bluuebunny/crossref_metadata_2025_split huggingface dataset:
The
Staphylococcus aureus lrgAB
Operon Modulates Murein Hydrolase Activity and Penicillin Tolerance
Authors: Groicher Kajetan H. , Firek Brian A. , Fujimoto David F. , Bayles Kenneth W. 
Published: July 2002

ABSTRACT

Previous studies in our laboratory have shown that the
Staphylococcus aureus
LytSR two-component regulatory system affects murein hydrolase activity and autolysis. A LytSR-regulated dicistronic operon has also been identified and shown to encode two potential membrane-associated proteins, designated LrgA and LrgB, hypothesized to be involved in the control of murein hydrolase activity. In the present study, a
lrgAB
mutant strain was generated and analyzed to test this hypothesis. Zymographic and quantitative analysis of murein hydrolase activity revealed that the
lrgAB
mutant produced increased extracellular murein hydrolase activity compared to that of the wild-type strain. Complementation of the
lrgAB
defect by providing the
lrgAB
genes in
trans
restored the wild-type phenotype, indicating that these genes confer negative control on extracellular murein hydrolase activity. In addition to these effects, the influence of the
lrgAB
mutation on penicillin-induced lysis and killing was examined. These studies demonstrated that the
lrgAB
mutation enhanced penicillin-induced killing of cells approaching the stationary phase of growth, the time at which the
lrgAB
operon was shown to be maximally expressed. This effect of the
lrgAB
mutation on penicillin-induced killing was shown to be independent of cell lysis. In contrast, the
lrgAB
mutation did not affect penicillin-induced killing of cells growing in early-exponential phase, a time in which
lrgAB
expression was shown to be minimal. However, expression of the
lrgAB
operon in early-exponential-phase cells inhibited penicillin-induced killing, again independent of cell lysis. The data generated by this study suggest that penicillin-induced killing of
S. aureus
involves a novel regulator of murein hydrolase activity.
Characterization of the
Mycobacterium tuberculosis iniBAC
Promoter, a Promoter That Responds to Cell Wall Biosynthesis Inhibition
Authors: Alland David , Steyn Andries J. , Weisbrod Torin , Aldrich Kate , Jacobs William R. 
Published: July 2002

ABSTRACT

The cell wall provides an attractive target for antibiotics against
Mycobacterium tuberculosis
. Agents such as isoniazid and ethambutol that work by inhibiting cell wall biosynthesis are among the most highly effective antibiotics against this pathogen. Although considerable progress has been made identifying the targets for cell wall active antibiotics, little is known about the intracellular mechanisms that are activated as a consequence of cell wall injury. These mechanisms are likely to have an important role in growth regulation and in the induction of cell death by antibiotics. We previously discovered three isoniazid-induced genes (
iniB
,
iniA
, and
iniC
) organized in tandem on the
M. tuberculosis
genome. Here, we investigate the unique features of the putative
iniBAC
promoter. This promoter was specifically induced by a broad range of inhibitors of cell wall biosynthesis but was not inducible by other conditions that are toxic to mycobacteria via other mechanisms. Induction required inhibitory concentrations of antibiotics and could be detected only in actively growing cells. Analysis of the
iniBAC
promoter sequence revealed both a regulatory element upstream and a potential repressor binding region downstream of the transcriptional start site. The induction phenotype and structure of the
iniBAC
promoter suggest that a complex intracellular response occurs when cell wall biosynthesis is inhibited in
M. tuberculosis
and other mycobacteria.
Characterization of In53, a Class 1 Plasmid- and Composite Transposon-Located Integron ofEscherichia coliWhich Carries an Unusual Array of Gene Cassettes
Authors: Naas Thierry , Mikami Yuzuru , Imai Tamae , Poirel Laurent , Nordmann Patrice 
Published: July 2002

ABSTRACTFurther characterization of the genetic environment of the gene encoding theEscherichia coliextended-spectrum β-lactamase,blaVEB-1, revealed the presence of a plasmid-located class 1 integron, In53, which carried eight functional resistance gene cassettes in addition toblaVEB-1. While theaadBand thearr-2gene cassettes were identical to those previously described, the remaining cassettes were novel: (i) a novel nonenzymatic chloramphenicol resistance gene of thecmlAfamily, (ii) aqacallele encoding a member of the small multidrug resistance family of proteins, (iii) a cassette,aacA1b/orfG,which encodes a novel 6′-N-acetyltransferase, and (iv) a fused gene cassette,oxa10/aadA1, which is made of two cassettes previously described as single cassettes. In addition,oxa10andaadA1genes were expressed from their own promoter sequence present upstream of theoxa10cassette.arr-2coded for a protein that shared 54% amino acid identity with the rifampin ADP-ribosylating transferase encoded by thearr-1gene fromMycobacterium smegmatisDSM43756. While inM. smegmatis, the main inactivated compound was 23-ribosyl-rifampin, the inactivated antibiotic recovered fromE. coliculture was 23-O-ADP-ribosyl-rifampin. The integrase gene of In53 was interrupted by an IS26insertion sequence, which was also present in the 3′ conserved segment. Thus, In53 is a truncated integron located on a composite transposon, named Tn2000, bounded by two IS26elements in opposite orientations. Target site duplication at both ends of the transposon indicated that the integron likely was inserted into the plasmid through a transpositional process. This is the first description of an integron located on a composite transposon.
Characterization of
fsr
, a Regulator Controlling Expression of Gelatinase and Serine Protease in
Enterococcus faecalis
OG1RF
Authors: Qin Xiang , Singh Kavindra V. , Weinstock George M. , Murray Barbara E. 
Published: July 2002

ABSTRACT

We have previously identified a locus,
fsr
, a homologue of staphylococcal
agr
loci, which positively regulates the expression of gelatinase and serine protease (encoded by
gelE
and
sprE
, respectively) in
Enterococcus faecalis
OG1RF. The expression of the three genes in the
fsr
locus,
fsrA
,
fsrB
, and
fsrC
, appears to be autoregulated, and we have shown that mutants with insertion disruptions in each of these three genes were significantly attenuated in a mouse peritonitis model compared to the parent strain. In the present study, we showed that
fsrB
and
fsrC
are highly expressed in the postexponential growth phase and that their expression is cell density dependent. Reverse transcriptase PCR using primers covering the intergenic regions in the
fsr
/
gelE
loci confirmed that
fsrB
and
fsrC
, as well as
gelE
and
sprE
, are cotranscribed. We also showed, using a nonpolar
fsrB
deletion mutant, that
fsrB
, the homologue of
agrB
of staphylococci with unknown function, is required for the regulatory function of
fsr
. Primer extension and analysis of transcriptional fusions indicated the presence of promoters immediately upstream of
fsrA
, of
fsrB
, and of
gelE
and that the
fsrB
and
gelE
promoters are
fsr
dependent, while the
fsrA
promoter is an
fsr
-independent weak constitutive promoter. Two conserved 7-bp direct repeats were found immediately upstream of the
fsrB
and
gelE
promoters, similar to the repeats found upstream of P2 and P3 promoters of the
agr
locus; deletions and mutations in the repeated sequences completely abolished the
fsrB
and
gelE
promoter activities, suggesting that the repeats are important for the regulatory function in the
fsrB
and
gelE
promoter regions.
Identification of a Dedicated Recycling Pathway for Anhydro-
N
-Acetylmuramic Acid and
N
-Acetylglucosamine Derived from
Escherichia coli
Cell Wall Murein
Authors: Park James T. 
Published: July 2002

ABSTRACT

Turnover and recycling of the cell wall murein represent a major metabolic pathway of
Escherichia coli
. It is known that
E. coli
efficiently reuses, i.e., recycles, its murein tripeptide,
l
-alanyl-γ-
d
-glutamyl-
meso
-diaminopimelate, to form new murein. However, the question of whether the cells also recycle the amino sugar moieties of cell wall murein has remained unanswered. It is demonstrated here that
E. coli
recycles the
N
-acetylglucosamine present in cell wall murein degradation products for de novo murein and lipopolysaccharide synthesis. Furthermore,
E. coli
also recycles the anhydro-
N
-acetylmuramic acid moiety by first converting it into
N
-acetylglucosamine. Based on the results obtained by studying mutants unable to recycle amino sugars, the pathway for recycling is revealed.
Alginate Overproduction AffectsPseudomonas aeruginosaBiofilm Structure and Function
Authors: Hentzer Morten , Teitzel Gail M. , Balzer Grant J. , Heydorn Arne , Molin Søren , Givskov Michael , Parsek Matthew R. 
Published: July 2002

ABSTRACTDuring the course of chronic cystic fibrosis (CF) infections,Pseudomonas aeruginosaundergoes a conversion to a mucoid phenotype, which is characterized by overproduction of the exopolysaccharide alginate. ChronicP. aeruginosainfections involve surface-attached, highly antibiotic-resistant communities of microorganisms organized in biofilms. Although biofilm formation and the conversion to mucoidy are both important aspects of CF pathogenesis, the relationship between them is at the present unclear. In this study, we report that the overproduction of alginate affects biofilm development on an abiotic surface. Biofilms formed by an alginate-overproducing strain exhibit a highly structured architecture and are significantly more resistant to the antibiotic tobramycin than a biofilm formed by an isogenic nonmucoid strain. These results suggest that an important consequence of the conversion to mucoidy is an altered biofilm architecture that shows increasing resistance to antimicrobial treatments.
Multiple Origins and Replication Proteins Influence Biological Properties of β-Lactamase-Producing Plasmids from
Neisseria gonorrhoeae
Authors: Pagotto Franco , Dillon Jo-Anne R. 
Published: July 2002

ABSTRACT

The β-lactamase-producing Asia-type plasmid pJD4 of
Neisseria gonorrhoeae
is a 7.4-kb, broad-host-range plasmid. It is part of a family of plasmids which are structurally related yet vary in size, found in both
N. gonorrhoeae
and
Haemophilus ducreyi
. Branch-point analysis by electron microscopy indicates that pJD4 carries three clustered but distinguishable origins of replication, which we named
ori1, ori2
, and
ori3
. Although pJD4 belongs to incompatibility (Inc) group W, it also carries a silent IncFII determinant which is expressed when
ori2
and
ori3
are absent. The Africa-type plasmid pJD5, a naturally occurring deletion derivative of pJD4, carries only
ori1
, belongs to the IncFII group, and, in contrast to pJD4, requires DNA polymerase I (Pol I) for replication. Plasmids constructed from pJD4 which lack
ori1
but carry
ori2
and
ori3
do not require Pol I and are incompatible with IncW plasmids, suggesting that the
ori2
or
ori3
region contains the IncW determinant. We have cloned a replication initiation protein (RepB) that is necessary for
ori2
and
ori3
to function. This Rep protein is distinct from RepA, which is necessary for
ori1.
Thus, pJD4 is unique because it is the smallest plasmid characterized containing three origins of replication and two unique Rep proteins.
Type II Secretion by
Aeromonas salmonicida
: Evidence for Two Periplasmic Pools of Proaerolysin
Authors: Burr Sarah E. , Diep Dzung B. , Buckley J. Thomas 
Published: July 2002

ABSTRACT

Aeromonas salmonicida
containing the cloned gene for proaerolysin secretes the protein via the type II secretory pathway. Here we show that altering a region near the beginning of
aerA
led to a dramatic increase in the amount of proaerolysin that was produced and that a large amount of the protein was cell associated. All of the cell-associated protein had crossed the cytoplasmic membrane, because the signal sequence had been removed, and all of it was accessible to processing by trypsin during osmotic shock. Enlargement of the periplasm was observed by electron microscopy in overproducing cells, likely caused by the osmotic effect of the very large concentrations of accumulated proaerolysin. Immunogold electron microscopy localized nearly all of the proaerolysin in the enlarged periplasm; however, only half of the protoxin was released from the cells by osmotic shocking. Cross-linking studies showed that this fraction contained normal dimeric proaerolysin but that proaerolysin in the fraction that was not shockable had not dimerized, although it appeared to be correctly folded. Both periplasmic fractions were secreted by the cells; however, the nonshockable fraction was secreted much more slowly than the shockable fraction. We estimated a rate for maximal secretion of proaerolysin from the bacteria that was much lower than the rates that have been estimated for inner membrane transit, which suggests that transit across the outer membrane is rate limiting and may account for the periplasmic accumulation of the protein. Finally, we show that overproduction of proaerolysin inhibited the release of the protease that is secreted by
A. salmonicida
.
Functional Analysis of Genes for Biosynthesis of Pyocyanin and Phenazine-1-Carboxamide from
Pseudomonas aeruginosa
PAO1
Authors: Mavrodi Dmitri V. , Bonsall Robert F. , Delaney Shannon M. , Soule Marilyn J. , Phillips Greg , Thomashow Linda S. 
Published: July 2002

ABSTRACT

Two seven-gene phenazine biosynthetic loci were cloned from
Pseudomonas aeruginosa
PAO1. The operons, designated
phzA1B1C1D1E1F1G1
and
phzA2B2C2D2E2F2G2
, are homologous to previously studied phenazine biosynthetic operons from
Pseudomonas fluorescens
and
Pseudomonas aureofaciens
. Functional studies of phenazine-nonproducing strains of fluorescent pseudomonads indicated that each of the biosynthetic operons from
P. aeruginosa
is sufficient for production of a single compound, phenazine-1-carboxylic acid (PCA). Subsequent conversion of PCA to pyocyanin is mediated in
P. aeruginosa
by two novel phenazine-modifying genes,
phzM
and
phzS
, which encode putative phenazine-specific methyltransferase and flavin-containing monooxygenase, respectively. Expression of
phzS
alone in
Escherichia coli
or in enzymes, pyocyanin-nonproducing
P. fluorescens
resulted in conversion of PCA to 1-hydroxyphenazine.
P. aeruginosa
with insertionally inactivated
phzM
or
phzS
developed pyocyanin-deficient phenotypes. A third phenazine-modifying gene,
phzH
, which has a homologue in
Pseudomonas chlororaphis
, also was identified and was shown to control synthesis of phenazine-1-carboxamide from PCA in
P. aeruginosa
PAO1. Our results suggest that there is a complex pyocyanin biosynthetic pathway in
P. aeruginosa
consisting of two core loci responsible for synthesis of PCA and three additional genes encoding unique enzymes involved in the conversion of PCA to pyocyanin, 1-hydroxyphenazine, and phenazine-1-carboxamide.
MexR Repressor of the
mexAB-oprM
Multidrug Efflux Operon of
Pseudomonas aeruginosa
: Identification of MexR Binding Sites in the
mexA-mexR
Intergenic Region
Authors: Evans Kelly , Adewoye Lateef , Poole Keith 
Published: July 2002

ABSTRACT

The MexR repressor of the
mexAB-oprM
multidrug efflux operon of
Pseudomonas aeruginosa
was purified as a C-terminal histidine-tagged protein by metal chelate affinity chromatography. The purified protein was shown to bind ca. 200 bp upstream of
mexA
, at two sites, each of which contains a repeat of the nucleotide sequence GTTGA in inverse orientation. DNA sequence analysis identified
mexA
and
mexR
promoters within the MexR binding regions, consistent with the previously observed negative regulation of
mexR
and
mexAB-oprM
expression by MexR. Transcription of
mexA
from the promoter originating within the MexR binding site II was confirmed and shown to be markedly enhanced in a
nalB
(i.e.,
mexR
) mutant of
P. aeruginosa
. A second
mexA
promoter was also identified, ca. 70 bp upstream of
mexAB-oprM
, and transcription from this promoter appeared to occur in both the wild type and a
nalB
mutant. Production of MexAB-OprM in wild-type cells may be due to expression from a constitutively expressed proximal promoter, while MexAB-OprM hyperexpression in
nalB
mutants is due to the additional expression from a MexR-regulated distal promoter.
Bap, a
Staphylococcus aureus
Surface Protein Involved in Biofilm Formation
Authors: Cucarella Carme , Solano Cristina , Valle Jaione , Amorena Beatriz , Lasa Íñigo , Penadés José R. 
Published: July 2002

ABSTRACT

Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing
Staphylococcus aureus
isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (
bap
[for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (
Pseudomonas aeruginosa
and
Salmonella enterica
serovar Typhi) and gram-positive (
Enteroccocus faecalis
) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids.
bap
was present in a small fraction of bovine mastitis isolates (5% of the 350
S. aureus
isolates tested), but it was absent from the 75 clinical human
S. aureus
isolates analyzed. All staphylococcal isolates harboring
bap
were highly adherent and strong biofilm producers. In a mouse infection model
bap
was involved in pathogenesis, causing a persistent infection.
Overexpression of Two Different GTPases Rescues a Null Mutation in a Heat-Induced rRNA Methyltransferase
Authors: Tan Jacqueline , Jakob Ursula , Bardwell James C. A. 
Published: July 2002

ABSTRACT

The
Escherichia coli
RrmJ (FtsJ) heat shock protein functions as an rRNA methyltransferase that modifies position U2552 of 23S rRNA in intact 50S ribosomal subunits. An in-frame deletion of the
rrmJ
(
ftsJ
) gene leads to severe growth disadvantages under all temperatures tested and causes significant accumulation of ribosomal subunits at the expense of functional 70S ribosomes. To investigate whether overexpression of other
E. coli
genes can restore the severe growth defect observed in
rrmJ
null mutants, we constructed an overexpression library from the
rrmJ
deletion strain and cloned and identified the
E. coli
genes that were capable of rescuing the
rrmJ
mutant phenotype. Our intention was to identify other methylases whose specificities overlapped enough with that of RrmJ to allow complementation when overexpressed. To our great surprise, no methylases were found by this method; rather, two small GTPases, Obg (YhbZ) and EngA, when overexpressed in the
rrmJ
deletion strains, were found to restore the otherwise severely impaired ribosome assembly process and/or stability of 70S ribosomes. 50S ribosomal subunits prepared from these overexpressing strains were shown to still serve as in vitro substrates for purified RrmJ, indicating that the 23S rRNA likely was still lacking the highly conserved Um2552 modification. The apparent lack of this modification, however, no longer caused ribosome defects or a growth disadvantage. Massive overexpression of another related small GTPase, Era, failed to rescue the growth defects of an
rrmJ
strain. These findings suggest a hitherto unexpected connection between rRNA methylation and GTPase function, specifically that of the two small GTPases Obg and EngA.
Behavior of Sister Copies of Mini-F Plasmid after Synchronized Plasmid Replication inEscherichia coliCells
Authors: Onogi Toshinari , Miki Takeyoshi , Hiraga Sota 
Published: July 2002

ABSTRACTTo clarify whether sister copies of mini-F plasmid are immediately separated from each other after replication, we analyzed the behavior of sister mini-F copies after synchronized replication of mini-F. Sister copies of mini-F were separated immediately or shortly after replication, in contrast to sisteroriCcopies of theEscherichia colichromosome.
Northern, Morphological, and Fermentation Analysis of
spo0A
Inactivation and Overexpression in
Clostridium acetobutylicum
ATCC 824
Authors: Harris Latonia M. , Welker Neil E. , Papoutsakis Eleftherios T. 
Published: July 2002

ABSTRACT

The
Clostridium acetobutylicum
ATCC 824
spo0A
gene was cloned, and two recombinant strains were generated, an
spo0A
inactivation strain (SKO1) and an
spo0A
overexpression strain [824(pMPSOA)]. SKO1 was developed by targeted gene inactivation with a replicative plasmid capable of double-crossover chromosomal integration—a technique never used before with solventogenic clostridia. SKO1 was severely deficient in solvent formation: it produced only 2 mM acetone and 13 mM butanol, compared to the 92 mM acetone and 172 mM butanol produced by the parental strain. After 72 h of growth on solid media, SKO1 formed long filaments of rod-shaped cells that failed to septate. SKO1 cells never achieved the swollen clostridial form typical of the parental strain and did not form endospores. No
spo0A
transcripts were detected in SKO1, while transcription of two solvent formation operons (
aad-ctfA-ctfB
and
adc
; both containing 0A boxes in their promoter regions) was limited. Strain 824(pMSPOA) produced higher butanol concentrations than the control strain [824(pIMP1)] and dramatically elevated
spo0A
transcript levels and displayed a bimodal pattern of
spo0A
transcription similar to that of
B. subtilis.
Microscopic studies indicated that sporulation was both enhanced and accelerated due to
spo0A
overexpression compared to that of both the 824(pIMP1) and parental strains. Consistent with that, expression of the key solvent formation genes (
aad-ctfA-ctfB
and
adc
) and three sporulation-specific genes (
spoIIGA
,
sigE
, and
sigG
) was observed earlier in strain 824(pMSPOA) than in the plasmid control. These data support the hypothesis that Spo0A is a transcriptional regulator that positively controls sporulation and solvent production. Its effect on solvent formation is a balancing act in regulating sporulation versus solvent gene expression: its overexpression apparently tips the balance in favor of accelerated and enhanced sporulation at the expense of overall solvent production.
Toxic Waste Disposal in
Escherichia coli
Authors: Helling Robert B. , Janes Brian K. , Kimball Heather , Tran Timothy , Bundesmann Michael , Check Pietra , Phelan Darcy , Miller Charles 
Published: July 2002

ABSTRACT

About 10% of the nalidixic acid-resistant (Nal
r
) mutants in a transposition-induced library exhibited a growth factor requirement as the result of
cysH
,
icdA
,
metE
, or
purB
mutation. Resistance in all of these mutants required a functional AcrAB-TolC efflux pump, but the EmrAB-TolC pump played no obvious role. Transcription of
acrAB
was increased in each type of Nal
r
mutant. In the
icdA
and
purB
mutants, each of the known signaling pathways appeared to be used in activating the AcrAB-TolC pump. The metabolites that accumulate upstream of the blocks caused by the mutations are hypothesized to increase the levels of the AcrAB-TolC pump, thereby removing nalidixic acid from the organism.