bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evaluation of a Structural Model of
Pseudomonas aeruginosa
Outer Membrane Protein OprM, an Efflux Component Involved in Intrinsic Antibiotic Resistance
Authors: Wong Kendy K. Y. , Brinkman Fiona S. L. , Benz Roland S. , Hancock Robert E. W. 
Published: July 2002

ABSTRACT

The outer membrane protein OprM of
Pseudomonas aeruginosa
is involved in intrinsic and mutational multiple-antibiotic resistance as part of two resistance-nodulation-division efflux systems. The crystal structure of TolC, a homologous protein in
Escherichia coli
, was recently published (V. Koronakis, A. Sharff, E. Koronakis, B. Luisl, and C. Hughes, Nature 405:914–919, 2000), demonstrating a distinctive architecture comprising outer membrane β-barrel and periplasmic helical-barrel structures, which assemble differently from the common β-barrel-only conformation of porins. Based on their sequence similarity, a similar content of α-helical and β-sheet structure determined by circular dichroism spectroscopy, and our observation that OprM, like TolC, reconstitutes channels in planar bilayer membranes, OprM and TolC were considered to be structurally homologous, and a model of OprM was constructed by threading its sequence to the TolC crystal structure. Residues thought to be important for the TolC structure were conserved in space in this OprM model. Analyses of deletion mutants and previously isolated insertion mutants of OprM in the context of this model allowed us to propose roles for different protein domains. Our data indicate that the helical barrel of the protein is critical for both the function and the integrity of the protein, while a C-terminal domain localized around the equatorial plane of this helical barrel is dispensable. Extracellular loops appear to play a lesser role in substrate specificity for this efflux protein compared to classical porins, and there appears to be a correlation between the change in antimicrobial activity for OprM mutants and the pore size. Our model and channel formation studies support the “iris” mechanism of action for TolC and permit us now to form more focused hypotheses about the functional domains of OprM and its related family of efflux proteins.
Catabolite Repression of the Citrate Fermentation Genes in
Klebsiella pneumoniae
: Evidence for Involvement of the Cyclic AMP Receptor Protein
Authors: Meyer Margareta , Dimroth Peter , Bott Michael 
Published: July 2002

ABSTRACT

Klebsiella pneumoniae
is able to grow anaerobically with citrate as a sole carbon and energy source by a fermentative pathway involving the Na
+
-dependent citrate carrier CitS, citrate lyase, and oxaloacetate decarboxylase. The corresponding genes are organized in the divergent
citC
and
citS
operons, whose expression is strictly dependent on the citrate-sensing CitA-CitB two-component system. Evidence is provided here that the citrate fermentation genes are subject to catabolite repression, since anaerobic cultivation with a mixture of citrate and glucose or citrate and gluconate resulted in diauxic growth. Glucose, gluconate, and also glycerol decreased the expression of a chromosomal
citS-lacZ
fusion by 60 to 75%, whereas a direct inhibition of the citrate fermentation enzymes was not observed. The purified cyclic AMP (cAMP) receptor protein (CRP) of
K. pneumoniae
bound to two sites in the
citC-citS
intergenic region, which were centered at position −41.5 upstream of the
citC
and
citS
transcriptional start sites. Binding was apparently stimulated by the response regulator CitB. These data indicate that catabolite repression of the citrate fermentation genes is exerted by CRP and that in the absence of repressing carbon sources the cAMP-CRP complex serves to enhance the basal, CitB-dependent transcription level.
Polymer Stability Plays an Important Role in the Positional Regulation of FtsZ
Authors: Levin Petra Anne , Schwartz Rachel L. , Grossman Alan D. 
Published: July 2002

ABSTRACT

We conducted a series of experiments examining the effect of polymer stability on FtsZ localization dynamics in
Bacillus subtilis.
A loss-of-function mutation in
ezrA
, a putative polymer-destabilizing factor, suppresses the defects in FtsZ polymer stability associated with
minCD
overexpression. In addition, a mutation that is predicted to stabilize the FtsZ polymer leads to the formation of polar FtsZ rings. These data support the hypothesis that carefully balanced polymer stability is important for the assembly and localization of FtsZ during the bacterial cell cycle.
Enhancer-Binding Proteins HrpR and HrpS Interact To Regulatehrp-Encoded Type III Protein Secretion inPseudomonas syringaeStrains
Authors: Hutcheson Steven W. , Bretz Jamie , Sussan Thomas , Jin Songmu , Pak Kyong 
Published: July 2002

ABSTRACTInPseudomonas syringaestrains, thehrp-hrcpathogenicity island consists of an HrpL-dependent regulon that encodes a type III protein translocation complex and translocated effector proteins required for pathogenesis. HrpR and HrpS function as positive regulatory factors for thehrpLpromoter, but their mechanism of action has not been established. Both HrpR and HrpS are structurally related to enhancer-binding proteins, but they lack receiver domains and do not appear to require a cognate protein kinase for activity.hrpRandhrpSwere shown to be expressed as an operon: a promoter was identified 5′ tohrpR, and reverse transcriptase PCR detected the presence of anhrpRStranscript. ThehrpRpromoter and coding sequence were conserved amongP. syringaestrains. The coding sequences forhrpRandhrpSwere cloned into compatible expression vectors, and their activities were monitored inEscherichia colitransformants carrying anhrpL′-lacZfusion. HrpS could function as a weak activator of thehrpLpromoter, but the activity was only 2.5% of the activity detected when both HrpR and HrpS were expressed in the reporter strain. This finding is consistent with a requirement for both HrpR and HrpS in the activation of thehrpLpromoter. By using a yeast two-hybrid assay, an interaction between HrpR and HrpS was detected, suggestive of the formation of a heteromeric complex. Physical interaction of HrpR and HrpS was confirmed by column-binding experiments. The results show that HrpR and HrpS physically interact to regulate the ς54-dependenthrpLpromoter inP. syringaestrains.
Binding Specificity of the
Porphyromonas gingivalis
Heme and Hemoglobin Receptor HmuR, Gingipain K, and Gingipain R1 for Heme, Porphyrins, and Metalloporphyrins
Authors: Olczak Teresa , Dixon Dabney White , Genco Caroline Attardo 
Published: July 2002

ABSTRACT

Previous genetic and biochemical studies have confirmed that hemoglobin and hemin utilization in
Porphyromonas gingivalis
is mediated by the outer membrane hemoglobin and heme receptor HmuR, as well as gingipain K (Kgp), a lysine-specific cysteine protease, and gingipain R1 (HRgpA), one of two arginine-specific cysteine proteases. In this study we report on the binding specificity of the recombinant
P. gingivalis
HmuR protein and native gingipains for hemoglobin, hemin, various porphyrins, and metalloporphyrins as assessed by spectrophotometric assays, by affinity chromatography, and by enzyme-linked immunosorbent assay. Protoporphyrin, mesoporphyrin, deuteroporphyrin, hematoporphyrin, and some of their iron, copper, and zinc derivatives were examined to evaluate the role of both the central metal ion and the peripheral substituents on binding to recombinant HmuR and soluble gingipains. Scatchard analysis of hemin binding to
Escherichia coli
cells expressing recombinant membrane-associated six-His-tagged HmuR yielded a linear plot with a binding affinity of 2.4 × 10
−5
M. Recombinant
E. coli
cells bound the iron, copper, and zinc derivatives of protoporphyrin IX (PPIX) with similar affinities, and approximately four times more tightly than PPIX itself, which suggests that the active site of HmuR contains a histidine that binds the metal ion in the porphyrin ring. Furthermore, we found that recombinant HmuR prefers the ethyl and vinyl side chains of the PPIX molecule to either the larger hydroxyethyl or smaller hydrogen side chains. Kgp and HRgpA were demonstrated to bind various porphyrins and metalloporphyrins with affinities similar to those for hemin, indicating that the binding of Kgp and HRgpA to these porphyrins does not require a metal within the porphyrin ring. We did not detect the binding of RgpB, the arginine-specific cysteine protease that lacks a C-terminal hemagglutinin domain, to hemoglobin, porphyrins, or metalloporphyrins. Kgp and HRgpA, but not RgpB, were demonstrated to bind directly to soluble recombinant six-His-tagged HmuR. Several possible mechanisms for the cooperation between outer membrane receptor HmuR and proteases Kgp and HRgpA in hemin and hemoglobin binding and utilization are discussed.
Functional Characterization of Three GlnB Homologs in the Photosynthetic Bacterium
Rhodospirillum rubrum
: Roles in Sensing Ammonium and Energy Status
Authors: Zhang Yaoping , Pohlmann Edward L. , Ludden Paul W. , Roberts Gary P. 
Published: July 2002

ABSTRACT

The GlnB (P
II
) protein, the product of
glnB
, has been characterized previously in the photosynthetic bacterium
Rhodospirillum rubrum
. Here we describe identification of two other P
II
homologs in this organism, GlnK and GlnJ. Although the sequences of these three homologs are very similar, the molecules have both distinct and overlapping functions in the cell. While GlnB is required for activation of NifA activity in
R. rubrum
, GlnK and GlnJ do not appear to be involved in this process. In contrast, either GlnB or GlnJ can serve as a critical element in regulation of the reversible ADP ribosylation of dinitrogenase reductase catalyzed by the dinitrogenase reductase ADP-ribosyl transferase (DRAT)/dinitrogenase reductase-activating glycohydrolase (DRAG) regulatory system. Similarly, either GlnB or GlnJ is necessary for normal growth on a variety of minimal and rich media, and any of the proteins is sufficient for normal posttranslational regulation of glutamine synthetase. Surprisingly, in their regulation of the DRAT/DRAG system, GlnB and GlnJ appeared to be responsive not only to changes in nitrogen status but also to changes in energy status, revealing a new role for this family of regulators in central metabolic regulation.
Biofilms and Planktonic Cells of
Pseudomonas aeruginosa
Have Similar Resistance to Killing by Antimicrobials
Authors: Spoering Amy L. , Lewis Kim 
Published: July 2002

ABSTRACT

Biofilms are considered to be highly resistant to antimicrobial agents. Strictly speaking, this is not the case—biofilms do not grow in the presence of antimicrobials any better than do planktonic cells. Biofilms are indeed highly resistant to killing by bactericidal antimicrobials, compared to logarithmic-phase planktonic cells, and therefore exhibit tolerance. It is assumed that biofilms are also significantly more tolerant than stationary-phase planktonic cells. A detailed comparative examination of tolerance of biofilms versus stationary- and logarithmic-phase planktonic cells with four different antimicrobial agents was performed in this study. Carbenicillin appeared to be completely ineffective against both stationary-phase cells and biofilms. Killing by this β-lactam antibiotic depends on rapid growth, and this result confirms the notion of slow-growing biofilms resembling the stationary state. Ofloxacin is a fluoroquinolone antibiotic that kills nongrowing cells, and biofilms and stationary-phase cells were comparably tolerant to this antibiotic. The majority of cells in both populations were eradicated at low levels of ofloxacin, leaving a fraction of essentially invulnerable persisters. The bulk of the population in both biofilm and stationary-phase cultures was tolerant to tobramycin. At very high tobramycin concentrations, a fraction of persister cells became apparent in stationary-phase culture. Stationary-phase cells were more tolerant to the biocide peracetic acid than were biofilms. In general, stationary-phase cells were somewhat more tolerant than biofilms in all of the cases examined. We concluded that, at least for
Pseudomonas aeruginosa
, one of the model organisms for biofilm studies, the notion that biofilms have greater resistance than do planktonic cells is unwarranted. We further suggest that tolerance to antibiotics in stationary-phase or biofilm cultures is largely dependent on the presence of persister cells.
Modulation of Anaerobic Energy Metabolism of
Bacillus subtilis
by
arfM
(
ywiD
)
Authors: Marino Marco , Ramos Hugo Cruz , Hoffmann Tamara , Glaser Philippe , Jahn Dieter 
Published: July 2002

ABSTRACT

Bacillus subtilis
grows under anaerobic conditions utilizing nitrate ammonification and various fermentative processes. The two-component regulatory system ResDE and the redox regulator Fnr are the currently known parts of the regulatory system for anaerobic adaptation. Mutation of the open reading frame
ywiD
located upstream of the respiratory nitrate reductase operon
narGHJI
resulted in elimination of the contribution of nitrite dissimilation to anaerobic nitrate respiratory growth. Significantly reduced nitrite reductase (NasDE) activity was detected, while respiratory nitrate reductase activity was unchanged. Anaerobic induction of
nasDE
expression was found to be significantly dependent on intact
ywiD,
while anaerobic
narGHJI
expression was
ywiD
independent. Anaerobic transcription of
hmp,
encoding a flavohemoglobin-like protein, and of the fermentative operons
lctEP
and
alsSD,
responsible for lactate and acetoin formation, was partially dependent on
ywiD
. Expression of
pta,
encoding phosphotransacetylase involved in fermentative acetate formation, was not influenced by
ywiD
. Transcription of the
ywiD
gene was anaerobically induced by the redox regulator Fnr via the conserved Fnr-box (TGTGA-6N-TCACT) centered 40.5 bp upstream of the transcriptional start site. Anaerobic induction of
ywiD
by
resDE
was found to be indirect via
resDE
-dependent activation of
fnr
. The
ywiD
gene is subject to autorepression and nitrite repression. These results suggest a ResDE → Fnr → YwiD regulatory cascade for the modulation of genes involved in the anaerobic metabolism of
B. subtilis
. Therefore,
ywiD
was renamed
arfM
for anaerobic respiration and fermentation modulator.
The Mere Lack of rT Modification in Initiator tRNA Does Not Facilitate Formylation-Independent Initiation in
Escherichia coli
Authors: Thanedar Swapna , Dineshkumar T. K. , Varshney Umesh 
Published: July 2002

ABSTRACT

Formylation of initiator methionyl-tRNA is essential for normal growth of eubacteria. However, under special conditions, it has been possible to initiate protein synthesis with unformylated initiator tRNA even in eubacteria. Earlier studies suggested that the lack of ribothymidine (rT) modification in initiator tRNA may facilitate initiation in the absence of formylation. In this report we show, by using
trmA
strains of
Escherichia coli
(defective for rT modification) and a sensitive in vivo initiation assay system, that the lack of rT modification in the initiators is not sufficient to effect formylation-independent initiation of protein synthesis.
New Family of Regulators in the Environmental Signaling Pathway Which Activates the General Stress Transcription Factor ς
B
of
Bacillus subtilis
Authors: Akbar Samina , Gaidenko Tatiana A. , Kang Choong Min , O'Reilly Mary , Devine Kevin M. , Price Chester W. 
Published: July 2002

ABSTRACT

Expression of the general stress regulon of
Bacillus subtilis
is controlled by the alternative transcription factor ς
B
, which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to ς
B
, and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated
B. subtilis
genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the ς
B
environmental signaling pathway, either singly or in combination. The two exceptions were
yetI
and
yezB
, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases ς
B
activity more than 400-fold in unstressed cells.
Modulation of Actinorhodin Biosynthesis in
Streptomyces lividans
by Glucose Repression of
afsR2
Gene Transcription
Authors: Kim Eung-Soo , Hong Hee-Jeon , Choi Cha-Yong , Cohen Stanley N. 
Published: July 2002

ABSTRACT

While the biosynthetic gene cluster encoding the pigmented antibiotic actinorhodin (ACT) is present in the two closely related bacterial species,
Streptomyces lividans
and
Streptomyces coelicolor
, it normally is expressed only in
S. coelicolor
—generating the deep-blue colonies responsible for the
S. coelicolor
name. However, multiple copies of the two regulatory genes,
afsR
and
afsR2
, activate ACT production in
S. lividans
, indicating that this streptomycete encodes a functional ACT biosynthetic pathway. Here we report that the occurrence of ACT biosynthesis in
S. lividans
is determined conditionally by the carbon source used for culture. We found that the growth of
S. lividans
on solid media containing glucose prevents ACT production in this species by repressing the synthesis of
afsR2
mRNA; a shift to glycerol as the sole carbon source dramatically relieved this repression, leading to extensive ACT synthesis and obliterating this phenotypic distinction between
S. lividans
and
S. coelicolor
. Transcription from the
afsR2
promoter during growth in glycerol was dependent on
afsR
gene function and was developmentally regulated, occurring specifically at the time of aerial mycelium formation and coinciding temporally with the onset of ACT production. In liquid media, where morphological differentiation does not occur, ACT production in the absence of glucose increased as
S. lividans
cells entered stationary phase, but unlike ACT biosynthesis on solid media, occurred by a mechanism that did not require either
afsR
or
afsR2
. Our results identify parallel medium-dependent pathways that regulate ACT biosynthesis in
S. lividans
and further demonstrate that the production of this antibiotic in
S. lividans
grown on agar can be modulated by carbon source through the regulation of
afsR2
mRNA synthesis.
GcpE Is Involved in the 2-
C
-Methyl-
d
-Erythritol 4-Phosphate Pathway of Isoprenoid Biosynthesis in
Escherichia coli
Authors: Altincicek Boran , Kollas Ann-Kristin , Sanderbrand Silke , Wiesner Jochen , Hintz Martin , Beck Ewald , Jomaa Hassan 
Published: July 2002

ABSTRACT

In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-
C
-methyl-
d
-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the
gcpE
gene of
Escherichia coli
is involved in this pathway.
E. coli
cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential
gcpE
gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of
gcpE
.
The ToxR-Mediated Organic Acid Tolerance Response of
Vibrio cholerae
Requires OmpU
Authors: Merrell D. Scott , Bailey Camella , Kaper James B. , Camilli Andrew 
Published: July 2002

ABSTRACT

It was previously demonstrated that the intestinal pathogen
Vibrio cholerae
could undergo an adaptive stress response known as the acid tolerance response (ATR). The ATR is subdivided into two branches, inorganic ATR and organic ATR. The transcriptional regulator ToxR, while not involved in inorganic ATR, is required for organic ATR in a ToxT-independent manner. Herein, we investigate the effect of organic acid stress on global protein synthesis in
V. cholerae
and show by two-dimensional gel electrophoresis that the stress response alters the expression of more than 100 polypeptide species. The expression of more than 20 polypeptide species is altered in a
toxR
strain compared to the wild type. Despite this, ectopic expression of the porin OmpU from an inducible promoter is shown to be sufficient to bypass the
toxR
organic ATR defect. Characterization of the effect of organic acid stress on
ompU
and
ompT
transcription reveals that while
ompU
transcription remains virtually unaffected,
ompT
transcription is repressed in a ToxR-independent manner. These transcript levels are similarly reflected in the extent of accumulation of OmpU and OmpT. Possible roles for OmpU in organic acid resistance are discussed.
Genes Essential to Iron Transport in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Katoh Hirokazu , Hagino Natsu , Grossman Arthur R. , Ogawa Teruo 
Published: July 2002

ABSTRACT

Genes encoding polypeptides of an ATP binding cassette (ABC)-type ferric iron transporter that plays a major role in iron acquisition in
Synechocystis
sp. strain PCC 6803 were identified. These genes are
slr1295, slr0513, slr0327
, and recently reported
sll1878
(Katoh et al., J. Bacteriol. 182:6523–6524, 2000) and were designated
futA1, futA2, futB
, and
futC
, respectively, for their involvement in ferric iron uptake. Inactivation of these genes individually or
futA1
and
futA2
together greatly reduced the activity of ferric iron uptake in cells grown in complete medium or iron-deprived medium. All the
fut
genes are expressed in cells grown in complete medium, and expression was enhanced by iron starvation. The
futA1
and
futA2
genes appear to encode periplasmic proteins that play a redundant role in iron binding. The deduced products of
futB
and
futC
genes contain nucleotide-binding motifs and belong to the ABC transporter family of inner-membrane-bound and membrane-associated proteins, respectively. These results and sequence similarities among the four genes suggest that the Fut system is related to the Sfu/Fbp family of iron transporters. Inactivation of
slr1392
, a homologue of
feoB
in
Escherichia coli
, greatly reduced the activity of ferrous iron transport. This system is induced by intracellular low iron concentrations that are achieved in cells exposed to iron-free medium or in the
fut
-less mutants grown in complete medium.