bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification, Evolution, and Essentiality of the Mevalonate Pathway for Isopentenyl Diphosphate Biosynthesis in Gram-Positive Cocci
Authors: Wilding E. Imogen , Brown James R. , Bryant Alexander P. , Chalker Alison F. , Holmes David J. , Ingraham Karen A. , Iordanescu Serban , So Chi Y. , Rosenberg Martin , Gwynn Michael N.
Published: July 2002

The mevalonate pathway and the glyceraldehyde 3-phosphate (GAP)–pyruvate pathway are alternative routes for the biosynthesis of the central isoprenoid precursor, isopentenyl diphosphate. Genomic analysis revealed that the staphylococci, streptococci, and enterococci possess genes predicted to encode all of the enzymes of the mevalonate pathway and not the GAP-pyruvate pathway, unlike
Bacillus subtilis
and most gram-negative bacteria studied, which possess only components of the latter pathway. Phylogenetic and comparative genome analyses suggest that the genes for mevalonate biosynthesis in gram-positive cocci, which are highly divergent from those of mammals, were horizontally transferred from a primitive eukaryotic cell. Enterococci uniquely encode a bifunctional protein predicted to possess both 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and acetyl-CoA acetyltransferase activities. Genetic disruption experiments have shown that five genes encoding proteins involved in this pathway (HMG-CoA synthase, HMG-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase) are essential for the in vitro growth of
Streptococcus pneumoniae
under standard conditions. Allelic replacement of the HMG-CoA synthase gene rendered the organism auxotrophic for mevalonate and severely attenuated in a murine respiratory tract infection model. The mevalonate pathway thus represents a potential antibacterial target in the low-G+C gram-positive cocci.
A Protease-Resistant Catalase, KatA, Released upon Cell Lysis during Stationary Phase Is Essential for Aerobic Survival of a
Pseudomonas aeruginosa oxyR
Mutant at Low Cell Densities
Authors: Hassett Daniel J. , Alsabbagh Eyad , Parvatiyar Kislay , Howell Michael L. , Wilmott Robert W. , Ochsner Urs A.
Published: July 2002

A
Pseudomonas aeruginosa oxyR
mutant was dramatically sensitive to H
2
O
2
, despite possessing wild-type catalase activity. Oxygen-dependent
oxyR
phenotypes also included an inability to survive aerobic serial dilution in Luria broth and to resist aminoglycosides. Plating the
oxyR
mutant after serial dilution in its own spent culture supernatant, which contained the major catalase KatA, or under anaerobic conditions allowed for survival. KatA was resistant to sodium dodecyl sulfate, proteinase K, pepsin, trypsin, chymotrypsin and the neutrophil protease cathepsin G. When provided in
trans
and expressed constitutively, the OxyR-regulated genes
katB
,
ahpB
, and
ahpCF
could not restore both the serial dilution defect and H
2
O
2
resistance; only
oxyR
itself could do so. The aerobic dilution defect could be complemented, in part, by only
ahpB
and
ahpCF
, suggesting that the latter gene products could possess a catalase-like activity. Aerobic Luria broth was found to generate ∼1.2 μM H
2
O
2
min
−1
via autoxidation, a level sufficient to kill serially diluted
oxyR
and
oxyR katA
bacteria and explain the molecular mechanism behind the aerobic serial dilution defect. Taken together, our results indicate that inactivation of OxyR renders
P. aeruginosa
exquisitely sensitive to both H
2
O
2
and aminoglycosides, which are clinically and environmentally important antimicrobials.
Chitin Synthesis in a
gas1
Mutant of
Saccharomyces cerevisiae
Authors: Valdivieso M-Henar , Ferrario Laura , Vai Marina , Duran Angel , Popolo Laura
Published: July 2002

The existence of a compensatory mechanism in response to cell wall damage has been proposed in yeast cells. The increase of chitin accumulation is part of this response. In order to study the mechanism of the stress-related chitin synthesis, we tested chitin synthase I (CSI), CSII, and CSIII in vitro activities in the cell-wall-defective mutant
gas1
Δ. CSI activity increased twofold with respect to the control, a finding in agreement with an increase in the expression of the
CHS1
gene. However, deletion of the
CHS1
gene did not affect the phenotype of the
gas1
Δ mutant and only slightly reduced the chitin content. Interestingly, in
chs1 gas1
double mutants the lysed-bud phenotype, typical of
chs1
null mutant, was suppressed, although in
gas1
cells there was no reduction in chitinase activity.
CHS3
expression was not affected in the
gas1
mutant. Deletion of the
CHS3
gene severely compromised the phenotype of
gas1
cells, despite the fact that CSIII activity, assayed in membrane fractions, did not change. Furthermore, in
chs3 gas1
cells the chitin level was about 10% that of
gas1
cells. Thus, CSIII is the enzyme responsible for the hyperaccumulation of chitin in response to cell wall stress. However, the level of enzyme or the in vitro CSIII activity does not change. This result suggests that an interaction with a regulatory molecule or a posttranslational modification, which is not preserved during membrane fractionation, could be essential in vivo for the stress-induced synthesis of chitin.
Lantibiotic Biosynthesis: Interactions between Prelacticin 481 and Its Putative Modification Enzyme, LctM
Authors: Uguen Patricia , Le Pennec Jean-Paul , Dufour Alain
Published: July 2002

Class AII and AIII lantibiotics and mersacidin are antibacterial peptides containing unusual residues obtained by posttranslational modifications of prepeptides, presumably catalyzed by LanM. LctM, the LanM for lacticin 481, is essential for the production of this class AII lantibiotic. Using the yeast two-hybrid system, we showed direct contact between the prelacticin 481 and LctM, supporting the proposed LctM function. Sixteen domains are conserved between the 10 known LanM proteins, whereas three additional domains were found only in class AII LanM proteins and in MrsM, the LanM for mersacidin. All the truncated LctM proteins that we tested presented impaired LctA-binding activity.
Identification in
Listeria monocytogenes
of MecA, a Homologue of the
Bacillus subtilis
Competence Regulatory Protein
Authors: Borezee Elise , Msadek Tarek , Durant Lionel , Berche Patrick
Published: July 2002

We identified in
Listeria monocytogenes
a gene encoding a protein homologous to MecA, a regulatory protein acting with ClpC and ComK in the competence pathway of
Bacillus subtilis
. In
L. monocytogenes
, MecA is involved, along with ClpC and ClpP, in the downregulation of a 64-kDa secreted protein. In
B. subtilis
, the MecA protein of
L. monocytogenes
behaves as a regulatory protein, controlling the transcription of
comK
and
comG
. Complete or disrupted ComK homologues were also found in
L. monocytogenes
. However, we failed to detect competence in various strains of
L. monocytogenes
, including those with intact ComK. Our results suggest that the functions of MecA in the saprophytes
L. monocytogenes
and
B. subtilis
have presumably diverged in response to their respective ecological niches.
Regulation of Ferritin-Mediated Cytoplasmic Iron Storage by the Ferric Uptake Regulator Homolog (Fur) of
Helicobacter pylori
Authors: Bereswill Stefan , Greiner Stefan , van Vliet Arnoud H. M. , Waidner Barbara , Fassbinder Frank , Schiltz Emile , Kusters Johannes G. , Kist Manfred
Published: July 2002

Homologs of the ferric uptake regulator Fur and the iron storage protein ferritin play a central role in maintaining iron homeostasis in bacteria. The gastric pathogen
Helicobacter pylori
contains an iron-induced prokaryotic ferritin (Pfr) which has been shown to be involved in protection against metal toxicity and a Fur homolog which has not been functionally characterized in
H. pylori
. Analysis of an isogenic
fur
-negative mutant revealed that
H. pylori
Fur is required for metal-dependent regulation of ferritin. Iron starvation, as well as medium supplementation with nickel, zinc, copper, and manganese at nontoxic concentrations, repressed synthesis of ferritin in the wild-type strain but not in the
H. pylori fur
mutant. Fur-mediated regulation of ferritin synthesis occurs at the mRNA level. With respect to the regulation of ferritin expression, Fur behaves like a global metal-dependent repressor which is activated under iron-restricted conditions but also responds to different metals. Downregulation of ferritin expression by Fur might secure the availability of free iron in the cytoplasm, especially if iron is scarce or titrated out by other metals.
The Bulged Nucleotide in the
Escherichia coli
Minimal Selenocysteine Insertion Sequence Participates in Interaction with SelB: a Genetic Approach
Authors: Li Chuang , Reches Myriam , Engelberg-Kulka Hanna
Published: July 2002

The UGA codon, which usually acts as a stop codon, can also direct the incorporation into a protein of the amino acid selenocysteine. This UGA decoding process requires a
cis
-acting mRNA element called the selenocysteine insertion sequence (SECIS), which can form a stem-loop structure. In
Escherichia coli
, selenocysteine incorporation requires only the 17-nucleotide-long upper stem-loop structure of the
fdhF
SECIS. This structure carries a bulged nucleotide U at position 17. Here we asked whether the single bulged nucleotide located in the upper stem-loop structure of the
E. coli fdhF
SECIS is involved in the in vivo interaction with SelB. We used a genetic approach, generating and characterizing
selB
mutations that suppress mutations of the bulged nucleotide in the SECIS. All the
selB
suppressor mutations isolated were clustered in a region corresponding to 28 amino acids in the SelB C-terminal subdomain 4b. These
selB
suppressor mutations were also found to suppress mutations in either the loop or the upper stem of the
E. coli
SECIS. Thus, the
E. coli
SECIS upper stem-loop structure can be considered a “single suppressible unit,” suggesting that there is some flexibility to the nature of the interaction between this element and SelB.
Comparative Genetic Analysis of
Mycobacterium ulcerans
and
Mycobacterium marinum
Reveals Evidence of Recent Divergence
Authors: Stinear Timothy P. , Jenkin Grant A. , Johnson Paul D. R. , Davies John K.
Published: July 2002

Previous studies of the 16S rRNA genes from
Mycobacterium ulcerans
and
Mycobacterium marinum
have suggested a very close genetic relationship between these species (99.6% identity). However, these organisms are phenotypically distinct and cause diseases with very different pathologies. To investigate this apparent paradox, we compared 3,306 nucleotides from the partial sequences of eight housekeeping and structural genes derived from 18
M. ulcerans
strains and 22
M. marinum
strains. This analysis confirmed the close genetic relationship inferred from the 16S rRNA data, with nucleotide sequence identity ranging from 98.1 to 99.7%. The multilocus sequence analysis also confirmed previous genotype studies of
M. ulcerans
that have identified distinct genotypes within a geographical region. Single isolates of both
M. ulcerans
and
M. marinum
that were shown by the sequence analysis to be the most closely related were then selected for further study. One- and two-dimensional pulsed-field gel electrophoresis was employed to compare the architecture and size of the genome from each species. Genome sizes of approximately 4.4 and 4.6 Mb were obtained for
M. ulcerans
and
M. marinum
, respectively. Significant macrorestriction fragment polymorphism was observed between the species. However, hybridization analysis of DNA cleaved with more frequently cutting enzymes identified significant preservation of the flanking sequence at seven of the eight loci sequenced. The exception was the 16S rRNA locus. Two high-copy-number insertion sequences, IS
2404
and IS
2606
, have recently been reported in
M. ulcerans
, and significantly, these elements are not present in
M. marinum
. Hybridization of the
Ase
I restriction fragments from
M. ulcerans
with IS
2404
and IS
2606
indicated widespread genome distribution for both of these repeated sequences. Taken together, these data strongly suggest that
M. ulcerans
has recently diverged from
M. marinum
by the acquisition and concomitant loss of DNA in a manner analogous to the emergence of
M. tuberculosis
, where species diversity is being driven mainly by the activity of mobile DNA elements.
Silencing and Activation of ClyA Cytotoxin Expression in
Escherichia coli
Authors: Westermark Marie , Oscarsson Jan , Mizunoe Yoshimitsu , Urbonaviciene Jurate , Uhlin Bernt Eric
Published: July 2002

Cytolysin A (ClyA) is a pore-forming cytotoxic protein encoded by the
clyA
gene of
Escherichia coli
K-12. Genetic analysis suggested that
clyA
is silenced by the nucleoid protein H-NS. Purified H-NS protein showed preferential binding to
clyA
sequences in the promoter region, as evidenced by DNase I footprinting and gel mobility shift assays. Transcriptional derepression and activation of a chromosomal
clyA
::
luxAB
operon fusion were seen under conditions of H-NS deficiency and SlyA overproduction, respectively. In H-NS-deficient bacteria neither the absence nor the overproduction of SlyA affected the derepressed ClyA expression any further. Therefore, we suggest that overproduction of SlyA in

hns
+
E. coli

derepresses
clyA
transcription by counteracting H-NS. The cyclic AMP receptor protein (CRP) was required for ClyA expression, and it interacted with a predicted, albeit suboptimal, CRP binding site in the
clyA
upstream region. Site-specific alterations of the CRP binding site to match the consensus resulted in substantially higher levels of ClyA expression, while alterations that were predicted to reduce CRP binding reduced ClyA expression. During anaerobic growth the fumarate and nitrate reduction regulator (FNR) was important for ClyA expression, and the
clyA
gene could be activated by overexpression of FNR. A major
clyA
transcript having its 5′ end (+1) located 72 bp upstream of the translational start codon and 61 bp downstream of the CRP-FNR binding site was detected in the absence of H-NS. The
clyA
promoter was characterized as a class I promoter that could be transcriptionally activated by CRP and/or FNR. According to DNA bending analyses, the
clyA
promoter region has high intrinsic curvature. We suggest that it represents a regulatory region which is particularly susceptible to H-NS silencing, and its features are discussed in relation to regulation of other silenced operons.
Characterization of the Collagen-Binding S-Layer Protein CbsA of
Lactobacillus crispatus
Authors: Sillanpää Jouko , Martínez Beatriz , Antikainen Jenni , Toba Takahiro , Kalkkinen Nisse , Tankka Sanna , Lounatmaa Kari , Keränen Jaakko , Höök Magnus , Westerlund-Wikström Benita , Pouwels Peter H. , Korhonen Timo K.
Published: July 2002

The
cbsA
gene of
Lactobacillus crispatus
strain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in
Escherichia coli
. The
cbsA
open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. The
cbsA
gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three other
Lactobacillus
S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in
cbsA
where deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed
cbsB
that was 44% identical in sequence to
cbsA
. RNA analysis showed that
cbsA
, but not
cbsB
, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of
L. crispatus
.
Characterization of SepL of Enterohemorrhagic
Escherichia coli
Authors: Kresse Andreas U. , Beltrametti Fabrizio , Müller Astrid , Ebel Frank , Guzmán Carlos A.
Published: July 2002

The
sepL
gene is expressed in the locus of enterocyte effacement and therefore is most likely implicated in the attaching and effacing process, as are the products encoded by open reading frames located up- and downstream of this gene. In this study, the
sepL
gene of the enterohemorrhagic
Escherichia coli
(EHEC) strain EDL933 was analyzed and the corresponding polypeptide was characterized. We found that
sepL
is transcribed monocistronically and independently from the
esp
operon located downstream, which codes for the secreted proteins EspA, -D, and -B. Primer extension analysis allowed us to identify a single start of transcription 83 bp upstream of the
sepL
start codon. The analysis of the upstream regions led to the identification of canonical promoter sequences between positions −5 and −36. Translational fusions using
lacZ
as a reporter gene demonstrated that
sepL
is activated in the exponential growth phase by stimuli that are characteristic for the intestinal niche, e.g., a temperature of 37°C, a nutrient-rich environment, high osmolarity, and the presence of Mn
2+
. Protein localization studies showed that SepL was present in the cytoplasm and associated with the bacterial membrane fraction. To analyze the functional role of the SepL protein during infection of eukaryotic cells, an in-frame deletion mutant was generated. This
sepL
mutant was strongly impaired in its ability to attach to HeLa cells and induce a local accumulation of actin. These defects were partially restored by providing the
sepL
gene in
trans
. The EDL933Δ
sepL
mutant also exhibited an impaired secretion but not biosynthesis of Esp proteins, which was fully complemented by providing
sepL
in
trans
. These results demonstrate the crucial role played by SepL in the biological cycle of EHEC.
Terminal Oxidases of
Bacillus subtilis
Strain 168: One Quinol Oxidase, Cytochrome
aa
3
or Cytochrome
bd
, Is Required for Aerobic Growth
Authors: Winstedt Lena , von Wachenfeldt Claes
Published: July 2002

The gram-positive endospore-forming bacterium
Bacillus subtilis
has, under aerobic conditions, a branched respiratory system comprising one quinol oxidase branch and one cytochrome oxidase branch. The system terminates in one of four alternative terminal oxidases. Cytochrome
caa
3
is a cytochrome
c
oxidase, whereas cytochrome
bd
and cytochrome
aa
3
are quinol oxidases. A fourth terminal oxidase, YthAB, is a putative quinol oxidase predicted from DNA sequence analysis. None of the terminal oxidases are, by themselves, essential for growth. However, one quinol oxidase (cytochrome
aa
3
or cytochrome
bd
) is required for aerobic growth of
B. subtilis
strain 168. Data indicating that cytochrome
aa
3
is the major oxidase used by exponentially growing cells in minimal and rich medium are presented. We show that one of the two heme-copper oxidases, cytochrome
caa
3
or cytochrome
aa
3
, is required for efficient sporulation of
B. subtilis
strain 168 and that deletion of YthAB in a strain lacking cytochrome
aa
3
makes the strain sporulation deficient.
Characterization of
Borrelia burgdorferi
BlyA and BlyB Proteins: a Prophage-Encoded Holin-Like System
Authors: Damman Christopher J. , Eggers Christian H. , Samuels D. Scott , Oliver Donald B.
Published: July 2002

The conserved cp32 plasmid family of
Borrelia burgdorferi
was recently shown to be packaged into a bacteriophage particle (C. H. Eggers and D. S. Samuels, J. Bacteriol. 181:7308–7313, 1999). This plasmid encodes BlyA, a 7.4-kDa membrane-interactive protein, and BlyB, an accessory protein, which were previously proposed to comprise a hemolysis system. Our genetic and biochemical evidence suggests that this hypothesis is incorrect and that BlyA and BlyB function instead as a prophage-encoded holin or holin-like system for this newly described bacteriophage. An
Escherichia coli
mutant containing the
blyAB
locus that was defective for the normally cryptic host hemolysin SheA was found to be nonhemolytic, suggesting that induction of
sheA
by
blyAB
expression was responsible for the hemolytic activity observed previously. Analysis of the structural features of BlyA indicated greater structural similarity to bacteriophage-encoded holins than to hemolysins. Consistent with holin characteristics, subcellular localization studies with
E. coli
and
B. burgdorferi
indicated that BlyA is solely membrane associated and that BlyB is a soluble protein. Furthermore, BlyA exhibited a holin-like function by promoting the endolysin-dependent lysis of an induced lambda lysogen that was defective in the holin gene. Finally, induction of the cp32 prophage in
B. burgdorferi
dramatically stimulated
blyAB
expression. Our results provide the first evidence of a prophage-encoded holin within
Borrelia
.
Role of ς
B
in Adaptation of
Listeria monocytogenes
to Growth at Low Temperature
Authors: Becker Lynne A. , Evans Stefanie N. , Hutkins Robert W. , Benson Andrew K.
Published: July 2002

The activity of ς
B
in
Listeria monocytogenes
is stimulated by high osmolarity and is necessary for efficient uptake of osmoprotectants. Here we demonstrate that, during cold shock, ς
B
contributes to adaptation in a growth phase-dependent manner and is necessary for efficient accumulation of betaine and carnitine as cryoprotectants.
Novel Phospholipase A Activity Secreted by
Legionella
Species
Authors: Flieger Antje , Gong Shimei , Faigle Marion , Deeg Martin , Bartmann Peter , Neumeister Birgid
Published: July 2002

Bacterial phospholipases are regarded as a major virulence factor in infection. In bacteria associated with pneumonia, destruction of lung surfactant and host cell membranes by bacterial phospholipases secreted during infection is thought to contribute to the disease. Phospholipase C (PLC) activity has been described in several
Legionella
species (W. B. Baine, J. Gen. Microbiol. 134:489–498, 1988; W. B. Baine, J. Gen. Microbiol. 131:1383–1391, 1985). By using detection methods such as thin-layer chromatography and mass spectrometry, PLC activity could not be detected in several strains of
Legionella pneumophila
. Instead, phospholipid degradation was identified to be caused by a novel PLA activity. We could demonstrate that PLA secretion starts at the mid-exponential-growth phase when bacteria were grown in liquid culture. Several
Legionella
species secreted different amounts of PLA.
Legionella
PLA may act as a powerful agent in the mediation of pathogenicity due to destruction of lung surfactant and epithelial cells.
Capsule Biosynthesis and Basic Metabolism in
Streptococcus pneumoniae
Are Linked through the Cellular Phosphoglucomutase
Authors: Hardy Gail G. , Caimano Melissa J. , Yother Janet
Published: July 2002

Synthesis of the type 3 capsular polysaccharide of
Streptococcus pneumoniae
requires UDP-glucose (UDP-Glc) and UDP-glucuronic acid (UDP-GlcUA) for production of the [3)-β-
d
-GlcUA-(1→4)-β-
d
-Glc-(1→]

n

polymer. The generation of UDP-Glc proceeds by conversion of Glc-6-P to Glc-1-P to UDP-Glc and is mediated by a phosphoglucomutase (PGM) and a Glc-1-P uridylyltransferase, respectively. Genes encoding both a Glc-1-P uridylyltransferase (
cps3U
) and a PGM homologue (
cps3M
) are present in the type 3 capsule locus, but these genes are not essential for capsule production. In this study, we characterized a mutant that produces fourfold less capsule than the type 3 parent. The spontaneous mutation resulting in this phenotype was not contained in the type 3 capsule locus but was instead located in a distant gene (
pgm
) encoding a second PGM homologue. The function of this gene product as a PGM was demonstrated through enzymatic and complementation studies. Insertional inactivation of
pgm
reduced capsule production to less than 10% of the parental level. The loss of PGM activity in the insertion mutants also caused growth defects and a strong selection for isolates containing second-site suppressor mutations. These results demonstrate that most of the PGM activity required for type 3 capsule biosynthesis is derived from the cellular PGM.
Characterization of Hydrogenase II from the Hyperthermophilic Archaeon
Pyrococcus furiosus
and Assessment of Its Role in Sulfur Reduction
Authors: Ma Kesen , Weiss Robert , Adams Michael W. W.
Published: July 2002

The fermentative hyperthermophile
Pyrococcus furiosus
contains an NADPH-utilizing, heterotetrameric (αβγδ), cytoplasmic hydrogenase (hydrogenase I) that catalyzes both H
2
production and the reduction of elemental sulfur to H
2
S. Herein is described the purification of a second enzyme of this type, hydrogenase II, from the same organism. Hydrogenase II has an
M
r
of 320,000 ± 20,000 and contains four different subunits with
M
r
s of 52,000 (α), 39,000 (β), 30,000 (γ), and 24,000 (δ). The heterotetramer contained Ni (0.9 ± 0.1 atom/mol), Fe (21 ± 1.6 atoms/mol), and flavin adenine dinucleotide (FAD) (0.83 ± 0.1 mol/mol). NADPH and NADH were equally efficient as electron donors for H
2
production with

K
m

values near 70 μM and
k
cat
/

K
m

values near 350 min
−1
mM
−1
. In contrast to hydrogenase I, hydrogenase II catalyzed the H
2
-dependent reduction of NAD (

K
m

, 128 μM;
k
cat
/

K
m

, 770 min
−1
mM
−1
). Ferredoxin from
P. furiosus
was not an efficient electron carrier for either enzyme. Both H
2
and NADPH served as electron donors for the reduction of elemental sulfur (S
0
) and polysulfide by hydrogenase I and hydrogenase II, and both enzymes preferentially reduce polysulfide to sulfide rather than protons to H
2
using NADPH as the electron donor. At least two [4Fe-4S] and one [2Fe-2S] cluster were detected in hydrogenase II by electron paramagnetic resonance spectroscopy, but amino acid sequence analyses indicated a total of five [4Fe-4S] clusters (two in the β subunit and three in the δ subunit) and one [2Fe-2S] cluster (in the γ subunit), as well as two putative nucleotide-binding sites in the γ subunit which are thought to bind FAD and NAD(P)(H). The amino acid sequences of the four subunits of hydrogenase II showed between 55 and 63% similarity to those of hydrogenase I. The two enzymes are present in the cytoplasm at approximately the same concentration. Hydrogenase II may become physiologically relevant at low S
0
concentrations since it has a higher affinity than hydrogenase I for both S
0
and polysulfide.
Isolation and Characterization of
vicH
, Encoding a New Pleiotropic Regulator in
Vibrio cholerae
Authors: Tendeng Christian , Badaut Cyril , Krin Evelyne , Gounon Pierre , Ngo Saravuth , Danchin Antoine , Rimsky Sylvie , Bertin Philippe
Published: July 2002

During the last decade, the
hns
gene and its product, the H-NS protein, have been extensively studied in
Escherichia coli
. H-NS-like proteins seem to be widespread in gram-negative bacteria. However, unlike in
E. coli
and in
Salmonella enterica
serovar Typhimurium, little is known about their role in the physiology of those organisms. In this report, we describe the isolation of
vicH
, an
hns
-like gene in
Vibrio cholerae
, the etiological agent of cholera. This gene was isolated from a
V. cholerae
genomic library by complementation of different phenotypes associated with an
hns
mutation in
E. coli
. It encodes a 135-amino-acid protein showing approximately 50% identity with both H-NS and StpA in
E. coli
. Despite a low amino acid conservation in the N-terminal part, VicH is able to cross-react with anti-H-NS antibodies and to form oligomers in vitro. The
vicH
gene is expressed as a single gene from two promoters in tandem and is induced by cold shock. A
V. cholerae
wild-type strain expressing a
vicHΔ92
gene lacking its 3′ end shows pleiotropic alterations with regard to mucoidy and salicin metabolism. Moreover, this strain is unable to swarm on semisolid medium. Similarly, overexpression of the
vicH
wild-type gene results in an alteration of swarming behavior. This suggests that VicH could be involved in the virulence process in
V. cholerae
, in particular by affecting flagellum biosynthesis.
OmpR Regulates the Stationary-Phase Acid Tolerance Response of
Salmonella enterica
Serovar Typhimurium
Authors: Bang Iel Soo , Kim Bae Hoon , Foster John W. , Park Yong Keun
Published: July 2002

Tolerance to acidic environments is an important property of free-living and pathogenic enteric bacteria.
Salmonella enterica
serovar Typhimurium possesses two general forms of inducible acid tolerance. One is evident in exponentially growing cells exposed to a sudden acid shock. The other is induced when stationary-phase cells are subjected to a similar shock. These log-phase and stationary-phase acid tolerance responses (ATRs) are distinct in that genes identified as participating in log-phase ATR have little to no effect on the stationary-phase ATR (I. S. Lee, J. L. Slouczewski, and J. W. Foster, J. Bacteriol. 176:1422–1426, 1994). An insertion mutagenesis strategy designed to reveal genes associated with acid-inducible stationary-phase acid tolerance (stationary-phase ATR) yielded two insertions in the response regulator gene
ompR
. The
ompR
mutants were defective in stationary-phase ATR but not log-phase ATR. EnvZ, the known cognate sensor kinase, and the porin genes known to be controlled by OmpR,
ompC
and
ompF
, were not required for stationary-phase ATR. However, the alternate phosphodonor acetyl phosphate appears to play a crucial role in OmpR-mediated stationary-phase ATR and in the OmpR-dependent acid induction of
ompC
. This conclusion was based on finding that a mutant form of OmpR, which is active even though it cannot be phosphorylated, was able to suppress the acid-sensitive phenotype of an
ack pta
mutant lacking acetyl phosphate. The data also revealed that acid shock increases the level of
ompR
message and protein in stationary-phase cells. Thus, it appears that acid shock induces the production of OmpR, which in its phosphorylated state can trigger expression of genes needed for acid-induced stationary-phase acid tolerance.
Osmotic and Chill Activation of Glycine Betaine Porter II in
Listeria monocytogenes
Membrane Vesicles
Authors: Gerhardt Paul N. M. , Tombras Smith Linda , Smith Gary M.
Published: July 2002

Listeria monocytogenes
is a foodborne pathogen known for its tolerance to conditions of osmotic and chill stress. Accumulation of glycine betaine has been found to be important in the organism's tolerance to both of these stresses. A procedure was developed for the purification of membranes from
L. monocytogenes
cells in which the putative ATP-driven glycine betaine permease glycine betaine porter II (Gbu) is functional. As is the case for the
L. monocytogenes
sodium-driven glycine betaine uptake system (glycine betaine porter I), uptake in this vesicle system was dependent on energization by ascorbate-phenazine methosulfate. Vesicles lacking the
gbu
gene product had no uptake activity. Transport by this porter did not require sodium ion and could be driven only weakly by artificial gradients. Uptake rates could be manipulated under conditions not affecting secondary transport but known to affect ATPase activity. The system was shown to be both osmotically activated and cryoactivated. Under conditions of osmotic activation, the system exhibited Arrhenius-type behavior although the uptake rates were profoundly affected by the physical state of the membrane, with breaks in Arrhenius curves at approximately 10 and 18°C. In the absence of osmotic activation, the permease could be activated by decreasing temperature within the range of 15 to 4°C. Kinetic analyses of the permease at 30°C revealed

K
m

values for glycine betaine of 1.2 and 2.9 μM with
V
max
values of 2,200 and 3,700 pmol/min · mg of protein under conditions of optimal osmotic activation as mediated by KCl and sucrose, respectively.
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

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

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

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

InPseudomonas 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

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

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

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

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

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.