bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Expression of over 60 Chromosomal Genes in
Escherichia coli
by Constitutive Expression of MarA
Authors: Barbosa Teresa M. , Levy Stuart B.
Published: July 2002

In
Escherichia coli
, the MarA protein controls expression of multiple chromosomal genes affecting resistance to antibiotics and other environmental hazards. For a more-complete characterization of the
mar
regulon, duplicate macroarrays containing 4,290 open reading frames of the
E. coli
genome were hybridized to radiolabeled cDNA populations derived from
mar
-deleted and
mar
-expressing
E. coli
. Strains constitutively expressing MarA showed altered expression of more than 60 chromosomal genes: 76% showed increased expression and 24% showed decreased expression. Although some of the genes were already known to be MarA regulated, the majority were newly determined and belonged to a variety of functional groups. Some of the genes identified have been associated with iron transport and metabolism; other genes were previously known to be part of the
soxRS
regulon. Northern blot analysis of selected genes confirmed the results obtained with the macroarrays. The findings reveal that the
mar
locus mediates a global stress response involving one of the largest networks of genes described.
Organization and Cell-Cell Interaction in Starved
Saccharomyces cerevisiae
Colonies
Authors: Varon Mazal , Choder Mordechai
Published: July 2002

Cell growth in yeast colonies is a complex process, the control of which is largely unknown. Here we present scanning electron micrographs of
Saccharomyces cerevisiae
colonies, showing changes in the pattern of cell organization and cell-cell interactions during colony development. In young colonies (≤36 h), cell density is relatively low, and the cells seem to divide in a random orientation. However, as the colonies age, cell density increases and the cells seem to be oriented in a more orderly fashion. Unexpectedly, cells in starved colonies form connecting fibrils. A single connecting fibril 180 ± 50 nm wide is observed between any two neighboring cells, and the fibrils appear to form a global network. The results suggest a novel type of communication between cells within a colony that may contribute to the ability of the community to cope with starvation.
comH
, a Novel Gene Essential for Natural Transformation of
Helicobacter pylori
Authors: Smeets Leonard C. , Bijlsma Jetta J. E. , Boomkens Sacha Y. , Vandenbroucke-Grauls Christina M. J. E. , Kusters Johannes G.
Published: July 2002

Helicobacter pylori
is naturally competent for transformation, but the DNA uptake system of this bacterium is only partially characterized, and nothing is known about the regulation of competence in
H. pylori
. To identify other components involved in transformation or competence regulation in this species, we screened a mutant library for competence-deficient mutants. This resulted in the identification of a novel,
Helicobacter
-specific competence gene (
comH
) whose function is essential for transformation of
H. pylori
with chromosomal DNA fragments as well as with plasmids. Complementation of
comH
mutants in
trans
completely restored competence. Unlike other transformation genes of
H. pylori
,
comH
does not belong to a known family of orthologous genes. Moreover, no significant homologs of
comH
were identified in currently available databases of bacterial genome sequences. The
comH
gene codes for a protein with an N-terminal leader sequence and is present in both highly competent and less-efficient transforming
H. pylori
strains. A
comH
homolog was found in
Helicobacter acinonychis
but not in
Helicobacter felis
and
Helicobacter mustelae.
Slow Polymerization of
Mycobacterium tuberculosis
FtsZ
Authors: White E. Lucile , Ross Larry J. , Reynolds Robert C. , Seitz Lainne E. , Moore Georgia D. , Borhani David W.
Published: July 2002

The essential cell division protein, FtsZ, from
Mycobacterium tuberculosis
has been expressed in
Escherichia coli
and purified. The recombinant protein has GTPase activity typical of tubulin and other FtsZs. FtsZ polymerization was studied using 90° light scattering. The mycobacterial protein reaches maximum polymerization much more slowly (∼10 min) than
E. coli
FtsZ. Depolymerization also occurs slowly, taking 1 h or longer under most conditions. Polymerization requires both Mg
2+
and GTP. The minimum concentration of FtsZ needed for polymerization is 3 μM. Electron microscopy shows that polymerized
M. tuberculosis
FtsZ consists of strands that associate to form ordered aggregates of parallel protofilaments. Ethyl 6-amino-2,3-dihydro-4-phenyl-1H-pyrido[4,3-b][1,4]diazepin-8-ylcarbamate (SRI 7614), an inhibitor of tubulin polymerization synthesized at Southern Research Institute, inhibits
M. tuberculosis
FtsZ polymerization, inhibits GTP hydrolysis, and reduces the number and sizes of FtsZ polymers.
The Serotype of Type Ia and III Group B Streptococci Is Determined by the Polymerase Gene within the Polycistronic Capsule Operon
Authors: Chaffin Donald O. , Beres Stephen B. , Yim Harry H. , Rubens Craig E.
Published: July 2002

Streptococcus agalactiae
is a primary cause of neonatal morbidity and mortality. Essential to the virulence of this pathogen is the production of a type-specific capsular polysaccharide (CPS) that enables the bacteria to evade host immune defenses. The identification, cloning, sequencing, and functional characterization of seven genes involved in type III capsule production have been previously reported. Here, we describe the cloning and sequencing of nine additional adjacent genes,

cps
III
FGHIJKL

,

neu
III
B

, and

neu
III
C

. Sequence comparisons suggested that these genes are involved in sialic acid synthesis, pentasaccharide repeating unit formation, and oligosaccharide transport and polymerization. The type III CPS (
cps
III) locus was comprised of 16 genes within 15.5 kb of contiguous chromosomal DNA. Primer extension analysis and investigation of mRNA from mutants with polar insertions in their
cps
III loci supported the hypothesis that the operon is transcribed as a single polycistronic message. The translated
cps
III sequences were compared to those of the
S. agalactiae cps
Ia locus, and the primary difference between the operons was found to reside in

cps
III
H

, the putative CPS polymerase gene. Expression of

cps
III
H

in a type Ia strain resulted in suppression of CPS Ia synthesis and in production of a CPS which reacted with type III-specific polyclonal antibody. Likewise, expression of the putative type Ia polymerase gene in a type III strain reduced synthesis of type III CPS with production of a type Ia immunoreactive capsule. Based on the similar structures of the oligosaccharide repeating units of the type Ia and III capsules, our observations demonstrated that

cps
Ia
H

and

cps
III
H

encoded the type Ia and III CPS polymerases, respectively. Additionally, these findings suggested that a single gene can confer serotype specificity in organisms that produce complex polysaccharides.
Microarray-Based Identification of a Novel
Streptococcus pneumoniae
Regulon Controlled by an Autoinduced Peptide
Authors: de Saizieu Antoine , Gardès Christophe , Flint Nicholas , Wagner Christian , Kamber Markus , Mitchell Timothy J. , Keck Wolfgang , Amrein Kurt E. , Lange Roland
Published: July 2002

We have identified in the
Streptococcus pneumoniae
genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (
blpAB
) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of
blpC
, a small gene located downstream of
blpAB
, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the
blp
genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended −10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.
Domain Structure of
Salmonella
FlhB, a Flagellar Export Component Responsible for Substrate Specificity Switching
Authors: Minamino Tohru , Macnab Robert M.
Published: July 2002

We have investigated the properties of the cytoplasmic domain (FlhB
C
) of the 383-amino-acid
Salmonella
membrane protein FlhB, a component of the type III flagellar export apparatus. FlhB, along with the hook-length control protein FliK, mediates the switching of export specificity from rod- and hook-type substrates to filament-type substrates during flagellar morphogenesis. Wild-type FlhB
C
was unstable (half-life, ca. 5 min), being specifically cleaved at Pro-270 into two polypeptides, FlhB
CN
and FlhB
CC
, which retained the ability to interact with each other after cleavage. Full-length wild-type FlhB was also subject to cleavage. Coproduction of the cleavage products, FlhB
ΔCC
(i.e., the N-terminal transmembrane domain FlhB
TM
plus FlhB
CN
) and FlhB
CC
, resulted in restoration of both motility and flagellar protein export to an
flhB
mutant host, indicating that the two polypeptides were capable of productive association. Mutant FlhB proteins that can undergo switching of substrate specificity even in the absence of FliK were much more resistant to cleavage (half-lives, 20 to 60 min). The cleavage products of wild-type FlhB
C
, existing as a FlhB
CN
–FlhB
CC
complex on an affinity blot membrane, bound the rod- and hook-type substrate FlgD more strongly than the filament-type substrate FliC. In contrast, the intact form of FlhB
C
(mutant or wild type) or the FlhB
CC
polypeptide alone bound FlgD and FliC to about the same extent. FlhB
CN
by itself did not bind substrates appreciably. We propose that FlhB
C
has two substrate specificity states and that a conformational change, mediated by the interaction between FlhB
CN
and FlhB
CC
, is responsible for the specificity switching process. FliK itself is an export substrate; its binding properties for FlhB
C
resemble those of FlgD and do not provide any evidence for a physical interaction beyond that of the export process.
Recognition of Overlapping Nucleotides by AraC and the Sigma Subunit of RNA Polymerase
Authors: Dhiman Anjali , Schleif Robert
Published: July 2002

The
Escherichia coli
promoter

p
BAD

, under the control of the AraC protein, drives the expression of mRNA encoding the AraB, AraA, and AraD gene products of the arabinose operon. The binding site of AraC at

p
BAD

overlaps the RNA polymerase −35 recognition region by 4 bases, leaving 2 bases of the region not contacted by AraC. This overlap raises the question of whether AraC substitutes for the sigma subunit of RNA polymerase in recognition of the −35 region or whether both AraC and sigma make important contacts with the DNA in the −35 region. If sigma does not contact DNA near the −35 region,

p
BAD

activity should be independent of the identity of the bases in the hexamer region that are not contacted by AraC. We have examined this issue in the

p
BAD

promoter and in a second promoter where the AraC binding site overlaps the −35 region by only 2 bases. In both cases promoter activity is sensitive to changes in bases not contacted by AraC, showing that despite the overlap, sigma does read DNA in the −35 region. Since sigma and AraC are thus closely positioned at

p
BAD

, it is possible that AraC and sigma contact one another during transcription initiation. DNA migration retardation assays, however, showed that there exists only a slight degree of DNA binding cooperativity between AraC and sigma, thus suggesting either that the normal interactions between AraC and sigma are weak or that the presence of the entire RNA polymerase is necessary for significant interaction.
Plasmid-Located Pathogenicity Determinants of
Serratia entomophila
, the Causal Agent of Amber Disease of Grass Grub, Show Similarity to the Insecticidal Toxins of
Photorhabdus luminescens
Authors: Hurst Mark R. H. , Glare Travis R. , Jackson Trevor A. , Ronson Clive W.
Published: July 2002

Serratia entomophila
and
Serratia proteamaculans
cause amber disease in the grass grub
Costelytra zealandica
(Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. Larval disease symptoms include cessation of feeding, clearance of the gut, amber coloration, and eventual death. A 115-kb plasmid, pADAP, identified in
S. entomophila
is required for disease causation and, when introduced into
Escherichia coli
, enables that organism to cause amber disease. A 23-kb fragment of pADAP that conferred disease-causing ability on
E. coli
and a pADAP-cured strain of
S. entomophila
was isolated. Using insertion mutagenesis, the pathogenicity determinants were mapped to a 17-kb region of the clone. Sequence analysis of the 17-kb region showed that the predicted products of three of the open reading frames (
sepA
,
sepB
, and
sepC
) showed significant sequence similarity to components of the insecticidal toxin produced by the bacterium
Photorhabdus luminescens
. Transposon insertions in
sepA
,
sepB
, or
sepC
completely abolished both gut clearance and cessation of feeding on the 23-kb clone; when recombined back into pADAP, they abolished gut clearance but not cessation of feeding. These results suggest that SepA, SepB, and SepC together are sufficient for amber disease causation by
S. entomophila
and that another locus also able to exert a cessation-of-feeding effect is encoded elsewhere on pADAP.
The PspA Protein of
Escherichia coli
Is a Negative Regulator of ς
54
-Dependent Transcription
Authors: Dworkin Jonathan , Jovanovic Goran , Model Peter
Published: July 2002

In
Eubacteria
, expression of genes transcribed by an RNA polymerase holoenzyme containing the alternate sigma factor ς
54
is positively regulated by proteins belonging to the family of enhancer-binding proteins (EBPs). These proteins bind to upstream activation sequences and are required for the initiation of transcription at the ς
54
-dependent promoters. They are typically inactive until modified in their N-terminal regulatory domain either by specific phosphorylation or by the binding of a small effector molecule. EBPs lacking this domain, such as the PspF activator of the ς
54
-dependent
pspA
promoter, are constitutively active. We describe here the in vivo and in vitro properties of the PspA protein of
Escherichia coli
, which negatively regulates expression of the
pspA
promoter without binding DNA directly.
Genetics of Swarming Motility inSalmonella entericaSerovar Typhimurium: Critical Role for Lipopolysaccharide
Authors: Toguchi Adam , Siano Michael , Burkart Mark , Harshey Rasika M.
Published: July 2002

Salmonella entericaserovar Typhimurium can differentiate into hyperflagellated swarmer cells on agar of an appropriate consistency (0.5 to 0.8%), allowing efficient colonization of the growth surface. Flagella are essential for this form of motility. In order to identify genes involved in swarming, we carried out extensive transposon mutagenesis of serovar Typhimurium, screening for those that had functional flagella yet were unable to swarm. A majority of these mutants were defective in lipopolysaccharide (LPS) synthesis, a large number were defective in chemotaxis, and some had defects in putative two-component signaling components. While the latter two classes were defective in swarmer cell differentiation, representative LPS mutants were not and could be rescued for swarming by external addition of a biosurfactant. A mutation inwaaG(LPS core modification) secreted copious amounts of slime and showed a precocious swarming phenotype. We suggest that the O antigen improves surface “wettability” required for swarm colony expansion, that the LPS core could play a role in slime generation, and that multiple two-component systems cooperate to promote swarmer cell differentiation. The failure to identify specific swarming signals such as amino acids, pH changes, oxygen, iron starvation, increased viscosity, flagellar rotation, or autoinducers leads us to consider a model in which the external slime is itself both the signal and the milieu for swarming motility. The model explains the cell density dependence of the swarming phenomenon.
Multicomponent Transcriptional Regulation at the Complex Promoter of the Exopolysaccharide I Biosynthetic Operon of
Ralstonia solanacearum
Authors: Garg Ram P. , Huang Jianzhong , Yindeeyoungyeon Wandee , Denny Timothy P. , Schell Mark A.
Published: July 2002

High-level transcription of
eps
, an operon encoding biosynthesis of an exopolysaccharide virulence factor of the phytopathogen
Ralstonia
(
Pseudomonas
)
solanacearum
, requires the products of at least seven regulatory genes (
phcA
,
phcB
,
xpsR
,
vsrA-vsrD
, and
vsrB-vsrC
), which are organized in three converging signal transduction cascades. Because
xpsR
and the
vsrB-vsrC
two-component system are the most downstream cascade components required for activation of
eps
, we explored how these components control transcription from the
eps
promoter (P

eps

). Deletion and PCR mutagenesis identified an upstream region of P

eps

(nucleotides −82 to −62) that is critical for transcription activation by VsrB-VsrC and XpsR and also is required for negative control of P

eps

by the putative
eps
regulator EpsR. Using PCR mutagenesis we generated the
vsrC1
allele that encodes a response regulator that constitutively activates P

eps

in the absence of its cognate sensor, VsrB. However, activation of P

eps

by
vsrC1
still required
xpsR
. Unexpectedly, the amino acid substitution conferring the constitutive phenotype on VsrC1 is 12 residues from its C terminus, outside the known functional domains of response regulators. Finally, a modified DNase I footprinting method was used to demonstrate specific binding of both VsrC1 and VsrC to the −72 to −62 upstream region of P

eps

.
Functional Analysis of PvdS, an Iron Starvation Sigma Factor of
Pseudomonas aeruginosa
Authors: Leoni Livia , Orsi Nicola , de Lorenzo Victor , Visca Paolo
Published: July 2002

In
Pseudomonas aeruginosa
, iron modulates gene expression through a cascade of negative and positive regulatory proteins. The master regulator Fur is involved in iron-dependent repression of several genes. One of these genes,
pvdS
, was predicted to encode a putative sigma factor responsible for the transcription of a subset of genes of the Fur regulon. PvdS appears to belong to a structurally and functionally distinct subgroup of the extracytoplasmic function family of alternative sigma factors. Members of this subgroup, also including PbrA from
Pseudomonas fluorescens
, PfrI and PupI from
Pseudomonas putida
, and FecI from
Escherichia coli
, are controlled by the Fur repressor, and they activate transcription of genes for the biosynthesis or the uptake of siderophores. Evidence is provided that the PvdS protein of
P. aeruginosa
is endowed with biochemical properties of eubacterial sigma factors, as it spontaneously forms 1:1 complexes with the core fraction of RNA polymerase (RNAP, α
2
ββ′ subunits), thereby promoting in vitro binding of the PvdS-RNAP holoenzyme to the promoter region of the
pvdA
gene. These functional features of PvdS are consistent with the presence of structural domains predicted to be involved in core RNAP binding, promoter recognition, and open complex formation. The activity of pyoverdin biosynthetic (
pvd
) promoters was significantly lower in
E. coli
overexpressing the multicopy
pvdS
gene than in wild-type
P. aeruginosa
PAO1 carrying the single gene copy, and
pvd
::
lacZ
transcriptional fusions were silent in both
pfrI
(the
pvdS
homologue) and
pfrA
(a positive regulator of pseudobactin biosynthetic genes) mutants of
P. putida
WCS358, while they are expressed at PAO1 levels in wild-type WCS358. Moreover, the PvdS-RNAP holoenzyme purified from
E. coli
lacked the ability to generate in vitro transcripts from the
pvdA
promoter. These observations suggest that at least one additional positive regulator could be required for full activity of the PvdS-dependent transcription complex both in vivo and in vitro. This is consistent with the presence of a putative activator binding site (the iron starvation box) at variable distance from the transcription initiation sites of promoters controlled by the iron starvation sigma factors PvdS, PfrI, and PbrA of fluorescent pseudomonads.
CelE, a Multidomain Cellulase from
Clostridium cellulolyticum
: a Key Enzyme in the Cellulosome?
Authors: Gaudin Christian , Belaich Anne , Champ Stéphanie , Belaich Jean-Pierre
Published: July 2002

CelE, one of the three major proteins of the cellulosome of
Clostridium cellulolyticum
, was characterized. The amino acid sequence of the protein deduced from
celE
DNA sequence led us to the supposition that CelE is a three-domain protein. Recombinant CelE and a truncated form deleted of the putative cellulose binding domain (CBD) were obtained. Deletion of the CBD induces a total loss of activity. Exhibiting rather low levels of activity on soluble, amorphous, and crystalline celluloses, CelE is more active on
p
-nitrophenyl–cellobiose than the other cellulases from this organism characterized to date. The main product of its action on Avicel is cellobiose (more than 90% of the soluble sugars released), and its attack on carboxymethyl cellulose is accompanied by a relatively small decrease in viscosity. All of these features suggest that CelE is a cellobiohydrolase which has retained a certain capacity for random attack mode. We measured saccharification of Avicel and bacterial microcrystalline cellulose by associations of CelE with four other cellulases from
C. cellulolyticum
and found that CelE acts synergistically with all tested enzymes. The positive influence of CelE activity on the activities of other cellulosomal enzymes may explain its relative abundance in the cellulosome.
Genome-Wide Transcriptional Profiling of the
Escherichia coli
Responses to Superoxide Stress and Sodium Salicylate
Authors: Pomposiello Pablo J. , Bennik Marjon H. J. , Demple Bruce
Published: July 2002

Escherichia coli
responds to oxidative stress by activating sets of coregulated genes that help the cell to maintain homeostasis. Identified previously by genetic and biochemical approaches, the
soxRS
system mediates the induction of 18 of these redox-inducible genes (including the
soxS
gene itself). An overlapping set of genes is activated by an assortment of structurally unrelated molecules with antibiotic activities; many genes in this response are controlled by the
marRAB
system. The activation of either the
soxRS
or the
marRAB
system results in enhanced resistance to both superoxide-generating agents and multiple antibiotics. In order to probe the extent of these regulatory networks, we have measured whole-genome transcriptional profiles of the
E. coli
response to the superoxide-generating agent paraquat (PQ), an inducer of the
soxRS
system, and to the weak acid salt sodium salicylate (NaSal), an inducer of the
marRA
system. A total of 112 genes was modulated in response to PQ, while 134 genes were modulated in response to NaSal. We have also obtained transcriptional profiles of the SoxS and MarA regulons in the absence of global stress, in order to establish the regulatory hierarchies within the global responses. Several previously unrelated genes were shown to be under SoxS or MarA control. The genetic responses to both environmental insults revealed several common themes, including the activation of genes coding for functions that replenish reducing potential; regulate iron transport and storage; and participate in sugar and amino acid transport, detoxification, protein modification, osmotic protection, and peptidoglycan synthesis. A large number of PQ- and NaSal-responsive genes have no known function, suggesting that many adaptive metabolic changes that ensue after stress remain uncharacterized.
Design of a Protein-Targeting System for Lactic Acid Bacteria
Authors: Dieye Y. , Usai S. , Clier F. , Gruss A. , Piard J.-C.
Published: July 2002

We designed an expression and export system that enabled the targeting of a reporter protein (the staphylococcal nuclease Nuc) to specific locations inLactococcus lactiscells, i.e., cytoplasm, cell wall, or medium. Optimization of protein secretion and of protein cell wall anchoring was performed withL.lactiscells by modifying the signals located at the N and C termini, respectively, of the reporter protein. Efficient translocation of precursor (∼95%) is obtained using the signal peptide from the lactococcal Usp45 protein and provided that the mature protein is fused to overall anionic amino acids at its N terminus; those residues prevented interactions of Nuc with the cell envelope. Nuc could be covalently anchored to the peptidoglycan by using the cell wall anchor motif of theStreptococcus pyogenesM6 protein. However, the anchoring step proved to not be totally efficient inL. lactis, as considerable amounts of protein remained membrane associated. Our results may suggest that the defect is due to limiting sortase in the cell. The optimized expression and export vectors also allowed secretion and cell wall anchoring of Nuc in food-fermenting and commensal strains ofLactobacillus. In all strains tested, both secreted and cell wall-anchored Nuc was enzymatically active, suggesting proper enzyme folding in the different locations. These results provide the first report of a targeting system in lactic acid bacteria in which the final location of a protein is controlled and biological activity is maintained.
Treponema pallidum
3-Phosphoglycerate Mutase Is a Heat-Labile Enzyme That May Limit the Maximum Growth Temperature for the Spirochete
Authors: Benoit Stéphane , Posey James E. , Chenoweth Matthew R. , Gherardini Frank C.
Published: July 2002

In the causative agent of syphilis,
Treponema pallidum
, the gene encoding 3-phosphoglycerate mutase,
gpm
, is part of a six-gene operon (
tro
operon) that is regulated by the Mn-dependent repressor TroR. Since substrate-level phosphorylation via the Embden-Meyerhof pathway is the principal way to generate ATP in
T. pallidum
and Gpm is a key enzyme in this pathway, Mn could exert a regulatory effect on central metabolism in this bacterium. To study this,
T. pallidum gpm
was cloned, Gpm was purified from
Escherichia coli
, and antiserum against the recombinant protein was raised. Immunoblots indicated that Gpm was expressed in freshly extracted infective
T. pallidum
. Enzyme assays indicated that Gpm did not require Mn
2+
while 2,3-diphosphoglycerate (DPG) was required for maximum activity. Consistent with these observations, Mn did not copurify with Gpm. The purified Gpm was stable for more than 4 h at 25°C, retained only 50% activity after incubation for 20 min at 34°C or 10 min at 37°C, and was completely inactive after 10 min at 42°C. The temperature effect was attenuated when 1 mM DPG was added to the assay mixture. The recombinant Gpm from pSLB2 complemented
E. coli
strain PL225 (
gpm
) and restored growth on minimal glucose medium in a temperature-dependent manner. Increasing the temperature of cultures of
E. coli
PL225 harboring pSLB2 from 34 to 42°C resulted in a 7- to 11-h period in which no growth occurred (compared to wild-type
E. coli
). These data suggest that biochemical properties of Gpm could be one contributing factor to the heat sensitivity of
T. pallidum
.
Transduction by φBB-1, a Bacteriophage of
Borrelia burgdorferi
Authors: Eggers Christian H. , Kimmel Betsy J. , Bono James L. , Elias Abdallah F. , Rosa Patricia , Samuels D. Scott
Published: July 2002

We previously described a bacteriophage of the Lyme disease agent
Borrelia burgdorferi
designated φBB-1. This phage packages the host complement of the 32-kb circular plasmids (cp32s), a group of homologous molecules found throughout the genus
Borrelia
. To demonstrate the ability of φBB-1 to package and transduce DNA, a kanamycin resistance cassette was inserted into a cloned fragment of phage DNA, and the resulting construct was transformed into
B. burgdorferi
CA-11.2A cells. The
kan
cassette recombined into a resident cp32 and was stably maintained. The cp32 containing the
kan
cassette was packaged by φBB-1 released from this
B. burgdorferi
strain. φBB-1 has been used to transduce this antibiotic resistance marker into naive CA-11.2A cells, as well as two other strains of
B. burgdorferi
. This is the first direct evidence of a mechanism for lateral gene transfer in
B. burgdorferi
.
Cytological Evidence for Association of the Ends of the Linear Chromosome in
Streptomyces coelicolor
Authors: Yang Melody C. , Losick Richard
Published: July 2002

The chromosome of the filamentous bacterium
Streptomyces coelicolor
is linear, but the genetic map is circular. We present cytological evidence based on the use of fluorescence in situ hybridization showing that the ends of the chromosome frequently colocalize, in agreement with the idea that the ends are held together, effectively forming a circular chromosome. These observations provide a possible explanation for how a linear bacterial chromosome can exhibit a circular genetic map.
Specificity and Genetic Polymorphism of the
Bacillus
Competence Quorum-Sensing System
Authors: Tortosa P. , Logsdon L. , Kraigher B. , Itoh Y. , Mandic-Mulec I. , Dubnau D.
Published: July 2002

A quorum-sensing mechanism involving the pheromone ComX and the ComP-ComA two-component system controls natural competence in
Bacillus subtilis
. ComX is expressed as a cytoplasmic inactive precursor that is released into the extracellular medium as a cleaved, modified decapeptide. This process requires the product of
comQ
. In the presence of ComX, the membrane-localized ComP histidine kinase activates the response regulator ComA. We compared the sequences of the quorum-sensing genes from four closely related bacilli, and we report extensive genetic polymorphism extending through
comQ
,
comX
, and the 5′ two-thirds of
comP
. This part of ComP encodes the membrane-localized and linker domains of the sensor protein. We also determined the sequences of the
comX
genes of four additional wild-type bacilli and tested the in vivo activities of all eight pheromones on isogenic strains containing four different ComP receptor proteins. A striking pattern of specificity was discovered, providing strong evidence that the pheromone contacts ComP directly. Furthermore, we show that coexpression of
comQ
and
comX
in
Escherichia coli
leads to the production of active pheromone in the medium, demonstrating that
comQ
is the only dedicated protein required for the processing, modification, and release of active competence pheromone. Some of the implications of these findings for the evolution and the mechanism of the quorum-sensing system are discussed.
Characterization ofgrvA, an Antivirulence Gene on the Gifsy-2 Phage inSalmonella entericaSerovar Typhimurium
Authors: Ho Theresa D. , Slauch James M.
Published: July 2002

The lambdoid phage Gifsy-2 contributes significantly toSalmonella entericaserovar Typhimurium virulence. The phage carries the periplasmic superoxide dismutase gene,sodCI, and other unidentified virulence factors. We have characterized the genegrvA, a single open reading frame inserted in the opposite orientation in the tail operon of the Gifsy-2 phage. Contrary to what is observed with classic virulence genes,grvAnull mutants were more virulent than wild type as measured by intraperitoneal competition assays in mice. We have termed this effect antivirulence. Wild-typegrvAin single copy complemented this phenotype. However,grvA+on a multicopy plasmid also conferred the antivirulence phenotype. Neither agrvAnull mutation nor thegrvA+plasmid conferred a growth advantage or disadvantage in laboratory media. The antivirulence phenotype conferred by thegrvAnull mutation and thegrvA+plasmid required wild-typesodCIbut was independent of other virulence factors encoded on Gifsy-2. These results suggest that in a wild-type situation, GrvA decreases the pathogenicity of serovar Typhimurium in the host, most likely by affecting resistance to toxic oxygen species. These virulence phenotypes were independent of functional Gifsy-2 phage production. Our data suggest that the contribution of Gifsy-2 is a complicated sum of both positive virulence factors such assodCIand antivirulence factors such asgrvA.
Escherichia coli
Minicell Membranes Are Enriched in Cardiolipin
Authors: Koppelman Cecile-Marie , Den Blaauwen Tanneke , Duursma Marc C. , Heeren Ron M. A. , Nanninga Nanne
Published: July 2002

The phospholipid composition of
Escherichia coli
minicells has been studied as a model for the cell division site. Minicells appeared to be enriched in cardiolipin at the expense of phosphatidylglycerol. Mass spectrometry showed no differences between the gross acyl chain compositions of minicells and wild-type cells.
Pseudomonas aeruginosa
PAO1 Kills
Caenorhabditis elegans
by Cyanide Poisoning
Authors: Gallagher Larry A. , Manoil Colin
Published: July 2002

In this report we describe experiments to investigate a simple virulence model in which
Pseudomonas aeruginosa
PAO1 rapidly paralyzes and kills the nematode
Caenorhabditis elegans
. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by
P. aeruginosa
that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (
hcnC
) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if
hcnC
mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (
egl-9
) resistant to
P. aeruginosa
was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of
P. aeruginosa
and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.
Deletion of
lolB
, Encoding an Outer Membrane Lipoprotein, Is Lethal for
Escherichia coli
and Causes Accumulation of Lipoprotein Localization Intermediates in the Periplasm
Authors: Tanaka Kimie , Matsuyama Shin-Ichi , Tokuda Hajime
Published: July 2002

Outer membrane lipoproteins of
Escherichia coli
are released from the inner membrane upon the formation of a complex with a periplasmic chaperone, LolA, followed by localization to the outer membrane. In vitro biochemical analyses revealed that the localization of lipoproteins to the outer membrane generally requires an outer membrane lipoprotein, LolB, and occurs via transient formation of a LolB-lipoprotein complex. On the other hand, a mutant carrying the chromosomal
lolB
gene under the control of the
lac
promoter-operator grew normally in the absence of LolB induction if the mutant did not possess the major outer membrane lipoprotein Lpp, suggesting that LolB is only important for the localization of Lpp in vivo. To examine the in vivo function of LolB, we constructed a chromosomal
lolB
null mutant harboring a temperature-sensitive helper plasmid carrying the
lolB
gene. At a nonpermissive temperature, depletion of the LolB protein due to loss of the
lolB
gene caused cessation of growth and a decrease in the number of viable cells irrespective of the presence or absence of Lpp. LolB-depleted cells accumulated the LolA-lipoprotein complex in the periplasm and the mature form of lipoproteins in the inner membrane. Taken together, these results indicate that LolB is the first example of an essential lipoprotein for
E. coli
and that its depletion inhibits the upstream reactions of lipoprotein trafficking.
Alkyl Hydroperoxide Reductase Is the Primary Scavenger of Endogenous Hydrogen Peroxide in
Escherichia coli
Authors: Seaver Lauren Costa , Imlay James A.
Published: July 2002

Hydrogen peroxide is generated during aerobic metabolism and is capable of damaging critical biomolecules. However, mutants of
Escherichia coli
that are devoid of catalase typically exhibit no adverse phenotypes during growth in aerobic media. We discovered that catalase mutants retain the ability to rapidly scavenge H
2
O
2
whether it is formed internally or provided exogenously. Analysis of candidate genes revealed that the residual activity is due to alkyl hydroperoxide reductase (Ahp). Mutants that lack both Ahp and catalase could not scavenge H
2
O
2
. These mutants excreted substantial amounts of H
2
O
2
, and they grew poorly in air. Ahp is kinetically a more efficient scavenger of trace H
2
O
2
than is catalase and therefore is likely to be the primary scavenger of endogenous H
2
O
2
. Accordingly, mutants that lack Ahp accumulated sufficient hydrogen peroxide to induce the OxyR regulon, whereas the OxyR regulon remained off in catalase mutants. Catalase still has an important role in wild-type cells, because the activity of Ahp is saturated at a low (10
−5
M) concentration of H
2
O
2
. In contrast, catalase has a high
K

m

, and it therefore becomes the predominant scavenger when H
2
O
2
concentrations are high. This arrangement is reasonable because the cell cannot provide enough NADH for Ahp to rapidly degrade large amounts of H
2
O
2
. In sum,
E. coli
does indeed generate substantial H
2
O
2
, but damage is averted by the scavenging activity of Ahp.
Cellulosomal Scaffoldin-Like Proteins from
Ruminococcus flavefaciens
Authors: Ding Shi-You , Rincon Marco T. , Lamed Raphael , Martin Jennifer C. , McCrae Sheila I. , Aurilia Vincenzo , Shoham Yuval , Bayer Edward A. , Flint Harry J.
Published: July 2002

Two tandem cellulosome-associated genes were identified in the cellulolytic rumen bacterium,
Ruminococcus flavefaciens
. The deduced gene products represent multimodular scaffoldin-related proteins (termed ScaA and ScaB), both of which include several copies of explicit cellulosome signature sequences. The
scaB
gene was completely sequenced, and its upstream neighbor
scaA
was partially sequenced. The sequenced portion of
scaA
contains repeating cohesin modules and a C-terminal dockerin domain. ScaB contains seven relatively divergent cohesin modules, two extremely long T-rich linkers, and a C-terminal domain of unknown function. Collectively, the cohesins of ScaA and ScaB are phylogenetically distinct from the previously described type I and type II cohesins, and we propose that they define a new group, which we designated here type III cohesins. Selected modules from both genes were overexpressed in
Escherichia coli
, and the recombinant proteins were used as probes in affinity-blotting experiments. The results strongly indicate that ScaA serves as a cellulosomal scaffoldin-like protein for several
R. flavefaciens
enzymes. The data are supported by the direct interaction of a recombinant ScaA cohesin with an expressed dockerin-containing enzyme construct from the same bacterium. The evidence also demonstrates that the ScaA dockerin binds to a specialized cohesin(s) on ScaB, suggesting that ScaB may act as an anchoring protein, linked either directly or indirectly to the bacterial cell surface. This study is the first direct demonstration in a cellulolytic rumen bacterium of a cellulosome system, mediated by distinctive cohesin-dockerin interactions.
Hfq Is Necessary for Regulation by the Untranslated RNA DsrA
Authors: Sledjeski Darren D. , Whitman Christina , Zhang Aixia
Published: July 2002

DsrA is an 85-nucleotide, untranslated RNA that has multiple regulatory activities at 30°C. These activities include the translational regulation of RpoS and H-NS, global transcriptional regulators inEscherichia coli. Hfq is anE. coliprotein necessary for the in vitro and in vivo replication of the RNA phage Qβ. Hfq also plays a role in the degradation of numerous RNA transcripts. Here we show that anhfqmutant strain is defective for DsrA-mediated regulation of bothrpoSandhns. The defect inrpoSexpression can be partially overcome by overexpression of DsrA. Hfq does not regulate the transcription of DsrA, and DsrA does not alter the accumulation of Hfq. However, in anhfqmutant, chromosome-expressed DsrA was unstable (half-life of 1 min) and truncated at the 3′ end. When expressed from a multicopy plasmid, DsrA was stable in both wild-type andhfqmutant strains, but it had only partial activity in thehfqmutant strain. Purified Hfq binds DsrA in vitro. These results suggest that Hfq acts as a protein cofactor for the regulatory activities of DsrA by either altering the structure of DsrA or forming an active RNA-protein complex.
DnaD Protein of
Bacillus subtilis
Interacts with DnaA, the Initiator Protein of Replication
Authors: Ishigo-oka Daisuke , Ogasawara Naotake , Moriya Shigeki
Published: July 2002

The yeast two-hybrid assay revealed that
Bacillus subtilis
DnaD, a possible component of the primosome and required for replication initiation, interacted with DnaA and DnaD itself. The mutant DnaD23 was incapable of interacting with DnaA but retained interaction with the wild-type DnaD. These results suggest that interaction between DnaD and DnaA is important for replication initiation.
Heteromeric Interactions among Nucleoid-Associated Bacterial Proteins: Localization of StpA-Stabilizing Regions in H-NS ofEscherichia coli
Authors: Johansson Jörgen , Eriksson Sven , Sondén Berit , Wai Sun Nyunt , Uhlin Bernt Eric
Published: July 2002

The nucleoid-associated proteins H-NS and StpA inEscherichia colibind DNA as oligomers and are implicated in gene regulatory systems. There is evidence for both homomeric and heteromeric H-NS–StpA complexes. The two proteins show differential turnover, and StpA was previously found to be subject to protease-mediated degradation by the Lon protease. We investigated which regions of the H-NS protein are able to prevent degradation of StpA. A set of truncated H-NS derivatives was tested for their ability to mediate StpA stability and to form heteromers in vitro. The data indicate that H-NS interacts with StpA at two regions and that the presence of at least one of the H-NS regions is necessary for StpA stability. Our results also suggest that a proteolytically stable form of StpA, StpAF21C, forms dimers, whereas wild-type StpA in the absence of H-NS predominantly forms tetramers or oligomers, which are more susceptible to proteolysis.
Characterization of the
sat
Operon in
Streptococcus mutans
: Evidence for a Role of Ffh in Acid Tolerance
Authors: Kremer Bas H. A. , van der Kraan Marieke , Crowley Paula J. , Hamilton Ian R. , Brady L. Jeannine , Bleiweis Arnold S.
Published: July 2002

An essential protein translocation pathway in
Escherichia coli
and
Bacillus subtilis
involves the signal recognition particle (SRP), of which the 54-kDa homolog (Ffh) is an essential component. In a previous study, we found that a transposon insertion in the
ylxM-ffh
intergenic region of the designated secretion and acid tolerance (
sat
) operon of
Streptococcus mutans
resulted in an acid-sensitive phenotype. In the present study, we further characterized this genomic region in
S. mutans
after construction of bonafide
sat
operon mutants and confirmed the role of the SRP pathway in acid resistance. Northern blot and primer extension analyses identified an acid-inducible promoter upstream of
ylxM
that was responsible for upregulating the coordinate expression of all five genes of the
sat
operon when cells were grown at acid pH. Two constitutive promoters, one immediately upstream of
satD
and one just 3′ to the acid-inducible promoter, were also identified. Except for Ffh, the functions of the
sat
operon gene products are unknown. SatC, SatD, and SatE have no homology to proteins with known functions, although YlxM may function as a transcriptional regulator linked to genes encoding SRP pathway proteins. Nonpolar mutations created in each of the five genes of the
sat
locus resulted in viable mutants. Most striking, however, was the finding that a mutation in
ffh
did not result in loss of cell viability, as is the case in all other microbial species in which this pathway has been described. This mutant also lacked immunologically detectable Ffh and was severely affected in resistance to acid. Complementation of the mutation resulted in restoration of acid tolerance and reappearance of cytoplasmic Ffh. These data provide evidence that the SRP pathway plays an important role in acid tolerance in
S. mutans.
Biogenesis of T Pili in
Agrobacterium tumefaciens
Requires Precise VirB2 Propilin Cleavage and Cyclization
Authors: Lai Erh-Min , Eisenbrandt Ralf , Kalkum Markus , Lanka Erich , Kado Clarence I.
Published: July 2002

VirB2 propilin is processed by the removal of a 47-amino-acid signal peptide to generate a 74-amino-acid peptide product in both
Escherichia coli
and
Agrobacterium tumefaciens
. The cleaved VirB2 protein is further cyclized to form the T pilin in
A. tumefaciens
but not in
E. coli
. Mutations in the signal peptidase cleavage sequence of VirB2 propilin cause the formation of aberrant T pilin and also severely attenuate virulence. No T pilus was observed in these mutants. The potential role of the exact VirB2 propilin cleavage and cyclization in T pilus biogenesis and virulence is discussed.
Regulation of theBacillus subtilis furandperRGenes by PerR: Not All Members of the PerR Regulon Are Peroxide Inducible
Authors: Fuangthong Mayuree , Herbig Andrew F. , Bsat Nada , Helmann John D.
Published: July 2002

PerR is a ferric uptake repressor (Fur) homolog that functions as the central regulator of the inducible peroxide stress response inBacillus subtilis. PerR has been previously demonstrated to regulate themrgA,katA,ahpCF,hemAXCDBL, andzosAgenes. We now demonstrate that PerR also mediates both the repression of its own gene and that offur. Whereas PerR-mediated repression of most target genes can be elicited by either manganese or iron, repression ofperRandfuris selective for manganese. Genetic studies indicate that repression of PerR regulon genes by either manganese or iron requires PerR and is generally independent of Fur. Indeed, in afurmutant, iron-mediated repression is enhanced. Unexpectedly, repression of thefurgene by manganese appears to require both PerR and Fur, but only PerR binds to thefurregulatory region in vitro. Thefurmutation appears to act indirectly by affecting cellular metal ion pools and thereby affecting PerR-mediated repression. While many components of theperRregulon are strongly induced by hydrogen peroxide, little, if any, induction offurandperRcould be demonstrated. Thus, not all components of the PerR regulon are components of the peroxide stimulon. We suggest that PerR exists in distinct metallated forms that differ in DNA target selectivity and in sensitivity to oxidation. This model is supported by the observation that the metal ion composition of the growth medium can greatly influence the transcriptional response of the various PerR regulon genes to hydrogen peroxide.
Catabolite Repression of
Escherichia coli
Biofilm Formation
Authors: Jackson Debra W. , Simecka Jerry W. , Romeo Tony
Published: July 2002

Biofilm formation was repressed by glucose in several species of
Enterobacteriaceae
. In
Escherichia coli
, this effect was mediated at least in part by cyclic AMP (cAMP)-cAMP receptor protein. A temporal role for cAMP in biofilm development was indicated by the finding that glucose addition after ∼24 h failed to repress and generally activated biofilm formation.