bluuebunny/crossref_metadata_2025_split huggingface dataset:
Posttranscriptional Control of Quorum-Sensing-Dependent Virulence Genes by DksA in
Pseudomonas aeruginosa
Authors: Jude Florence , Köhler Thilo , Branny Pavel , Perron Karl , Mayer Matthias P. , Comte Rachel , van Delden Christian 
Published: May 2003

ABSTRACT

Pseudomonas aeruginosa
controls the secretion of extracellular virulence factors, including rhamnolipids and LasB elastase, by the
las
and
rhl
quorum-sensing systems. Here, we mutated the
dksA
gene of
P. aeruginosa
by insertion of an Ω-Hg cassette. The mutant displayed growth rates similar to that of the wild type in rich medium but was impaired in growth in defined minimal medium. Production of rhamnolipids and LasB elastase by the
dksA
mutant was only 4 and 10%, respectively, of wild-type levels. These defects could be partially complemented by introduction of the plasmid-encoded
dksA
genes from
P. aeruginosa
or
Escherichia coli
. In the
dksA
mutant, the expression of
rhlI
was increased early during exponential growth, but expression of other quorum-sensing regulator genes—
lasR
,
lasI
, and
rhlR
—was not affected. Although the transcription of the
lasB
and
rhlAB
genes was comparable between the
dksA
mutant and the wild-type strain in peptone tryptic soy broth medium, we observed reduced translation of both genes in the
dksA
mutant. Similarly, we found that full translation of
lasB
and
rhlAB
genes in
E. coli
also requires the
dksA
gene. DksA is therefore a novel regulator involved in the posttranscriptional control of extracellular virulence factor production in
P. aeruginosa
.
Flagellar Phase Variation inSalmonella entericaIs Mediated by a Posttranscriptional Control Mechanism
Authors: Bonifield Heather R. , Hughes Kelly T. 
Published: May 2003

ABSTRACTSalmonella entericahas two antigenically distinct flagellin genes,fliCandfljB, that are alternatively expressed. ThefljAgene is cotranscribed withfljBand encodes a protein that has been characterized as a transcriptional repressor of the unlinkedfliCgene when FljB is expressed. In this study we report genetic evidence that FljA prevents the production of FliC protein through an interaction with the 5′-untranslated region of thefliCmRNA transcript. Studies with operon and gene fusions, Western analyses, and T2RNase protection assays were performed for strains with thefljBApromoter locked in either the on or the off orientation. β-Galactosidase assays offliCtranscriptional and translational fusions to thelacoperon demonstrated that while FljA inhibitsfliCtranscription fivefold in thefljBAONorientation, it has a 200-fold effect on bothfliCtranscription and translation, indicating that the FljA inhibitor might act at both the transcriptional and translational level. T2RNase protection assays also demonstrated a fivefold decrease infliCtranscript levels for cells locked in thefljBAONorientation compared to those in thefljBAOFForientation, and an eightfold decrease in FliC protein levels was observed by Western analysis. This reduction in FliC protein levels is greater than the decrease observed for the transcript. These results are consistent with a new model whereby FljA inhibits FliC expression by an attenuation or translational control mechanism.
Characterization of the Second LysR-Type Regulator in the Biphenyl-Catabolic Gene Cluster of
Pseudomonas pseudoalcaligenes
KF707
Authors: Watanabe Takahito , Fujihara Hidehiko , Furukawa Kensuke 
Published: May 2003

ABSTRACT

Pseudomonas pseudoalcaligenes
KF707 possesses a biphenyl-catabolic (
bph
) gene cluster consisting of
bphR1A1A2-
(
orf3
)
-bphA3A4BCX0X1X2X3D
. The
bphR1
(formerly
orf0
) gene product, which belongs to the GntR family, is a positive regulator for itself and
bphX0X1X2X3D
. Further analysis in this study revealed that a second regulator belonging to the LysR family (designated
bphR2
) is involved in the regulation of the
bph
genes in KF707. The
bphR2
gene was not located near the
bph
gene cluster, and its product (BphR2) exhibited a high level of similarity to NahR (the naphthalene- and salicylate-catabolic regulator belonging to the LysR family) in plasmid NAH7 of
Pseudomonas putida
. A strain containing a disrupted
bphR2
gene failed to grow on biphenyl as a sole source of carbon, and the BphD (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase) activity was significantly reduced compared to that of wild-type strain KF707. Furthermore, the same strain exhibited extremely low transcription of
bphR1
,
bphA1
,
bphC
,
bphX0
, and
bphD
. However, when the
bphR2
gene was provided in
trans
to the
bphR2
-disrupted strain, the transcription level of these genes was restored. These results indicate that
bphR2
regulates the
bph
genes positively as a second regulator together with BphR1.
Suppression of Temperature-Sensitive Chromosome Replication of anEscherichia coli dnaX(Ts) Mutant by Reduction of Initiation Efficiency
Authors: Blinkova Alexandra , Hermandson Mary Jo , Walker James R. 
Published: May 2003

ABSTRACTTemperature sensitivity of DNA polymerization and growth of adnaX(Ts) mutant is suppressible at 39 to 40°C by mutations in the initiator gene,dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20°C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression ofdnaX(Ts).OnednaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explaineddnaA(Cs,Sx) suppression ofdnaX, which codes for both the τ and γ subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of thednaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40°C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, adnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type anddnaX(Ts) strains growing at 39 and 34°C. These effects were shown to result specifically from the Cs,Sx mutations, because thednaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing thednaA(Cs,Sx)dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39°C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39°C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38°C. Fourth, thednaX(Ts) mutation can be suppressed also by introduction oforiCmutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing adnaA(Cos) allele, which causes overinitiation, into thednaX(Ts) mutant exacerbated its temperature sensitivity.
Escherichia coli
O157:H7 Shiga Toxin-Encoding Bacteriophages: Integrations, Excisions, Truncations, and Evolutionary Implications
Authors: Shaikh Nurmohammad , Tarr Phillip I. 
Published: May 2003

ABSTRACT

As it descended from
Escherichia coli
O55:H7, Shiga toxin (Stx)-producing
E. coli
(STEC) O157:H7 is believed to have acquired, in sequence, a bacteriophage encoding Stx2 and another encoding Stx1. Between these events, sorbitol-fermenting
E. coli
O157:H
−
presumably diverged from this clade. We employed PCR and sequence analyses to investigate sites of bacteriophage integration into the chromosome, using evolutionarily informative STEC to trace the sequence of acquisition of elements encoding Stx. Contrary to expectations from the two currently sequenced strains, truncated bacteriophages occupy
yehV
in almost all
E. coli
O157:H7 strains that lack

stx
1

(

stx
1

-negative strains). Two truncated variants were determined to contain either GTT or TGACTGTT sequence, in lieu of 20,214 or 18,895 bp, respectively, of the bacteriophage central region. A single-nucleotide polymorphism in the latter variant suggests that recombination in that element extended beyond the inserted octamer. An

stx
2

bacteriophage usually occupies
wrbA
in

stx
1

+
/

stx
2

+
E. coli
O157:H7, but
wrbA
is unexpectedly unoccupied in most

stx
1

-negative/

stx
2

+
E. coli
O157:H7 strains, the presumed progenitors of

stx
1

+
/

stx
2

+
E. coli
O157:H7. Trimethoprim-sulfamethoxazole promotes the excision of all, and ciprofloxacin and fosfomycin significantly promote the excision of a subset of complete and truncated
stx
bacteriophages from the
E. coli
O157:H7 strains tested; bile salts usually attenuate excision. These data demonstrate the unexpected diversity of the chromosomal architecture of
E. coli
O157:H7 (with novel truncated bacteriophages and multiple

stx
2

bacteriophage insertion sites), suggest that

stx
1

acquisition might be a multistep process, and compel the consideration of multiple exogenous factors, including antibiotics and bile, when chromosome stability is examined.
Molecular Characterization of Inulosucrase fromLeuconostoc citreum: a Fructosyltransferase within a Glucosyltransferase
Authors: Olivares-Illana Vanesa , López-Munguía Agustín , Olvera Clarita 
Published: May 2003

ABSTRACTThe gene coding for inulosucrase inLeuconostoc citreumCW28,islA, was cloned, sequenced, and expressed inEscherichia coli. The recombinant enzyme catalyzed inulin synthesis from sucrose like the wild-type enzyme. Inulosucrase presents an unusual structure: its N-terminal region is similar to the variable region of glucosyltransferases, its catalytic domain is similar to fructosyltransferases from various microorganisms, and its C-terminal domain presents similarity to the glucan binding domain from alternansucrase, a glucosyltransferase fromLeuconostoc mesenteroidesNRRL B-1355. From sequence comparison, it was found that this fructosyltransferase is a natural chimeric enzyme resulting from the substitution of the catalytic domain of alternansucrase by a fructosyltransferase. Two different forms of theislAgene truncated in the C-terminal glucan binding domain were successfully expressed inE. coliand retained their ability to synthesize inulin but lost thermal stability. This is the first report of an inulosucrase bearing structural features of both glucosyltransferases and fructosyltransferases.
Role of the
Enterococcus faecalis
GelE Protease in Determination of Cellular Chain Length, Supernatant Pheromone Levels, and Degradation of Fibrin and Misfolded Surface Proteins
Authors: Waters Christopher M. , Antiporta Michelle H. , Murray Barbara E. , Dunny Gary M. 
Published: May 2003

ABSTRACT

Gelatinase (GelE), a secreted Zn-metalloprotease of
Enterococcus faecalis
, has been implicated as a virulence factor by both epidemiological data and animal model studies. Expression of
gelE
is induced at a high cell density by the
fsr
quorum-sensing system. In the present study, GelE was shown to be responsible for the instability of a number of Asc10 (aggregation substance) mutant proteins, implying that GelE functions to clear the bacterial cell surface of misfolded proteins. Disruption of GelE production led to increased cell chain length of
E. faecalis
, from a typical diplococcus morphology to chains of 5 to 10 cells. This function of GelE was also exhibited when the protein was expressed in
Streptococcus pyogenes
. GelE-expressing
E. faecalis
strains were more autolytic, suggesting that GelE affects chain length through activation of an autolysin. GelE was also essential for degradation of polymerized fibrin. GelE expression reduced the titer of cCF10, the peptide pheromone that induces conjugation of pCF10, and pCF10 had increased conjugation into non-GelE-expressing strains. These new functions attributed to GelE suggest that it acts to increase the dissemination of
E. faecalis
in high-density environments.
Variation between Pathogenic Serovars within
Salmonella
Pathogenicity Islands
Authors: Amavisit P. , Lightfoot D. , Browning G. F. , Markham P. F. 
Published: May 2003

ABSTRACT

Although four of the five
Salmonella
pathogenicity islands (SPIs) have been characterized in detail for
Salmonella enterica
serovar Typhimurium, and the fifth has been characterized for
Salmonella enterica
serovar Dublin, there have been limited studies to examine them in detail in a range of pathogenic serovars of
S. enterica
. The aim of this study was to examine these regions, shown to be crucial in virulence, in pathogenic serovars to identify any major deletions or insertions that may explain variation in virulence and provide further understanding of the elements involved in the evolution of these regions. Multiple strains of each of the 13 serovars were compared by Southern blot hybridization using a series of probes that together encompassed the full length of all five SPIs. With the exception of serovar Typhimurium, all strains of the same serovar were identical in all five SPIs. Those serovars that differed from serovar Typhimurium in SPI-1 to SPI-4 and from serovar Dublin in SPI-5 were examined in more detail in the variant regions by PCR, and restriction endonuclease digestion and/or DNA sequencing. While most variation in hybridization patterns was attributable to loss or gain of single restriction endonuclease cleavage sites, three regions, in SPI-1, SPI-3, and SPI-5, had differences due to major insertions or deletions. In SPI-1 the
avrA
gene was replaced by a 200-base fragment in three serovars, as reported previously. In SPI-5, two serovars had acquired an insertion with similarity to the
pagJ
and
pagK
genes between
pipC
and
pipD.
In SPI-3 the genes
sugR
and
rhuM
were deleted in most serovars and in some were replaced by sequences that were very similar to either the
Escherichia coli
fimbrial operon, flanked by two distinct insertion sequence elements, or to the
E. coli
retron phage ΦR73. The distribution of these differences suggests that there have been a number of relatively recent horizontal transfers of genes into
S. enterica
and that in some cases the same event has occurred in multiple lineages of
S. enterica.
Thus, it seems that insertion sequences and retron phages are likely to be involved in continuing evolution of the pathogenicity islands of pathogenic
Salmonella
serovars.
Electron Microscopic Analysis of Membrane Assemblies Formed by the Bacterial Chemotaxis Receptor Tsr
Authors: Weis Robert M. , Hirai Teruhisa , Chalah Anas , Kessel Martin , Peters Peter J. , Subramaniam Sriram 
Published: May 2003

ABSTRACT

The serine receptor (Tsr) from
Escherichia coli
is representative of a large family of transmembrane receptor proteins that mediate bacterial chemotaxis by influencing cell motility through signal transduction pathways. Tsr and other chemotaxis receptors form patches in the inner membrane that are often localized at the poles of the bacteria. In an effort to understand the structural constraints that dictate the packing of receptors in the plane of the membrane, we have used electron microscopy to examine ordered assemblies of Tsr in membrane extracts isolated from cells engineered to overproduce the receptor. Three types of assemblies were observed: ring-like “micelles” with a radial arrangement of receptor subunits, two-dimensional crystalline arrays with approximate hexagonal symmetry, and “zippers,” which are receptor bilayers that result from the antiparallel interdigitation of cytoplasmic domains. The registration among Tsr molecules in the micelle and zipper assemblies was sufficient for identification of the receptor domains and for determination of their contributions to the total receptor length. The overall result of this analysis is compatible with an atomic model of the receptor dimer that was constructed primarily from the X-ray crystal structures of the periplasmic and cytoplasmic domains. Significantly, the micelle and zipper structures were also observed in fixed, cryosectioned cells expressing the Tsr receptor at high abundance, suggesting that the modes of Tsr assembly found in vitro are relevant to the situation in the cell.
Antisense RNA Downregulation of Coenzyme A Transferase Combined with Alcohol-Aldehyde Dehydrogenase Overexpression Leads to Predominantly Alcohologenic
Clostridium acetobutylicum
Fermentations
Authors: Tummala Seshu B. , Junne Stefan G. , Papoutsakis Eleftherios T. 
Published: May 2003

ABSTRACT

Plasmid pAADB1 for the overexpression of the alcohol-aldehyde dehydrogenase (
aad
) gene and downregulation of the coenzyme A transferase (CoAT) using antisense RNA (asRNA) against
ctfB
(the second CoAT gene on the polycistronic
aad-ctfA-ctfB
message) was used in order to increase the butanol/acetone ratio of
Clostridium acetobutylicum
ATCC 824 fermentations. Acetone and butanol levels were drastically reduced in 824(pCTFB1AS) (expresses only an asRNA against
ctfB
) compared to 824(pSOS95del) (plasmid control). Compared to strain 824(pCTFB1AS), 824(pAADB1) fermentations exhibited two profound differences. First, butanol levels were ca. 2.8-fold higher in 824(pAADB1) and restored back to plasmid control levels, thus supporting the hypothesis that asRNA downregulation of
ctfB
leads to degradation of the whole
aad-ctfA-ctfB
transcript. Second, ethanol titers in 824(pAADB1) were ca. 23-fold higher and the highest (ca. 200 mM) ever reported in
C. acetobutylicum
. Western blot analysis confirmed that CoAT was downregulated in 824(pAADB1) at nearly the same levels as in strain 824(pCTFB1AS). Butyrate depletion in 824(pAADB1) fermentations suggested that butyryl-CoA was limiting butanol production in 824(pAADB1). This was confirmed by exogenously adding butyric acid to 824(pAADB1) fermentations to increase the butanol/ethanol ratio. DNA microarray analysis showed that
aad
overexpression profoundly affects the large-scale transcriptional program of the cells. Several classes of genes were differentially expressed [strain 824(pAADB1) versus strain 824(pCTFB1AS)], including genes of the stress response, sporulation, and chemotaxis. The expression patterns of the CoAT genes (
ctfA
and
ctfB
) and
aad
were consistent with the overexpression of
aad
and asRNA downregulation of
ctfB
.
Oxidative Stress in
Synechococcus
sp
.
Strain PCC 7942: Various Mechanisms for H
2
O
2
Detoxification with Different Physiological Roles
Authors: Perelman Alexander , Uzan Avraham , Hacohen Dalia , Schwarz Rakefet 
Published: May 2003

ABSTRACT

This study focuses on the mechanisms for hydrogen peroxide detoxification in
Synechococcus
sp. strain PCC 7942. To gain better understanding of the role of different routes of hydrogen peroxide detoxification, we inactivated
tplA
(thioredoxin-peroxidase-like), which we recently identified. In addition, we inactivated the gene encoding catalase-peroxidase and examined the ability to detoxify H
2
O
2
and to survive oxidative stress in both of the single mutants and in the double mutant. Surprisingly, we observed that the double mutant survived H
2
O
2
concentrations that the single catalase-peroxidase mutant could not tolerate. This phenotype correlated with an increased ability of the double mutant to detoxify externally added H
2
O
2
compared to the catalase-peroxidase mutant. Therefore, our studies suggested the existence of a hydrogen peroxide detoxification activity in addition to catalase-peroxidase and thioredoxin-peroxidase. The rate of detoxification of externally added H
2
O
2
was similar in the wild-type and the TplA mutant cells, suggesting that, under these conditions, catalase-peroxidase activity was essential for this process and TplA was dispensable. However, during excessive radiation, conditions under which the cell might experience oxidative stress, TplA appears to be essential for growth, and cells lacking it cannot compete with the wild-type strain. Overall, these studies suggested different physiological roles for various cellular hydrogen peroxide detoxification mechanisms in
Synechococcus
sp. strain PCC 7942.
Trigger Factor-Mediated Prolyl Isomerization Influences Maturation of the
Streptococcus pyogenes
Cysteine Protease
Authors: Lyon William R. , Caparon Michael G. 
Published: May 2003

ABSTRACT

Trigger factor, a ribosome-associated chaperone and peptidyl-prolyl
cis
-
trans
isomerase (PPIase), is essential for the secretion and maturation of the cysteine protease of the pathogenic gram-positive bacterium
Streptococcus pyogenes
. In the absence of trigger factor, the nascent protease polypeptide is not targeted to the secretory pathway. Some partial-function mutations restore targeting. However, the secreted protease does not efficiently mature into an enzymatically active form, suggesting that trigger factor has an additional role in protease biogenesis. Here, we show that, while not required for targeting, the PPIase activity of trigger factor is essential for maturation of the protease following its secretion from the bacterial cell. Site-specific mutations introduced into
ropA
, the gene which encodes trigger factor in
S. pyogenes
, produced mutant proteins deficient in PPIase activity. When these mutant alleles were used to replace the wild-type gene on the streptococcal chromosome, analysis of protease biogenesis revealed that, although the protease was secreted normally, it did not efficiently mature to an active form. Furthermore, mutation of a single proline residue in the protease prodomain suppressed the requirement for PPIase activity, suggesting that this residue is the target of trigger factor. These data support a model in which trigger factor-mediated prolyl isomerization influences the conformation of the prodomain, which in turn directs the protease into one of several alternative folding pathways.
Involvement of Cyclic AMP (cAMP) and cAMP Receptor Protein in Anaerobic Respiration of
Shewanella oneidensis
Authors: Saffarini Daad A. , Schultz Ryan , Beliaev Alex 
Published: May 2003

ABSTRACT

Shewanella oneidensis
is a metal reducer that can use several terminal electron acceptors for anaerobic respiration, including fumarate, nitrate, dimethyl sulfoxide (DMSO), trimethylamine
N
-oxide (TMAO), nitrite, and insoluble iron and manganese oxides. Two
S. oneidensis
mutants, SR-558 and SR-559, with Tn
5
insertions in
crp
, were isolated and analyzed. Both mutants were deficient in Fe(III) and Mn(IV) reduction. They were also deficient in anaerobic growth with, and reduction of, nitrate, fumarate, and DMSO. Although nitrite reductase activity was not affected by the
crp
mutation, the mutants failed to grow with nitrite as a terminal electron acceptor. This growth deficiency may be due to the observed loss of cytochromes
c
in the mutants. In contrast, TMAO reduction and growth were not affected by loss of cyclic AMP (cAMP) receptor protein (CRP). Fumarate and Fe(III) reductase activities were induced in rich medium by the addition of cAMP to aerobically growing wild-type
S. oneidensis
. These results indicate that CRP and cAMP play a role in the regulation of anaerobic respiration, in addition to their known roles in catabolite repression and carbon source utilization in other bacteria.