bluuebunny/crossref_metadata_2025_split huggingface dataset:
Error-Prone DNA Polymerase IV Is Regulated by the Heat Shock Chaperone GroE in
Escherichia coli
Authors: Layton Jill C. , Foster Patricia L. 
Published: January 2005

ABSTRACT

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The
groE
operon consists of two genes,
groES
and
groEL
; point mutations in either gene conferred the same phenotype, reducing Lac
+
adaptive mutation 10- to 20-fold.
groE
mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In
recG
+
strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in
recG
mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.
The
LEE1
Promoters from both Enteropathogenic and Enterohemorrhagic
Escherichia coli
Can Be Activated by PerC-Like Proteins from Either Organism
Authors: Porter Megan E. , Mitchell Paul , Free Andrew , Smith David G. E. , Gally David L. 
Published: January 2005

ABSTRACT

The PerC protein of enteropathogenic
Escherichia coli
(EPEC), encoded by the pEAF plasmid, is an activator of the locus of enterocyte effacement (LEE) pathogenicity island via the
LEE1
promoter. It has been assumed that the related LEE-containing pathogen enterohemorrhagic
E. coli
(EHEC) lacks PerC-dependent activation due to utilization of an alternative
LEE1
promoter and lack of a
perC
gene. However, we show here that EPEC PerC can activate both the EPEC and EHEC
LEE1
promoters and that the major transcriptional start site is similarly located in both organisms. Moreover, a PerC-like protein family identified from EHEC genome analyses, PerC1 (also termed PchABC), can also activate both promoters in a manner similar to that of EPEC PerC. The
perC1
genes are carried by lambdoid prophages, which exist in multiple copies in different EHEC strains, and have a variable flanking region which may affect their expression. Although individual
perC1
copies appear to be poorly expressed, the total
perC1
expression level from a strain encoding multiple copies approaches that of
perC
in EPEC and may therefore contribute significantly to
LEE1
activation. Alignment of the protein sequences of these PerC homologues allows core regions of the PerC protein to be identified, and we show by site-directed mutagenesis that these core regions are important for function. However, purified PerC protein shows no in vitro binding affinity for the
LEE1
promoter, suggesting that other core
E. coli
proteins may be involved in its mechanism of activation. Our data indicate that the nucleoid-associated protein IHF is one such protein.
Mutation Analysis of the Histidine Residues in the Glycylglycine Endopeptidase ALE-1
Authors: Fujiwara Tamaki , Aoki Shin , Komatsuzawa Hitoshi , Nishida Tetsuya , Ohara Masaru , Suginaka Hidekazu , Sugai Motoyuki 
Published: January 2005

ABSTRACT

A novel staphylolytic enzyme, ALE-1, is a glycylglycine endopeptidase produced by
Staphylococcus capitis
EPK1. ALE-1 possesses seven histidines. Chemical modification studies using diethylpyrocarbonate and iodoacetic acid suggested that a histidine or tyrosine residue(s) in the molecule is important for the organism's staphylolytic activity. All of the histidine residues, one tyrosine, and one aspartic acid residue in the N-terminally truncated ALE-1 (ΔN-term ALE-1) were systematically altered by site-directed mutagenesis, and the enzyme activities and metal contents of the variants were measured. Our studies indicated that His-150, His-200, His-231, His-233, and Asp-154 are essential for the enzyme activity of ΔN-term ALE-1. Except for His-150 and Asp-154, all of these amino acids were located within the 38-amino-acid region conserved among 11 proteins, including 5 staphylolytic endopeptidases. Inductively coupled plasma-mass spectrometric analysis of ΔN-term ALE-1 revealed that it contains one atom of zinc per molecule. Measurement of the zinc content of the mutant ΔN-term ALE-1 suggested that His-150 and -233 are important for zinc binding; their loss in these variant enzymes coincided with the loss of staphylolytic activity. These results strongly suggest that ALE-1 is a novel member of zinc metalloproteases.
Posttranslational Regulation of Nitrate Assimilation in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Kobayashi Masaki , Takatani Nobuyuki , Tanigawa Mari , Omata Tatsuo 
Published: January 2005

ABSTRACT

Posttranslational regulation of nitrate assimilation was studied in the cyanobacterium
Synechocystis
sp. strain PCC 6803. The ABC-type nitrate and nitrite bispecific transporter encoded by the
nrtABCD
genes was completely inhibited by ammonium as in
Synechococcus elongatus
strain PCC 7942. Nitrate reductase was insensitive to ammonium, while it is inhibited in the
Synechococcus
strain. Nitrite reductase was also insensitive to ammonium. The inhibition of nitrate and nitrite transport required the P
II
protein (
glnB
gene product) and the C-terminal domain of NrtC, one of the two ATP-binding subunits of the transporter, as in the
Synechococcus
strain. Mutants expressing the P
II
derivatives in which Ala or Glu is substituted for the conserved Ser
49
, which has been shown to be the phosphorylation site in the
Synechococcus
strain, showed ammonium-promoted inhibition of nitrate uptake like that of the wild-type strain. The S49A and S49E substitutions in GlnB did not affect the regulation of the nitrate and nitrite transporter in
Synechococcus
either. These results indicated that the presence or absence of negative electric charge at the 49th position does not affect the activity of the P
II
protein to regulate the cyanobacterial ABC-type nitrate and nitrite transporter according to the cellular nitrogen status. This finding suggested that the permanent inhibition of nitrate assimilation by an S49A derivative of P
II
, as was previously reported for
Synechococcus elongatus
strain PCC 7942, is likely to have resulted from inhibition of nitrate reductase rather than the nitrate and nitrite transporter.
Probing Direct Interactions between CodY and theoppDPromoter ofLactococcus lactis
Authors: den Hengst Chris D. , Curley Peter , Larsen Rasmus , Buist Girbe , Nauta Arjen , van Sinderen Douwe , Kuipers Oscar P. , Kok Jan 
Published: January 2005

ABSTRACTCodY ofLactococcus lactisMG1363 is a transcriptional regulator that represses the expression of several genes encoding proteins of the proteolytic system. These genes includepepN,pepC,opp-pepO1, and probablyprtPM,pepX, andpepDA2, since the expression of the latter three genes relative to nitrogen availability is similar to that of the former. By means of in vitro DNA binding assays and DNase I footprinting techniques, we demonstrate thatL. lactisCodY interacts directly with a region upstream of the promoter of its major target known so far, theoppsystem. Our results indicate that multiple molecules of CodY interact with this promoter and that the amount of bound CodY molecules is affected by the presence of branched-chain amino acids and not by GTP. Addition of these amino acids strongly affects the extent of the region protected by CodY in DNase I footprints. Random and site-directed mutagenesis of the upstream region ofoppDyielded variants that were derepressed in a medium with an excess of nitrogen sources. Binding studies revealed the importance of specific bases in the promoter region required for recognition by CodY.
Intimin-Mediated Export of Passenger Proteins Requires Maintenance of a Translocation-Competent Conformation
Authors: Adams Thorsten M. , Wentzel Alexander , Kolmar Harald 
Published: January 2005

ABSTRACTIntimins from pathogenic bacteria promote intimate bacterial adhesion to epithelial cells. Several structurally similar domains form on the bacterial cell surface an extended rigid rod that exposes the carboxy-terminal domain, which interacts with the translocated intimin receptor. We constructed a series of intimin-derived fusion proteins consisting of carboxy-terminally truncated intimin and the immunoglobulin light-chain variable domain REIv, ubiquitin, calmodulin, β-lactamase inhibitor protein, or β-lactamase. By systematically investigating the intimin-mediated cell surface exposure of these passenger domains in the presence or absence of compounds that interfere with outer membrane stability or passenger domain folding, we acquired experimental evidence that intimin-mediated protein export across the outer membrane requires, prior to export, the maintenance of a translocation-competent conformation that may be distinct from the final protein structure. We propose that, during export, competition exists between productive translocation and folding of the passenger domain in the periplasm into a stable conformation that is not compatible with translocation through the bacterial outer membrane. These results may expand understanding of the mechanism by which intimins are inserted into the outer membrane and expose extracellular domains on the cell surface.
Analysis of the Precursor rRNA Fractions of Rapidly Growing Mycobacteria: Quantification by Methods That Include the Use of a Promoter (
rrnA
P1) as a Novel Standard
Authors: Menéndez María del Carmen , Rebollo María José , Núñez María del Carmen , Cox Robert A. , García María Jesús 
Published: January 2005

ABSTRACT

Mycobacterial species are able to control rRNA production through variations in the number and strength of promoters controlling their
rrn
operons.
Mycobacterium chelonae
and
M. fortuitum
are members of the rapidly growing mycobacterial group. They carry a total of five promoters each, encoded, respectively, by one and two
rrn
operons per genome. Quantification of precursor
rrn
transcriptional products (pre-
rrn)
has allowed detection of different promoter usage during cell growth. Bacteria growing in several culture media with different nutrient contents were compared. Balanced to stationary phases were analyzed. Most promoters were found to be used at different levels depending on the stage of bacterial growth and the nutrient content of the culture medium. Some biological implications are discussed. Sequences of the several promoters showed motifs that could be correlated to their particular level of usage. A product corresponding to the first
rrnA
promoter in both species, namely,
rrnA
P1, was found to contribute at a low and near-constant level to pre-rRNA synthesis, regardless of the culture medium used and the stage of growth analyzed. This product was used as a standard to quantitate rRNA gene expression by real-time PCR when
M. fortuitum
infected macrophages. It was shown that this bacterium actively synthesizes rRNA during the course of infection and that one of its
rrn
operons is preferentially used under such conditions.
Caulobacter crescentusRequires RodA and MreB for Stalk Synthesis and Prevention of Ectopic Pole Formation
Authors: Wagner Jennifer K. , Galvani Cheryl D. , Brun Yves V. 
Published: January 2005

ABSTRACTCaulobacter crescentuscells treated with amdinocillin, an antibiotic which specifically inhibits the cell elongation transpeptidase penicillin binding protein 2 inEscherichia coli, exhibit defects in stalk elongation and morphology, indicating that stalk synthesis may be a specialized form of cell elongation. In order to investigate this possibility further, we examined the roles of two other proteins important for cell elongation, RodA and MreB. We show that, inC. crescentus, therodAgene is essential and that RodA depletion leads to a loss of control over stalk and cell body diameter and a stalk elongation defect. In addition, we demonstrate that MreB depletion leads to a stalk elongation defect and conclude that stalk elongation is a more constrained form of cell elongation. Our results strongly suggest that MreB by itself does not determine the diameter of the cell body or stalk. Finally, we show that cells recovering from MreB depletion exhibit a strong budding and branching cell body phenotype and possess ectopic poles, as evidenced by the presence of multiple, misplaced, and sometimes highly branched stalks at the ends of these buds and branches. This phenotype is also seen to a lesser extent in cells recovering from RodA depletion and amdinocillin treatment. We conclude that MreB, RodA, and the target(s) of amdinocillin all contribute to the maintenance of cellular polarity inC. crescentus.
Staphylococcus aureus
Serves as an Iron Source for
Pseudomonas aeruginosa
during In Vivo Coculture
Authors: Mashburn Lauren M. , Jett Amy M. , Akins Darrin R. , Whiteley Marvin 
Published: January 2005

ABSTRACT

Pseudomonas aeruginosa
is a gram-negative opportunistic human pathogen often infecting the lungs of individuals with the heritable disease cystic fibrosis and the peritoneum of individuals undergoing continuous ambulatory peritoneal dialysis. Often these infections are not caused by colonization with
P. aeruginosa
alone but instead by a consortium of pathogenic bacteria. Little is known about growth and persistence of
P. aeruginosa
in vivo, and less is known about the impact of coinfecting bacteria on
P. aeruginosa
pathogenesis and physiology. In this study, a rat dialysis membrane peritoneal model was used to evaluate the in vivo transcriptome of
P. aeruginosa
in monoculture and in coculture with
Staphylococcus aureus
. Monoculture results indicate that approximately 5% of all
P. aeruginosa
genes are differentially regulated during growth in vivo compared to in vitro controls. Included in this analysis are genes important for iron acquisition and growth in low-oxygen environments. The presence of
S. aureus
caused decreased transcription of
P. aeruginosa
iron-regulated genes during in vivo coculture, indicating that the presence of
S. aureus
increases usable iron for
P. aeruginosa
in this environment. We propose a model where
P. aeruginosa
lyses
S. aureus
and uses released iron for growth in low-iron environments.
Introns in the Cytolethal Distending Toxin Gene ofActinobacillus actinomycetemcomitans
Authors: Tan Kai Soo , Ong Grace , Song Keang Peng 
Published: January 2005

ABSTRACTIn eukaryotic cells, genes are interrupted by intervening sequences called introns. Introns are transcribed as part of a precursor RNA that is subsequently removed by splicing, giving rise to mature mRNA. However, introns are rarely found in bacteria.Actinobacillus actinomycetemcomitansis a periodontal pathogen implicated in aggressive forms of periodontal disease. This organism has been shown to produce cytolethal distending toxin (CDT), which causes sensitive eukaryotic cells to become irreversibly blocked at the G2/M phase of the cell cycle. In this study, we report the presence of introns within thecdtgene ofA. actinomycetemcomitans. By use of reverse transcription-PCR,cdttranscripts of 2.123, 1.572, and 0.882 kb (RTA1, RTA2, and RTA3, respectively) were detected. In contrast, a single 2.123-kb amplicon was obtained by PCR with the genomic DNA. Similar results were obtained when a plasmid carryingcdtwas cloned intoEscherichia coli. Sequence analysis of RTA1, RTA2, and RTA3 revealed that RTA1 had undergone splicing, giving rise to RTA2 and RTA3. Two exon-intron boundaries, or splice sites, were identified at positions 863 to 868 and 1553 to 1558 of RTA1. Site-directed and deletion mutation studies of the splice site sequence indicated that sequence conservation was important in order for accurate splicing to occur. The catalytic region of thecdtRNA was located within thecdtCgene. This 0.56-kb RNA behaved independently as a catalytically active RNA molecule (a ribozyme) in vitro, capable of splicing heterologous RNA in bothcisandtransconfigurations.
Molecular Nature of Spontaneous Modifications ingacSWhich Cause Colony Phase Variation inPseudomonassp. Strain PCL1171
Authors: van den Broek Daan , Chin-A-Woeng Thomas F. C. , Bloemberg Guido V. , Lugtenberg Ben J. J. 
Published: January 2005

ABSTRACTPseudomonassp. strain PCL1171 displays colony phase variation between opaque phase I and translucent phase II colonies, thereby regulating the production of secondary metabolites and exoenzymes. Complementation and sequence analysis of 26 phase II mutants and of 13 wild-type phase II sectors growing out of phase I colonies showed that in all these cases the phase II phenotype is caused by spontaneous mutations ingacAor/andgacS. Mutation ofgacreduced both the length of the lag phase and the generation time. Isolation and sequencing of thegacSgenes from the phase II bacteria revealed one insertion as well as several random point mutations, deletions, and DNA rearrangements. Most phase II colonies reverted with a high frequency, resulting in wild-typegacAandgacSgenes and a phase I phenotype. Some phase II bacteria retained the phase II phenotype but changed genotypically as a result of (re)introduction of mutations in eithergacAorgacS. The reversion ofgacAorgacSto the wild type was not affected by mutation ofrecAandrecB. We conclude that inPseudomonassp. strain PCL1171, mutations ingacAandgacSare the basis for phase variation from phase I to phase II colonies and that, since these mutations are efficiently removed, mutations ingacresult in dynamic switches between the “wild-type” population and the subpopulations harboring spontaneous mutations ingacAand orgacS, thereby enabling both populations to be maintained.
Roles of Thioredoxin Reductase during the Aerobic Life of
Lactococcus
lactis
Authors: Vido Karin , Diemer Hélène , Van Dorsselaer Alain , Leize Emmanuelle , Juillard Vincent , Gruss Alexandra , Gaudu Philippe 
Published: January 2005

ABSTRACT

Thiol-disulfide bond balance is generally maintained in bacteria by thioredoxin reductase-thioredoxin and/or glutathione-glutaredoxin systems. Some gram-positive bacteria, including
Lactococcus
lactis
, do not produce glutathione, and the thioredoxin system is presumed to be essential. We constructed an
L. lactis
trxB1
mutant. The mutant was obtained under anaerobic conditions in the presence of dithiothreitol (DTT). Unexpectedly, the
trxB1
mutant was viable without DTT and under aerated static conditions, thus disproving the essentiality of this system. Aerobic growth of the
trxB1
mutant did not require glutathione, also ruling out the need for this redox maintenance system. Proteomic analyses showed that known oxidative stress defense proteins are induced in the
trxB1
mutant. Two additional effects of
trxB1
were not previously reported in other bacteria: (i) induction of proteins involved in fatty acid or menaquinone biosynthesis, indicating that membrane synthesis is part of the cellular response to a redox imbalance, and (ii) alteration of the isoforms of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GapB). We determined that the two GapB isoforms in
L. lactis
differed by the oxidation state of catalytic-site cysteine C
152
. Unexpectedly, a decrease specific to the oxidized, inactive form was observed in the
trxB1
mutant, possibly because of proteolysis of oxidized GapB. This study showed that thioredoxin reductase is not essential in
L. lactis
and that its inactivation triggers induction of several mechanisms acting at the membrane and metabolic levels. The existence of a novel redox function that compensates for
trxB1
deficiency is suggested.
Analysis of MinD Mutations Reveals Residues Required for MinE Stimulation of the MinD ATPase and Residues Required for MinC Interaction
Authors: Zhou Huaijin , Schulze Ryan , Cox Sandra , Saez Cristian , Hu Zonglin , Lutkenhaus Joe 
Published: January 2005

ABSTRACT
The MinD ATPase is critical to the oscillation of the Min proteins, which limits formation of the Z ring to midcell. In the presence of ATP, MinD binds to the membrane and recruits MinC, forming a complex that can destabilize the cytokinetic Z ring. MinE, which is also recruited to the membrane by MinD, displaces MinC and stimulates the MinD ATPase, resulting in the oscillation of the Min proteins. In this study we have investigated the role of lysine 11, present in the deviant Walker A motif of MinD, and the three residues in helix 7 (E146, S148, and D152) that interact electrostatically with lysine 11. Lysine 11 is required for interaction of MinD with the membrane, MinC, MinE, and itself. In contrast, the three residues in helix 7 that interact with lysine 11 are not required for binding to the membrane or activation of MinC. They are also not required for MinE binding; however, they are required for MinE to stimulate the MinD ATPase. Interestingly, the D152A mutant self-interacts, binds to the membrane, and recruits MinC and MinE in the presence of ADP as well as ATP. This mutant provides evidence that dimerization of MinD is sufficient for MinD to bind the membrane and recruit its partners.
The F-Plasmid TraI Protein Contains Three Functional Domains Required for Conjugative DNA Strand Transfer
Authors: Matson Steven W. , Ragonese Heather 
Published: January 2005

ABSTRACT
The F-plasmid-encoded TraI protein, also known as DNA helicase I, is a bifunctional protein required for conjugative DNA transfer. The enzyme catalyzes two distinct but functionally related reactions required for the DNA processing events associated with conjugation: the site- and strand-specific transesterification (relaxase) reaction that provides the nick required to initiate strand transfer and a processive 5′-to-3′ helicase reaction that provides the motive force for strand transfer. Previous studies have identified the relaxase domain, which encompasses the first ∼310 amino acids of the protein. The helicase-associated motifs lie between amino acids 990 and 1450. The function of the region between amino acids 310 and 990 and the region from amino acid 1450 to the C-terminal end is unknown. A protein lacking the C-terminal 252 amino acids (TraIΔ252) was constructed and shown to have essentially wild-type levels of transesterase and helicase activity. In addition, the protein was capable of a functional interaction with other components of the minimal relaxosome. However, TraIΔ252 was not able to support conjugative DNA transfer in genetic complementation experiments. We conclude that TraIΔ252 lacks an essential C-terminal domain that is required for DNA transfer. We speculate this domain may be involved in essential protein-protein interactions with other components of the DNA transfer machinery.