors: Canals Rocío , Altarriba Maria , Vilches Silvia , Horsburgh Gavin , Shaw Jonathan G. , Tomás Juan M. , Merino Susana 
Published: January 2006

ABSTRACTMesophilicAeromonasstrains express a polar flagellum in all culture conditions, and certain strains produce lateral flagella on semisolid media or on surfaces. AlthoughAeromonaslateral flagella have been described as a colonization factor, little is known about their organization and expression. Here we characterized the complete lateral flagellar gene cluster ofAeromonas hydrophilaAH-3 containing 38 genes, 9 of which (lafA-U) have been reported previously. Among theflgLLandlafAstructural genes we found a modification accessory factor gene (maf-5) that is involved in formation of lateral flagella; this is the first time that such a gene has been described for lateral flagellar gene systems. AllAeromonaslateral flagellar genes were located in a unique chromosomal region, in contrast toVibrio parahaemolyticus, in which the analogous genes are distributed in two different chromosomal regions. InA. hydrophilamutations inflhAL,lafK,fliJL,flgNL,flgEL, andmaf-5resulted in a loss of lateral flagella and reductions in adherence and biofilm formation, but they did not affect polar flagellum synthesis. Furthermore, we also cloned and sequenced theA. hydrophilaAH-3 alternative sigma factor σ54(rpoN); mutation of this factor suggested that it is involved in expression of both types of flagella.
Topology and Boundaries of the Aerotaxis Receptor Aer in the Membrane ofEscherichia coli
Authors: Amin Divya N. , Taylor Barry L. , Johnson Mark S. 
Published: January 2006

ABSTRACTEscherichia colichemoreceptors are type I membrane receptors that have a periplasmic sensing domain, a cytosolic signaling domain, and two transmembrane segments. The aerotaxis receptor, Aer, is different in that both its sensing and signaling regions are proposed to be cytosolic. This receptor has a 38-residue hydrophobic segment that is thought to form a membrane anchor. Most transmembrane prediction programs predict a single transmembrane-spanning segment, but such a topology is inconsistent with recent studies indicating that there is direct communication between the membrane flanking PAS and HAMP domains. We studied the overall topology and membrane boundaries of the Aer membrane anchor by a cysteine-scanning approach. The proximity of 48 cognate cysteine replacements in Aer dimers was determined in vivo by measuring the rate and extent of disulfide cross-linking after adding the oxidant copper phenanthroline, both at room temperature and to decrease lateral diffusion in the membrane, at 4°C. Membrane boundaries were identified in membrane vesicles using 5-iodoacetamidofluorescein and methoxy polyethylene glycol 5000 (mPEG). To map periplasmic residues, accessible cysteines were blocked in whole cells by pretreatment with 4-acetamido-4′-maleimidylstilbene-2, 2′ disulfonic acid before the cells were lysed in the presence of mPEG. The data were consistent with two membrane-spanning segments, separated by a short periplasmic loop. Although the membrane anchor contains a central proline residue that reaches the periplasm, its position was permissive to several amino acid and peptide replacements.
Peptidoglycan O Acetylation and Autolysin Profile ofEnterococcus faecalisin the Viable but Nonculturable State
Authors: Pfeffer John M. , Strating Hendrik , Weadge Joel T. , Clarke Anthony J. 
Published: January 2006

ABSTRACTThe O acetylation of peptidoglycan occurs specifically at the C-6 hydroxyl group of muramoyl residues. Using a combination of high-performance liquid chromatography-based organic acid analysis and carbohydrate analysis by high-pH anion-exchange chromatography, we determined that strains ofEntercoccus durans,E. faecalis,E. faecium, andE. hiraeproduce O-acetylated peptidoglycan. The levels of O acetylation ranged from 19% to 72% relative to the muramic acid content, and they were found to vary with the growth phase of the culture. Increases of 10 to 40% in O acetylation were observed with cultures entering the stationary phase. Cells ofE. faecalisin the viable but nonculturable (VBNC) state had the highest levels of peptidoglycan O acetylation. The presence of this modification to peptidoglycan was shown to inhibit the action of hen egg white lysozyme in a concentration-dependent manner. Zymography using sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels containing either O-acetylated or chemically de-O-acetylated peptidoglycan was used to monitor the production of specific autolysins inE. faecalis. Differences in the expression of specific autolysins were observed with the age of the culture, and VBNCE. faecalisproduced the highest levels of these enzymes. This technique also permitted classification of the enterococcal autolysins into enzymes that preferentially hydrolyze either O-acetylated or non-O-acetylated peptidoglycan and enzymes that show no apparent preference for either substrate type.
Function and Evolution of Plasmid-Borne Genes for Pyrimidine Biosynthesis inBorreliaspp
Authors: Zhong Jianmin , Skouloubris Stephane , Dai Qiyuan , Myllykallio Hannu , Barbour Alan G. 
Published: January 2006

ABSTRACTThethyXgene for thymidylate synthase of the Lyme borreliosis (LB) agentBorrelia burgdorferiis located in a 54-kb linear plasmid. In the present study, we identified an orthologous thymidylate synthase gene in the relapsing fever (RF) agentBorrelia hermsii, located it in a 180-kb linear plasmid, and demonstrated its expression. The functions of theB. hermsiiandB. burgdorferi thyXgene products were evaluated both in vivo, by complementation of a thymidylate synthase-deficientEscherichia colimutant, and in vitro, by testing their activities after purification. TheB. hermsii thyXgene complemented thethyAmutation inE. coli, and purifiedB. hermsiiThyX protein catalyzed the conversion of dTMP from dUMP. In contrast, theB. burgdorferiThyX protein had only weakly detectable activity in vitro, and theB. burgdorferi thyXgene did not provide complementation in vivo. The lack of activity ofB. burgdorferi's ThyX protein was associated with the substitution of a cysteine for a highly conserved arginine at position 91. TheB. hermsii thyXlocus was further distinguished by the downstream presence in the plasmid of orthologues ofnrdI,nrdE, andnrdF, which encode the subunits of ribonucleoside diphosphate reductase and which are not present in the LB agentsB. burgdorferiandBorrelia garinii. Phylogenetic analysis suggested that thenrdIEFcluster ofB. hermsiiwas acquired by horizontal gene transfer. These findings indicate thatBorreliaspp. causing RF have a greater capability for de novo pyrimidine synthesis than those causing LB, thus providing a basis for some of the biological differences between the two groups of pathogens.
Arginine-Dependent Gene Regulation via the ArgR Repressor Is Species Specific inChlamydia
Authors: Schaumburg Chris S. , Tan Ming 
Published: January 2006

ABSTRACTSome, but not all,Chlamydiaspp. are predicted to encode a homolog of ArgR, a master regulatory molecule that modulates arginine biosynthesis and catabolism in bacteria in response to intracellular arginine levels. While genes for arginine biosynthesis are apparently missing inChlamydia, a putative arginine transport system encoded byglnP,glnQ, andartJis present. We found that recombinantChlamydia pneumoniaeArgR functions as an arginine-dependent aporepressor that bound specifically to operator sequences upstream of theglnPQoperon. ArgR was able to repress transcription in a promoter-specific manner that was dependent on the concentration of the corepressorl-arginine. We were able to locate ArgR operators upstream ofglnPQinC. pneumoniaeandChlamydophila caviaebut notChlamydia trachomatis, which corresponded to the predicted presence or absence of ArgR in these chlamydial species. Our findings indicate that only some members of the familyChlamydiaceaehave an arginine-responsive mechanism of gene regulation that is predicted to control arginine uptake from the host cell. This is the first study to directly demonstrate a species-specific mechanism of transcriptional regulation inChlamydia.
Two DNA Invertases Contribute to Flagellar Phase Variation in
Salmonella enterica
Serovar Typhimurium Strain LT2
Authors: Kutsukake Kazuhiro , Nakashima Hisashi , Tominaga Akira , Abo Tatsuhiko 
Published: January 2006

ABSTRACT

Salmonella enterica
serovar Typhimurium strain LT2 possesses two nonallelic structural genes,
fliC
and
fljB
, for flagellin, the component protein of flagellar filaments. Flagellar phase variation occurs by alternative expression of these two genes. This is controlled by the inversion of a DNA segment, called the H segment, containing the
fljB
promoter. H inversion occurs by site-specific recombination between inverted repetitious sequences flanking the H segment. This recombination has been shown in vivo and in vitro to be mediated by a DNA invertase, Hin, whose gene is located within the H segment. However, a search of the complete genomic sequence revealed that LT2 possesses another DNA invertase gene that is located adjacent to another invertible DNA segment within a resident prophage, Fels-2. Here, we named this gene
fin
. We constructed
hin
and
fin
disruption mutants from LT2 and examined their phase variation abilities. The
hin
disruption mutant could still undergo flagellar phase variation, indicating that Hin is not the sole DNA invertase responsible for phase variation. Although the
fin
disruption mutant could undergo phase variation,
fin hin
double mutants could not. These results clearly indicate that both Hin and Fin contribute to flagellar phase variation in LT2. We further showed that a phase-stable serovar, serovar Abortusequi, which is known to possess a naturally occurring
hin
mutation, lacks Fels-2, which ensures the phase stability in this serovar.
FljA-Mediated Posttranscriptional Control of Phase 1 Flagellin Expression in Flagellar Phase Variation of
Salmonella enterica
Serovar Typhimurium
Authors: Yamamoto Shouji , Kutsukake Kazuhiro 
Published: January 2006

ABSTRACT

Flagellar phase variation of
Salmonella
is a phenomenon where two flagellin genes,
fliC
(phase 1) and
fljB
(phase 2), are expressed alternately. This is controlled by the inversion of a DNA segment containing the promoter for the
fljB
gene. The
fljB
gene constitutes an operon with the
fljA
gene, which encodes a negative regulator for
fliC
expression. Previous biochemical analysis suggested that phase variation might depend on alternative synthesis of phase-specific flagellin mRNA (H. Suzuki and T. Iino, J. Mol. Biol.
81:
57-70, 1973). However, recently reported results suggested that FljA-dependent inhibition might be mediated by a posttranscriptional control mechanism (H. R. Bonifield and K. T. Hughes, J. Bacteriol.
185:
3567-3574, 2003). In this study, we reexamined the mechanism of FljA-mediated inhibition of
fliC
expression more carefully. Northern blotting analysis revealed that no
fliC
mRNA was detected in phase 2 cells. However, only a moderate decrease in β-galactosidase activity was observed from the
fliC-lacZ
transcriptional fusion gene in phase 2 cells compared with that in phase 1 cells. In contrast, the expression of the
fliC-lacZ
translational fusion gene was severely impaired in phase 2 cells. The half-life of
fliC
mRNA was shown to be much shorter in phase 2 cells than in phase 1 cells. Purified His-tagged FljA protein was shown to bind specifically to
fliC
mRNA and inhibit the translation from
fliC
mRNA in vitro. On the basis of these results, we propose that in phase 2 cells, FljA binds to
fliC
mRNA and inhibits its translation, which in turn facilitates its degradation.
GTPase Activity, Structure, and Mechanical Properties of Filaments Assembled from Bacterial Cytoskeleton Protein MreB
Authors: Esue Osigwe , Wirtz Denis , Tseng Yiider 
Published: January 2006

ABSTRACT

MreB, a major component of the recently discovered bacterial cytoskeleton, displays a structure homologous to its eukaryotic counterpart actin. Here, we study the assembly and mechanical properties of
Thermotoga maritima
MreB in the presence of different nucleotides in vitro. We found that GTP, not ADP or GDP, can mediate MreB assembly into filamentous structures as effectively as ATP. Upon MreB assembly, both GTP and ATP release the gamma phosphate at similar rates. Therefore, MreB is an equally effective ATPase and GTPase. Electron microscopy and quantitative rheology suggest that the morphologies and micromechanical properties of filamentous ATP-MreB and GTP-MreB are similar. In contrast, mammalian actin assembly is favored in the presence of ATP over GTP. These results indicate that, despite high structural homology of their monomers,
T. maritima
MreB and actin filaments display different assembly, morphology, micromechanics, and nucleotide-binding specificity. Furthermore, the biophysical properties of
T. maritima
MreB filaments, including high rigidity and propensity to form bundles, suggest a mechanism by which MreB helical structure may be involved in imposing a cylindrical architecture on rod-shaped bacterial cells.