bluuebunny/crossref_metadata_2025_split huggingface dataset:
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

ABSTRACT

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

ABSTRACTWe 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACT

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

ABSTRACTDsrA 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

ABSTRACT

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

ABSTRACTThe 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

ABSTRACT

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

ABSTRACT

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.