bluuebunny/crossref_metadata_2025_split huggingface dataset:
TcaR, a Putative MarR-Like Regulator of
sarS
Expression
Authors: McCallum Nadine , Bischoff Markus , Maki Hideki , Wada Akihito , Berger-Bächi Brigitte 
Published: May 2004

ABSTRACT

TcaR, which shares sequence homology with MarR-like transcriptional regulators, has been identified as a novel
Staphylococcus aureus
regulator affecting the expression of the global regulatory element SarS (SarH1), as well as that of the cell surface-associated protein SasF (N315-SA2439). Microarray analysis, confirmatory Northern blots, and genetic complementation experiments showed that TcaR upregulates
sarS
and thus
spa
transcription. In addition, it attenuates whole-length transcription of
sasF
, thereby producing a truncated transcript lacking the 3′ terminus, which codes for the cell wall anchor motif. Hence, in strains containing an intact
tcaR
gene, TcaR is likely to decrease the amount of the surface-associated protein SasF and to increase that of the surface-associated protein A. The widely used laboratory strains derived from NCTC8325 were found to be natural, truncated mutants of
tcaR
, harboring an inactive TcaR and therefore expressing very low levels of
sarS
. The data presented here identified TcaR as a further activator of
sarS
, and a modulator of
sasF
expression that has to be taken into account in studies of virulence gene expression in
S. aureus
.
Assembly of the MexAB-OprM Multidrug Efflux System of
Pseudomonas aeruginosa
: Identification and Characterization of Mutations in
mexA
Compromising MexA Multimerization and Interaction with MexB
Authors: Nehme Dominic , Li Xian-Zhi , Elliot Rachel , Poole Keith 
Published: May 2004

ABSTRACT

The membrane fusion protein (MFP) component, MexA, of the MexAB-OprM multidrug efflux system of
P. aeruginosa
is proposed to link the inner (MexB) and outer (OprM) membrane components of this pump as a probable oligomer. A cross-linking approach confirmed the in vivo interaction of MexA and MexB, while a LexA-based assay for assessing protein-protein interaction similarly confirmed MexA multimerization. Mutations compromising the MexA contribution to antibiotic resistance but yielding wild-type levels of MexA were recovered and shown to map to two distinct regions within the N- and C-terminal halves of the protein. Most of the N-terminal mutations occurred at residues that are highly conserved in the MFP family (P68, G72, L91, A108, L110, and V129), consistent with these playing roles in a common feature of these proteins (e.g., oligomerization). In contrast, the majority of the C-terminal mutations occurred at residues poorly conserved in the MFP family (V264, N270, H279, V286, and G297), with many mapping to a region of MexA that corresponds to a region in the related MFP of
Escherichia coli
, AcrA, that is implicated in binding to its RND component, AcrB (C. A. Elkins and H. Nikaido, J. Bacteriol. 185:5349-5356, 2003). Given the noted specificity of MFP-RND interaction in this family of pumps, residues unique to MexA may well be important for and define the MexA interaction with its RND component, MexB. Still, all but one of the MexA mutations studied compromised MexA-MexB association, suggesting that native structure and/or proper assembly of the protein may be necessary for this.
Identification of
rhtX
and
fptX
, Novel Genes Encoding Proteins That Show Homology and Function in the Utilization of the Siderophores Rhizobactin 1021 by
Sinorhizobium meliloti
and Pyochelin by
Pseudomonas aeruginosa
, Respectively
Authors: Ó Cuív Páraic , Clarke Paul , Lynch Damien , O'Connell Michael 
Published: May 2004

ABSTRACT

Rhizobactin 1021 is a hydroxymate siderophore produced by the soil bacterium
Sinorhizobium meliloti
2011. A regulon comprising
rhtA
, encoding the outer membrane receptor protein for the ferrisiderophore; the biosynthesis operon
rhbABCDEF
; and
rhrA
, the Ara-C-like regulator of the receptor and biosynthesis genes has been previously described. We report the discovery of a gene, located upstream of
rhbA
and named
rhtX
(for “rhizobactin transport”), which is required, in addition to
rhtA
, to confer the ability to utilize rhizobactin 1021 on a strain of
S. meliloti
that does not naturally utilize the siderophore. Rhizobactin 1021 is structurally similar to aerobactin, which is transported in
Escherichia coli
via the IutA outer membrane receptor and the FhuCDB inner membrane transport system.
E. coli
expressing
iutA
and
fhuCDB
was found to also transport rhizobactin 1021. We demonstrated that RhtX alone could substitute for FhuCDB to transport rhizobactin 1021 in
E. coli
. RhtX shows similarity to a number of uncharacterized proteins which are encoded proximal to genes that are either known to be or predicted to be involved in iron acquisition. Among these is PA4218 of
Pseudomonas aeruginosa
, which is located close to the gene cluster that functions in pyochelin biosynthesis and outer membrane transport. PA4218 was mutated by allelic replacement, and the mutant was found to have a pyochelin utilization-defective phenotype. It is proposed that PA4218 be named
fptX
(for “ferripyochelin transport”). RhtX and FptX appear to be members of a novel family of permeases that function as single-subunit transporters of siderophores.
Transcription of the
Staphylococcus aureus cid
and
lrg
Murein Hydrolase Regulators Is Affected by Sigma Factor B
Authors: Rice Kelly C. , Patton Toni , Yang Soo-Jin , Dumoulin Alexis , Bischoff Markus , Bayles Kenneth W. 
Published: May 2004

ABSTRACT

The
Staphylococcus aureus lrg
and
cid
loci are homologous operons that have been shown to regulate murein hydrolase activity and affect sensitivity to penicillin. Although the mode of action of these operons has not been demonstrated, a model based on the similarities of the
lrgA
and
cidA
gene products to the bacteriophage holin family of proteins has been proposed. In this study, the transcription organization and regulation of these operons were examined by Northern blot analyses. Unexpectedly,
cidB
and a gene located immediately downstream, designated
cidC
, were found to be cotranscribed on a 2.7-kb transcript. Maximal
cidBC
transcription occurred during early exponential growth, and high-level transcription of
cidBC
was dependent on the
rsbU
-mediated activation of the alternative sigma factor B (σ
B
). In contrast,
lrgAB
transcription in stationary phase was negatively regulated by σ
B
. Although
cidABC
transcription was not detected by Northern blot analysis, reverse transcriptase PCR revealed that these genes are also cotranscribed as a single RNA message in early exponential growth. Primer extension analysis revealed the presence of two
cidBC
transcription start sites, but no apparent σ
B
-dependent promoter consensus sequence was identified in these regions. The
rsbU
gene was also shown to have a positive impact on murein hydrolase activity but a negligible effect on sensitivity to penicillin-induced killing. These results suggest that the
lrgAB
and
cidBC
genes may be part of the
S. aureus
σ
B
-controlled stress regulon.
Expression of
magA
in
Legionella pneumophila
Philadelphia-1 Is Developmentally Regulated and a Marker of Formation of Mature Intracellular Forms
Authors: Hiltz Margot F. , Sisson Gary R. , Brassinga Ann Karen C. , Garduno Elizabeth , Garduno Rafael A. , Hoffman Paul S. 
Published: May 2004

ABSTRACT

Legionella pneumophila
displays a biphasic developmental cycle in which replicating forms (RFs) differentiate postexponentially into highly infectious, cyst-like mature intracellular forms (MIFs). Using comparative protein profile analyses (MIFs versus RFs), we identified a 20-kDa protein, previously annotated as “Mip-like” protein, that was enriched in MIFs. However, this 20-kDa protein shared no similarity with Mip, a well-characterized peptidyl-prolyl isomerase of
L. pneumophila
, and for clarity we renamed it MagA (for “MIF-associated gene”). We monitored MagA levels across the growth cycle (in vitro and in vivo) by immunoblotting and established that MagA levels increased postexponentially in vitro (∼3-fold) and nearly 10-fold during MIF morphogenesis in HeLa cells. DNA sequence analysis of the
magA
locus revealed an upstream divergently transcribed gene,
msrA
, encoding a peptide methionine sulfoxide reductase and a shared promoter region containing direct and indirect repeat sequences as well as −10 hexamers often associated with stationary-phase regulation. While MagA has no known function, it contains a conserved CXXC motif commonly found in members of the thioredoxin reductase family and in AhpD reductases that are associated with alkylhydroperoxide reductase (AhpC), suggesting a possible role in protection from oxidative stress. MIFs from
L. pneumophila
strain Lp02 containing a
magA
deletion exhibited differences in Giménez staining, as well as an apparent increase in cytopathology to HeLa cells, but otherwise were unaltered in virulence traits. As demonstrated by this study, MagA appears to be a MIF-specific protein expressed late in intracellular growth that may serve as a useful marker of development.
Temperature Sensitivity Caused by Mutant Release Factor 1 Is Suppressed by Mutations That Affect 16S rRNA Maturation
Authors: Kaczanowska Magdalena , Rydén-Aulin Monica 
Published: May 2004

ABSTRACT

To study the effect of slow termination on the protein synthesizing machinery, we isolated suppressors to a temperature-sensitive release factor 1 (RF1). Of 26 independent clones, five complementation groups have been identified, two of which are presented here. The first mutation disrupts a base pair in the transcription terminator stem for the
rplM-rpsI
operon, which encodes ribosomal proteins L13 and S9. We have found that this leads to readthrough of the terminator and that lower levels of transcript (compared to the results seen with the wild type) are found in the cell. This probably leads to decreased expression of the two proteins. The second mutation is a small deletion of the
yrdC
open reading frame start site, and it is not likely that the protein is expressed. Both mutant strains show an increased accumulation of 17S rRNA (immature 16S rRNA). Maturation of 16S rRNA is dependent on proper assembly of the ribosomal proteins, a process that is disturbed when proteins are missing. The function of the YrdC protein is not known, but it is able to bind to double-stranded RNA; therefore, we suggest that it is an assembly factor important for 30S subunit biogenesis. On the basis of our findings, we propose that lesser amounts of S9 or a lack of YrdC causes the maturation defect. We have shown that as a consequence of the maturation defect, fewer 70S ribosomes and polysomes are formed. This and other results suggest that it is the lowered concentration of functional ribosomes that suppresses the temperature sensitivity caused by the mutant RF1.
Bacillus subtilisSalA (YbaL) Negatively Regulates Expression ofscoC, Which Encodes the Repressor for the Alkaline Exoprotease Gene,aprE
Authors: Ogura Mitsuo , Matsuzawa Atsushi , Yoshikawa Hirofumi , Tanaka Teruo 
Published: May 2004

ABSTRACTDuring the course of screening for exoprotease-deficient mutants amongBacillus subtilisgene disruptants, a strain showing such a phenotype was identified. The locus responsible for this phenotype was the previously unknown geneybaL, which we renamedsalA.The predicted gene product encoded bysalAbelongs to the Mrp family, which is widely conserved among archaea, prokaryotes, and eukaryotes. Disruption ofsalAresulted in a decrease in the expression of alacZfusion of theaprEgene encoding the major extracellular alkaline protease. The decrease was recovered by the clonedsalAgene on a plasmid, demonstrating that the gene is involved inaprEexpression. Determination of thecis-acting region of SalA on the upstream region ofaprE, together with epistatic analyses withscoC,abrB, andspo0Amutations that also affectaprEexpression, suggested thatsalAdeficiency affectsaprE-lacZexpression through the negative regulator ScoC. Northern and reverse transcription-PCR analyses revealed enhanced levels ofscoCtranscripts in thesalAmutant cells in the transition and early stationary phases. Concomitant with these observations, larger amounts of the ScoC protein were detected in the mutant cells by Western analysis. From these results we conclude that SalA negatively regulatesscoCexpression. It was also found that the expression of asalA-lacZfusion was increased bysalAdeficiency, suggesting thatsalAis autoregulated.
Instability of Pathogenicity Islands in Uropathogenic
Escherichia coli
536
Authors: Middendorf Barbara , Hochhut Bianca , Leipold Kristina , Dobrindt Ulrich , Blum-Oehler Gabriele , Hacker Jörg 
Published: May 2004

ABSTRACT

The uropathogenic
Escherichia coli
strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of
E. coli
536 with the counterselectable marker
sacB
to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II
536
and PAI III
536
were the most unstable islands followed by PAI I
536
and PAI V
536
, whereas PAI IV
536
was stable. In addition, we found that deletion of PAI II
536
and PAI III
536
was induced by several environmental stimuli. Whereas excision of PAI I
536
, PAI II
536
, and PAI V
536
was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III
536
was deleted either by site-specific recombination or by homologous recombination between two IS
100
-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II
536
and PAI III
536
were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic
E. coli
can be modulated by deletion of PAIs.
Distinctive Protein Signatures Provide Molecular Markers and Evidence for the Monophyletic Nature of the
Deinococcus-Thermus
Phylum
Authors: Griffiths Emma , Gupta Radhey S. 
Published: May 2004

ABSTRACT

The
Deinococcus-Thermus
group of species is currently recognized as a distinct phylum solely on the basis of their branching in 16S rRNA trees. No unique biochemical or molecular characteristics that can distinguish this group from all other bacteria are known at present. In this work, we describe eight conserved indels (viz., inserts or deletions) in seven widely distributed proteins that are distinctive characteristics of the
Deinococcus-Thermus
phylum but are not found in any other group of bacteria. The identified signatures include a 7-amino-acid (aa) insert in threonyl-tRNA synthetase, 1- and 3-aa inserts in the RNA polymerase β′ subunit, a 5-aa deletion in signal recognition particle (Ffh/SR54), a 2-aa insert in major sigma factor 70 (σ
70
), a 2-aa insert in seryl-tRNA synthetase (SerRS), a 1-aa insert in ribosomal protein L1, and a 2-aa insert in UvrA homologs. By using PCR primers for conserved regions, fragments of these genes were amplified from a number of
Deinococcus-Thermus
species, and all such fragments (except SerRS in
Deinococcus proteolyticus
) were found to contain the indicated signatures. The presence of these signatures in various species from all three known genera within this phylum, viz.,
Deinococcus
,
Thermus
, and
Meiothermus
, provide evidence that they are likely distinctive characteristics of the entire phylum which were introduced in a common ancestor of this group. The signature in SerRS, which is absent in
D. proteolyticus
, was likely introduced after the branching of this species. Phylogenetic studies as well as the nature of the inserts in some of these proteins (viz., σ
70
and SerRS) also support a sister group relationship between the
Thermus
and the
Meiothermus
genera. The identified signatures provide strong evidence for the monophyletic nature of the
Deinococcus-Thermus
phylum. These molecular markers should prove very useful in the identification of new species related to this group.
An Extracytoplasmic-Function Sigma Factor Is Involved in a Pathway Controlling β-Exotoxin I Production in
Bacillus thuringiensis
subsp.
thuringiensis
Strain 407-1
Authors: Espinasse Sylvain , Gohar Michel , Lereclus Didier , Sanchis Vincent 
Published: May 2004

ABSTRACT

β-Exotoxin I is an insecticidal nucleotide analogue secreted by various
Bacillus thuringiensis
strains. In this report, we describe the characterization and transcriptional analysis of a gene cluster, designated
sigW
-
ecfX
-
ecfY
, that is essential for β-exotoxin I production in
B. thuringiensis
subsp.
thuringiensis
strain 407-1. In this strain, the disruption of the
sigW
cluster resulted in nontoxic culture supernatants.
sigW
encodes a protein of 177 residues that is 97 and 94% identical to two putative RNA polymerase extracytoplasmic-function-type sigma factors from
Bacillus anthracis
strain Ames and
Bacillus cereus
strain ATCC 14579, respectively. It is also 50, 30, and 26% identical to SigW from
Clostridium perfringens
and SigW and SigX from
Bacillus subtilis
, respectively. EcfX, encoded by the gene following
sigW
, significantly repressed the expression of
sigW
when both genes were overtranscribed, suggesting that it could be the anti-sigma factor of SigW. Following the loss of its curable
cry
plasmid, strain 407 became unable to synthesize crystal toxins, in contrast to the mutant strain 407-1(Cry
−
)(Pig
+
), which overproduced this molecule in the absence of this plasmid. Transcriptional analysis of
sigW
indicated that this gene was expressed during the stationary phase and only in the 407-1(Cry
−
)(Pig
+
) mutant. This suggests that in the wild type-407(Cry
+
) strain, β-exotoxin I was produced from determinants located on a
cry
gene-bearing plasmid and that
sigW
is able to induce β-exotoxin I production in
B. thuringiensis
in the absence of
cry
gene
-
bearing plasmids. Although the signal responsible for this activation is unknown, these results indicate that β-exotoxin I production in
B. thuringiensis
can be restored or induced via an alternative pathway that requires
sigW
expression.
Toluene 3-Monooxygenase of
Ralstonia pickettii
PKO1 Is a
para
-Hydroxylating Enzyme
Authors: Fishman Ayelet , Tao Ying , Wood Thomas K. 
Published: May 2004

ABSTRACT

Oxygenases are promising biocatalysts for performing selective hydroxylations not accessible by chemical methods. Whereas toluene 4-monooxygenase (T4MO) of
Pseudomonas mendocina
KR1 hydroxylates monosubstituted benzenes at the
para
position and toluene
ortho
-monooxygenase (TOM) of
Burkholderia cepacia
G4 hydroxylates at the
ortho
position, toluene 3-monooxygenase (T3MO) of
Ralstonia pickettii
PKO1 was reported previously to hydroxylate toluene at the
meta
position, producing primarily
m
-cresol (R. H. Olsen, J. J. Kukor, and B. Kaphammer, J. Bacteriol. 176:3749-3756, 1994). Using gas chromatography, we have discovered that T3MO hydroxylates monosubstituted benzenes predominantly at the
para
position. TG1/pBS(Kan)T3MO cells expressing T3MO oxidized toluene at a maximal rate of 11.5 ± 0.33 nmol/min/mg of protein with an apparent

K
m

value of 250 μM and produced 90%
p
-cresol and 10%
m
-cresol. This product mixture was successively transformed to 4-methylcatechol. T4MO, in comparison, produces 97%
p
-cresol and 3%
m
-cresol.
Pseudomonas aeruginosa
PAO1 harboring pRO1966 (the original T3MO-bearing plasmid) also exhibited the same product distribution as that of TG1/pBS(Kan)T3MO. TG1/pBS(Kan)T3MO produced 66%
p
-nitrophenol and 34%
m
-nitrophenol from nitrobenzene and 100%
p
-methoxyphenol from methoxybenzene, as well as 62% 1-naphthol and 38% 2-naphthol from naphthalene; similar results were found with TG1/pBS(Kan)T4MO. Sequencing of the
tbu
locus from pBS(Kan)T3MO and pRO1966 revealed complete identity between the two, thus eliminating any possible cloning errors.
1
H nuclear magnetic resonance analysis confirmed the structural identity of
p
-cresol in samples containing the product of hydroxylation of toluene by pBS(Kan)T3MO.
Characterization of Monospecies Biofilm Formation by
Helicobacter pylori
Authors: Cole Sheri P. , Harwood Julia , Lee Richard , She Rosemary , Guiney Donald G. 
Published: May 2004

ABSTRACT

As all bacteria studied to date, the gastric pathogen
Helicobacter pylori
has an alternate lifestyle as a biofilm.
H. pylori
forms biofilms on glass surfaces at the air-liquid interface in stationary or shaking batch cultures. By light microscopy, we have observed attachment of individual, spiral
H. pylori
to glass surfaces, followed by division to form microcolonies, merging of individual microcolonies, and growth in the third dimension. Scanning electron micrographs showed
H. pylori
arranged in a matrix on the glass with channels for nutrient flow, typical of other bacterial biofilms. To understand the importance of biofilms to the
H. pylori
life cycle, we tested the effect of mucin on biofilm formation. Our results showed that 10% mucin greatly increased the number of planktonic
H. pylori
while not affecting biofilm bacteria, resulting in a decline in percent adherence to the glass. This suggests that in the mucus-rich stomach,
H. pylori
planktonic growth is favored over biofilm formation. We also investigated the effect of specific mutations in several genes, including the quorum-sensing gene,
luxS
, and the
cagE
type IV secretion gene. Both of these mutants were found to form biofilms approximately twofold more efficiently than the wild type in both assays. These results indicate the relative importance of these genes to the production of biofilms by
H. pylori
and the selective enhancement of planktonic growth in the presence of gastric mucin.
Generation of Enhanced Competitive Root-Tip-Colonizing
Pseudomonas
Bacteria through Accelerated Evolution
Authors: de Weert Sandra , Dekkers Linda C. , Kuiper Irene , Bloemberg Guido V. , Lugtenberg Ben J. J. 
Published: May 2004

ABSTRACT

A recently published procedure to enrich for efficient competitive root tip colonizers (I. Kuiper, G. V. Bloemberg, and B. J. J. Lugtenberg, Mol. Plant-Microbe Interact. 14:1197-1205) after bacterization of seeds was applied to isolate efficient competitive root tip colonizers for both the dicotyledenous plant tomato and the monocotyledenous plant grass from a random Tn
5luxAB
mutant bank of the good root colonizer
Pseudomonas fluorescens
WCS365. Unexpectedly, the best-colonizing mutant, strain PCL1286, showed a strongly enhanced competitive root-tip-colonizing phenotype. Sequence analyses of the Tn
5luxAB
flanking regions showed that the transposon had inserted in a
mutY
homolog. This gene is involved in the repair of A · G mismatches caused by spontaneous oxidation of guanine. We hypothesized that, since the mutant is defective in repairing its mismatches, its cells harbor an increased number of mutations and therefore can adapt faster to the environment of the root system. To test this hypothesis, we constructed another
mutY
mutant and analyzed its competitive root tip colonization behavior prior to and after enrichment. As a control, a nonmutated wild type was subjected to the enrichment procedure. The results of these analyses showed (i) that the enrichment procedure did not alter the colonization ability of the wild type, (ii) that the new
mutY
mutant was strongly impaired in its colonization ability, but (iii) that after three enrichment cycles it colonized significantly better than its wild type. Therefore it is concluded that both the
mutY
mutation and the selection procedure are required to obtain an enhanced root-tip-colonizing mutant.