bluuebunny/crossref_metadata_2025 huggingface dataset:
Biofilm Formation in a Hydrodynamic Environment by Novel FimH Variants and Ramifications for Virulence
Authors: Schembri Mark A. , Klemm Per
Published: July 2002

Type 1 fimbriae are surface-located adhesion organelles of
Escherichia coli
that are directly associated with virulence of the urinary tract. They mediate
d
-mannose-sensitive binding to different host surfaces by way of the minor fimbrial component FimH. Naturally occurring variants of FimH that bind strongly to terminally exposed monomannose residues have been associated with a pathogenicity-adaptive phenotype that enhances
E. coli
colonization of extraintestinal locations such as the urinary tract. The FimH adhesin also promotes biofilm formation in a mannose-inhibitable manner on abiotic surfaces under static growth conditions. In this study, we used random mutagenesis combined with a novel selection-enrichment technique to specifically identify mutations in the FimH adhesin that confer on
E. coli
the ability to form biofilms under hydrodynamic flow (HDF) conditions. We identified three FimH variants from our mutant library that could mediate an HDF biofilm formation phenotype to various degrees. This phenotype was induced by the cumulative effect of multiple changes throughout the receptor-binding region of the protein. Two of the HDF biofilm-forming FimH variants were insensitive to mannose inhibition and represent novel phenotypes not previously identified in naturally occurring isolates. Characterization of our enriched clones revealed some similarities to amino acid alterations that occur in urinary tract infection (UTI) strains. Subsequent screening of a selection of UTI FimH variants demonstrated that they too could promote biofilm formation on abiotic surfaces under HDF conditions. Interestingly, the same correlation was not observed for commensal FimH variants. FimH is a multifaceted protein prone to rapid microevolution. In addition to its previously documented roles in adherence and invasion, we have now demonstrated its function in biofilm formation on abiotic surfaces subjected to HDF conditions. The study indicates that UTI FimH variants possess adaptations that enhance biofilm formation and suggests a novel role for FimH in UTIs associated with medical implants such as catheters.
Marrow-Derived CD40-Positive Cells Are Required for Mice To ClearCryptosporidium parvumInfection
Authors: Hayward Anthony R. , Cosyns Mary , Jones Michelle , Ponnuraj Esther M.
Published: July 2002

To clear aCryptosporidium parvuminfection, mice need CD4+T cells, major histocompatibility complex class II, and an intact CD40-CD154 signaling pathway. CD40 is constitutively expressed on marrow-derived cells such as dendritic cells and B lymphocytes and is induced by gamma interferon (IFN-γ) on most somatic cells. To determine whether the CD40 needed to clear aC. parvuminfection has to be on marrow-derived mononuclear cells or on the epithelial cells that normally harbor the parasite, we transplanted CD40−/−mice with CD40+/−bone marrow and then infected them withC. parvum. These chimeras cleared theC. parvuminfection, while CD40+/−controls transplanted with CD40−/−marrow cells remained infected. CD40 expression on marrow-derived cells therefore suffices for aC. parvuminfection to be cleared, while CD40 expression on intestinal epithelial cells is not sufficient. There was no difference between the acquisition of CD69 and CD154 by mesenteric lymph node T cells ofC. parvum-infected animals with intact or disrupted CD40-CD154 pathways. CD4 T cells entered the intestinal laminae propriae ofC. parvum-infected animals whether or not the CD40 genes of these recipients were intact. These results suggest that, for aC. parvuminfection to be cleared, CD40 is not necessary for T-cell activation but may instead contribute to an effector pathway of marrow-derived cells.
Lactoferrin Peptide Increases the Survival of
Candida albicans
- Inoculated Mice by Upregulating Neutrophil and Macrophage Functions, Especially in Combination with Amphotericin B and Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Tanida Toyohiro , Rao Fu , Hamada Toshihiro , Ueta Eisaku , Osaki Tokio
Published: July 2002

To develop a new strategy to control candidiasis, we examined in vivo the anticandidal effects of a synthetic lactoferrin peptide, FKCRRWQWRM (peptide 2) and the peptide that mimics it, FKARRWQWRM (peptide 2′). Although all mice that underwent intraperitoneal injection of 5 × 10
8
Candida
cells with or without peptide 2′ died within 8 or 7 days, respectively, the survival times of mice treated with 5 to 100 μg of intravenous peptide 2 per day for 5 days after the candidal inoculation were prolonged between 8.4 ± 2.9 and 22.4 ± 3.6 days, depending on the dose of peptide 2. The prolongation of survival by peptide 2 was also observed in mice that were infected with 1.0 × 10
9
Candida albicans
cells (3.2 ± 1.3 days in control mice versus 8.2 ± 2.4 days in the mice injected with 10 μg of peptide 2 per day). In the high-dose inoculation, a combination of peptide 2 (10 μg/day) with amphotericin B (0.1 μg/day) and granulocyte-macrophage colony-stimulating factor (GM-CSF) (0.1 μg/day) brought prolonged survival. With a combination of these agents, 60% of the mice were alive for more than 22 days. Correspondingly, peptide 2 activated phagocytes inducing inducible NO synthase and the expression of p47
phox
and p67
phox
, and peptide 2 increased phagocyte
Candida
-killing activities up to 1.5-fold of the control levels upregulating the generation of superoxide, lactoferrin, and defensin from neutrophils and macrophages. These findings indicated that the anticandidal effects of peptide 2 depend not only on the direct
Candida
cell growth-inhibitory activity, but also on the phagocytes' upregulatory activity, and that combinations of peptide 2 with GM-CSF and antifungal drugs will help in the development of new strategies for control of candidiasis.
Invasive Candidiasis Stimulates Hepatocyte and Monocyte Production of Active Transforming Growth Factor β
Authors: Letterio John J. , Lehrnbecher Thomas , Pollack Greg , Walsh Thomas J. , Chanock Stephen J.
Published: July 2002

Candida albicansis an opportunistic fungal pathogen and a major cause of morbidity and mortality in patients with compromised immune function. The cytokine response to tissue invasion byC. albicanscan influence the differentiation and function of lymphocytes and other mononuclear cells that are critical components of the host response. While the production of transforming growth factor β (TGF-β) has been documented in mice infected withC. albicansand is known to suppress phagocyte function, the cellular source and role of this cytokine in the pathogenesis of systemic candidiasis are not well understood. We have investigated the source of production of TGF-β by immunohistochemical studies in tissue samples from patients with an uncommon complication of lymphoreticular malignancy, chronic disseminated candidiasis (CDC), and from a neutropenic-rabbit model of CDC. Liver biopsy specimens from patients with documented CDC demonstrated intense staining for extracellular matrix-associated TGF-β1 within inflammatory granulomas, as well as staining for TGF-β1 and TGF-β3 within adjacent hepatocytes. These results correlate with the immunolocalization of TGF-β observed in livers of infected neutropenic rabbits, using a neutralizing antibody that recognizes the mature TGF-β protein. Human peripheral blood monocytes incubated withC. albicansin vitro release large amounts of biologically active TGF-β1. The data demonstrate that local production of active TGF-βs by hepatocytes and by infected mononuclear cells is a component of the response toC. albicansinfection that most probably contributes to disease progression in the immunocompromised host.
Characterization of the Murine T-Lymphocyte Response toSalmonella entericaSerovar Typhimurium Infection
Authors: Mittrücker Hans-Willi , Köhler Anne , Kaufmann Stefan H. E.
Published: July 2002

Infection of mice withSalmonella entericaserotype Typhimurium induces a strong Th1 cell response that is central for the control of infection. We infected mice of a resistant background with a virulent strain ofS. entericaserovar Typhimurium and analyzed the kinetics and magnitude of the T-cell response. After infection, the majority of CD4+and CD8+splenocytes acquired an activated phenotype, as indicated by expression levels of CD44 and CD62L. In addition, after 3 to 4 weeks of infection, more than 20% of the CD4+and more than 30% of the CD8+T cells produced gamma interferon (IFN-γ) in response to short-term polyclonal stimulation. In contrast, we detected only a moderate (two- to threefold) expansion of both T-cell populations, and BrdU incorporation revealed that there was either no or only a limited increase in the in vivo proliferation of CD4+and CD8+T cells, respectively. Our results indicate that although an unexpectedly large population of both CD4+and CD8+T cells is activated and acquires the potential to secrete IFN-γ, this activation is not paralleled by substantial expansion of these T-cell populations.
Leishmania majorPromastigotes Inhibit Dendritic Cell Motility In Vitro
Authors: Jebbari Heather , Stagg Andrew J. , Davidson Robert N. , Knight Stella C.
Published: July 2002

Using an in vitro transwell migration assay, we have demonstrated that products secreted byLeishmania majorpromastigotes inhibit the motility of dendritic cells (DC) by up to 93%. Inhibition was dose dependent and reversible. By inhibiting DC migration in vivo,L. majormay therefore subvert DC from their potentially protective role during leishmaniasis.
Improved Immunogenicity and Efficacy of the Recombinant 19-Kilodalton Merozoite Surface Protein 1 by the Addition of Oligodeoxynucleotide and Aluminum Hydroxide Gel in a Murine Malaria Vaccine Model
Authors: Near Karen A. , Stowers Anthony W. , Jankovic Dragana , Kaslow David C.
Published: July 2002

Vaccination of mice with yeast-secretedPlasmodium yoelii-derived 19-kilodalton merozoite surface protein 1 (yMSP119) has been shown to afford protection from challenge with a lethal strain ofP. yoelii. Sterile immunity can be achieved when MSP119is emulsified in Freund adjuvant but not when it is adsorbed to aluminum hydroxide gel (alum). Because complete Freund adjuvant is not an acceptable adjuvant for use in humans, alternative adjuvants must be identified for formulating MSP119as a vaccine for use in humans. To determine whether oligodeoxynucleotides with CpG motifs (ODN), reported to be a powerful new class of adjuvants, could enhance the immunogenicity of yMSP119, C57BL/6 mice were vaccinated either with yMSP119formulated with Freund adjuvant, with alum, or with ODN plus alum and challenged intravenously withP. yoelii17XL asexual blood-stage parasites. Adsorption of immunogen and adjuvant to alum was optimized by adjusting buffer (phosphate versus acetate) and pH. We found that the adjuvant combination of ODN plus alum with yMSP119, injected intraperitoneally (i.p.), increased immunoglobulin G (IgG) yMSP119-specific antibody production 12-fold over Freund adjuvant given i.p., 3-fold over Freund adjuvant given subcutaneously (s.c.), 300-fold over alum given i.p., and 48-fold over alum given s.c. The predominant antibody isotype in the group receiving alum-ODN-yMSP119was IgG1. Increased antibody levels correlated to protection from a challenge withP. yoelii17XL. Supernatant cytokine levels of gamma interferon in yMSP119-stimulated splenocytes were dramatically elevated in the alum-ODN-yMSP119group. Interleukin-10 (IL-10) levels were also elevated; however, no IL-5 was detected. The cytokine profile, as well as the predominant IgG1 antibody isotype, suggests the protective immune response was a mixed Th1/Th2 response.
Characterization of the
spv
Locus in
Salmonella enterica
Serovar Arizona
Authors: Libby Stephen J. , Lesnick Marc , Hasegawa Patricia , Kurth Michael , Belcher Christopher , Fierer Joshua , Guiney Donald G.
Published: July 2002

Salmonella enterica
serovar Arizona (
S. enterica
subspecies IIIa) is a common
Salmonella
isolate from reptiles and can cause serious systemic disease in humans. The
spv
virulence locus, found on large plasmids in
Salmonella
subspecies I serovars associated with severe infections, was confirmed to be located on the chromosome of serovar Arizona. Sequence analysis revealed that the serovar Arizona
spv
locus contains homologues of
spvRABC
but lacks the
spvD
gene and contains a frameshift in
spvA
, resulting in a different C terminus. The SpvR protein functions as a transcriptional activator for the
spvA
promoter, and SpvB and SpvC are highly conserved. The analysis supports the proposal that the chromosomal
spv
sequence more closely corresponds to the ancestral locus acquired during evolution of
S. enterica
, with plasmid acquisition of
spv
genes in the subspecies I strains involving addition of
spvD
and polymorphisms in
spvA
.
A Study of the CopF Repressor of Plasmid pAMβ1 by Phage Display
Authors: d'Alençon Emmanuelle , Ehrlich S. Dusko
Published: July 2002

We studied DNA binding of a transcriptional repressor, CopF, displayed on a filamentous phage. Mutagenesis of a putative helix-turn-helix motif of CopF and of certain bases of the operator abolished the protein-DNA interaction, establishing the elements involved in CopF function and showing that phage display can be used to study repressor proteins.
Morphogenesis, Adhesive Properties, and Antifungal Resistance Depend on the Pmt6 Protein Mannosyltransferase in the Fungal Pathogen
Candida albicans
Authors: Timpel Claudia , Zink Sigrid , Strahl-Bolsinger Sabine , Schröppel Klaus , Ernst Joachim
Published: July 2002

Protein mannosyltransferases (Pmt proteins) initiate O glycosylation of secreted proteins in fungi. We have characterized
PMT6
, which encodes the second Pmt protein of the fungal pathogen
Candida albicans
. The residues of Pmt6p are 21 and 42% identical to those of
C. albicans
Pmt1p and
S. cerevisiae
Pmt6p, respectively. Mutants lacking one or two
PMT6
alleles grow normally and contain normal Pmt enzymatic activities in cell extracts but show phenotypes including a partial block of hyphal formation (dimorphism) and a supersensitivity to hygromycin B. The morphogenetic defect can be suppressed by overproduction of known components of signaling pathways, including Cek1p, Cph1p, Tpk2p, and Efg1p, suggesting a specific Pmt6p target protein upstream of these components. Mutants lacking both
PMT1
and
PMT6
are viable and show
pmt1
mutant phenotypes and an additional sensitivity to the iron chelator ethylenediamine-di(
o
-hydroxyphenylacetic acid). The lack of Pmt6p significantly reduces adherence to endothelial cells and overall virulence in a mouse model of systemic infection. The results suggest that Pmt6p regulates a more narrow subclass of proteins in
C. albicans
than Pmt1p, including secreted proteins responsible for morphogenesis and antifungal sensitivities.
Characterization of
devH
, a Gene Encoding a Putative DNA Binding Protein Required for Heterocyst Function in
Anabaena
sp. Strain PCC 7120
Authors: Hebbar Pratibha B. , Curtis Stephanie E.
Published: July 2002

The
devH
gene was identified in a screen for
Anabaena
sp. strain PCC 7120 sequences whose transcripts increase in abundance during a heterocyst development time course. The product of
devH
contains a helix-turn-helix motif similar to the DNA binding domain of members of the cyclic AMP receptor protein family, and the protein is most closely related to the cyanobacterial transcriptional activator NtcA.
devH
transcripts are barely detectable in vegetative cells and are induced approximately fivefold after nitrogen starvation. This induction is absent in the two developmental mutants
hetR
and
ntcA
. The gene is expressed as monocistronic transcripts with multiple 5′ termini, and the ∼500-bp region 5′ to
devH
was shown to have promoter activity in vivo. The
devH
gene was insertionally inactivated by the integration of plasmid sequences within the open reading frame. Nitrogen starvation of the
devH
mutant induces heterocysts of wild-type morphology, but the mutant is inviable in the absence of fixed nitrogen and unable to reduce acetylene aerobically.
Constitutive Expression of a Transcription Termination Factor by a Repressed Prophage: Promoters for Transcribing the Phage HK022
nun
Gene
Authors: King Rodney A. , Madsen Peter L. , Weisberg Robert A.
Published: July 2002

Lysogens of phage HK022 are resistant to infection by phage λ. Lambda resistance is caused by the action of the HK022 Nun protein, which prematurely terminates early λ transcripts. We report here that transcription of the
nun
gene initiates at a constitutive prophage promoter,
P
Nun
, located just upstream of the protein coding sequence. The 5′ end of the transcript was determined by primer extension analysis of RNA isolated from HK022 lysogens or RNA made in vitro by transcribing a template containing the promoter with purified
Escherichia coli
RNA polymerase. Inactivation of
P
Nun
by mutation greatly reduced Nun activity and Nun antigen in an HK022 lysogen. However, a low level of residual activity was detected, suggesting that a secondary promoter also contributes to
nun
expression. We found one possible secondary promoter,
P
Nun
′, just upstream of
P
Nun
. Neither promoter is likely to increase the expression of other phage genes in a lysogen because their transcripts should be terminated downstream of
nun
. We estimate that HK022 lysogens in stationary phase contain several hundred molecules of Nun per cell and that cells in exponential phase probably contain fewer.
Role of the
Escherichia coli
Nucleotide Excision Repair Proteins in DNA Replication
Authors: Moolenaar Geri F. , Moorman Celine , Goosen Nora
Published: July 2002

DNA polymerase I (PolI) functions both in nucleotide excision repair (NER) and in the processing of Okazaki fragments that are generated on the lagging strand during DNA replication.
Escherichia coli
cells completely lacking the PolI enzyme are viable as long as they are grown on minimal medium. Here we show that viability is fully dependent on the presence of functional UvrA, UvrB, and UvrD (helicase II) proteins but does not require UvrC. In contrast, Δ
polA
cells grow even better when the
uvrC
gene has been deleted. Apparently UvrA, UvrB, and UvrD are needed in a replication backup system that replaces the PolI function, and UvrC interferes with this alternative replication pathway. With specific mutants of UvrC we could show that the inhibitory effect of this protein is related to its catalytic activity that on damaged DNA is responsible for the 3′ incision reaction. Specific mutants of UvrA and UvrB were also studied for their capacity to support the PolI-independent replication. Deletion of the UvrC-binding domain of UvrB resulted in a phenotype similar to that caused by deletion of the
uvrC
gene, showing that the inhibitory incision activity of UvrC is mediated via binding to UvrB. A mutation in the N-terminal zinc finger domain of UvrA does not affect NER in vivo or in vitro. The same mutation, however, does give inviability in combination with the Δ
polA
mutation. Apparently the N-terminal zinc-binding domain of UvrA has specifically evolved for a function outside DNA repair. A model for the function of the UvrA, UvrB, and UvrD proteins in the alternative replication pathway is discussed.
Characterization of the
ccpA
Gene of
Enterococcus faecalis
: Identification of Starvation-Inducible Proteins Regulated by CcpA
Authors: Leboeuf Céline , Leblanc Laurence , Auffray Yanick , Hartke Axel
Published: July 2002

Inactivation of
ccpA
in
Enterococcus faecalis
leads to reduction of the growth rate, derepression of the
galKETR
operon in the presence of a mixture of glucose and galactose, and reduction of transcription of
ldh
in the presence of glucose. Moreover, the
E. faecalis ccpA
gene fully complements a
Bacillus subtilis ccpA
mutant, arguing for similar functions of these two homologous proteins. Protein comparison on two-dimensional gels from the wild-type cells and the
ccpA
mutant cells revealed a pleiotropic effect of the mutation on gene expression. The HPr protein of the carbohydrate-phosphotransferase system was identified by microsequencing, and a modification of its phosphorylation state was observed between the wild-type and the mutant strains. Moreover, at least 16 polypeptides are overexpressed in the mutant, and 6 are repressed. Interestingly, 13 of the 16 polypeptides whose synthesis is enhanced in the mutant were also identified as glucose starvation proteins. The N-terminal amino acid sequences of four of them match sequences deduced from genes coding for
l
-serine dehydratase, dihydroxyacetone kinase (two genes), and a protein of unknown function from
Deinococcus radiodurans.
Deletion of the
min
Operon Results in Increased Thermosensitivity of an
ftsZ84
Mutant and Abnormal FtsZ Ring Assembly, Placement, and Disassembly
Authors: Yu Xuan-Chuan , Margolin William
Published: July 2002

To investigate the interaction between FtsZ and the Min system during cell division of
Escherichia coli
, we examined the effects of combining a well-known thermosensitive mutation of
ftsZ
,
ftsZ84
, with Δ
minCDE
, a deletion of the entire
min
locus. Because the Min system is thought to down-regulate Z-ring assembly, the prediction was that removing
minCDE
might at least partially suppress the thermosensitivity of
ftsZ84
, which can form colonies below 42°C but not at or above 42°C. Contrary to expectations, the double mutant was significantly more thermosensitive than the
ftsZ84
single mutant. When shifted to the new lower nonpermissive temperature, the double mutant formed long filaments mostly devoid of Z rings, suggesting a likely cause of the increased thermosensitivity. Interestingly, even at 22°C, many Z rings were missing in the double mutant, and the rings that were present were predominantly at the cell poles. Of these, a large number were present only at one pole. These cells exhibited a higher than expected incidence of polar divisions, with a bias toward the newest pole. Moreover, some cells exhibited dramatically elongated septa that stained for FtsZ, suggesting that the double mutant is defective in Z-ring disassembly, and providing a possible mechanism for the polar bias. Thermoresistant suppressors of the double mutant arose that had modestly increased levels of FtsZ84. These cells also exhibited elongated septa and, in addition, produced a high frequency of branched cells. A thermoresistant suppressor of the
ftsZ84
single mutant also synthesized more FtsZ84 and produced branched cells. The evidence from this study indicates that removing the Min system exposes and exacerbates the inherent defects of the FtsZ84 protein, resulting in clear septation phenotypes even at low growth temperatures. Increasing levels of FtsZ84 can suppress some, but not all, of these phenotypes.
Characterization of Alginate Lyase from
Pseudomonas syringae
pv. syringae
Authors: Preston Lori A. , Wong T. Y. , Bender Carol L. , Schiller Neal L.
Published: July 2002

The gene encoding alginate lyase (
algL
) in
Pseudomonas syringae
pv. syringae was cloned, sequenced, and overexpressed in
Escherichia coli
. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42°C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from
P. syringae
showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.
Transcriptional Control of the Hydrogen Cyanide Biosynthetic Genes
hcnABC
by the Anaerobic Regulator ANR and the Quorum-Sensing Regulators LasR and RhlR in
Pseudomonas aeruginosa
Authors: Pessi Gabriella , Haas Dieter
Published: July 2002

Virulence factors of
Pseudomonas aeruginosa
include hydrogen cyanide (HCN). This secondary metabolite is maximally produced at low oxygen tension and high cell densities during the transition from exponential to stationary growth phase. The
hcnABC
genes encoding HCN synthase were identified on a genomic fragment complementing an HCN-deficient mutant of
P. aeruginosa
PAO1. The
hcnA
promoter was found to be controlled by the FNR-like anaerobic regulator ANR and by the quorum-sensing regulators LasR and RhlR. Primer extension analysis revealed two transcription starts, T1 and T2, separated by 29 bp. Their function was confirmed by transcriptional
lacZ
fusions. The promoter sequence displayed an FNR/ANR box at −42.5 bp upstream of T2 and a
lux
box centered around −42.5 bp upstream of T1. Expression of the
hcn
genes was completely abolished when this
lux
box was deleted or inactivated by two point mutations in conserved nucleotides. The
lux
box was recognized by both LasR [activated by
N
-(oxododecanoyl)-homoserine lactone] and RhlR (activated by
N
-butanoyl-homoserine lactone), as shown by expression experiments performed in quorum-sensing-defective
P. aeruginosa
mutants and in the
N
-acyl-homoserine lactone-negative heterologous host
P. fluorescens
CHA0. A second, less conserved
lux
box lying 160 bp upstream of T1 seems to account for enhanced quorum-sensing-dependent expression. Without LasR and RhlR, ANR could not activate the
hcn
promoter. Together, these data indicate that expression of the
hcn
promoter from T1 can occur under quorum-sensing control alone. Enhanced expression from T2 appears to rely on a synergistic action between LasR, RhlR, and ANR.
Evolution of Drug Resistance in Experimental Populations of
Candida albicans
Authors: Cowen Leah E. , Sanglard Dominique , Calabrese David , Sirjusingh Caroline , Anderson James B. , Kohn Linda M.
Published: July 2002

Adaptation to inhibitory concentrations of the antifungal agent fluconazole was monitored in replicated experimental populations founded from a single, drug-sensitive cell of the yeast
Candida albicans
and reared over 330 generations. The concentration of fluconazole was maintained at twice the MIC in six populations; no fluconazole was added to another six populations. All six replicate populations grown with fluconazole adapted to the presence of drug as indicated by an increase in MIC; none of the six populations grown without fluconazole showed any change in MIC. In all populations evolved with drug, increased fluconazole resistance was accompanied by increased resistance to ketoconazole and itraconazole; these populations contained ergosterol in their cell membranes and were amphotericin sensitive. The increase in fluconazole MIC in the six populations evolved with drug followed different trajectories, and these populations achieved different levels of resistance, with distinct overexpression patterns of four genes involved in azole resistance: the ATP-binding cassette transporter genes,
CDR1
and
CDR2
; the gene encoding the target enzyme of the azoles in the ergosterol biosynthetic pathway,
ERG11
; and the major facilitator gene,
MDR1
. Selective sweeps in these populations were accompanied by additional genomic changes with no known relationship to drug resistance: loss of heterozygosity in two of the five marker genes assayed and alterations in DNA fingerprints and electrophoretic karyotypes. These results show that chance, in the form of mutations that confer an adaptive advantage, is a determinant in the evolution of azole drug resistance in experimental populations of
C. albicans.
Intra- and Interspecies Signaling between
Streptococcus salivarius
and
Streptococcus pyogenes
Mediated by SalA and SalA1 Lantibiotic Peptides
Authors: Upton M. , Tagg J. R. , Wescombe P. , Jenkinson H. F.
Published: July 2002

Streptococcus salivarius
20P3 produces a 22-amino-acid residue lantibiotic, designated salivaricin A (SalA), that inhibits the growth of a range of streptococci, including all strains of
Streptococcus pyogenes
. Lantibiotic production is associated with the
sal
genetic locus comprising
salA
, the lantibiotic structural gene;
salBCTX
genes encoding peptide modification and export machinery proteins; and
salYKR
genes encoding a putative immunity protein and two-component sensor-regulator system. Insertional inactivation of
salB
in
S. salivarius
20P3 resulted in abrogation of SalA peptide production, of immunity to SalA, and of
salA
transcription. Addition of exogenous SalA peptide to
salB
mutant cultures induced dose-dependent expression of
salA
mRNA (0.2 kb), demonstrating that SalA production was normally autoregulated. Inactivation of
salR
encoding the response regulator of the SalKR two-component system led to reduced production of, and immunity to, SalA. The
sal
genetic locus was also present in
S. pyogenes
SF370 (M type 1), but because of a deletion across the
salBCT
genes, the corresponding lantibiotic peptide, designated SalA1, was not produced. However, in
S. pyogenes
T11 (M type 4) the
sal
locus gene complement was apparently complete, and active SalA1 peptide was synthesized. Exogenously added SalA1 peptide from
S. pyogenes
T11 induced
salA1
transcription in
S. pyogenes
SF370 and in an isogenic
S. pyogenes
T11
salB
mutant and
salA
transcription in
S. salivarius
20P3
salB
. Thus, SalA and SalA1 are examples of streptococcal lantibiotics whose production is autoregulated. These peptides act as intra- and interspecies signaling molecules, modulating lantibiotic production and possibly influencing streptococcal population ecology in the oral cavity.
ISAfe1, an ISL3 Family Insertion Sequence fromAcidithiobacillus ferrooxidansATCC 19859
Authors: Holmes David S. , Zhao Hui-Ling , Levican Gloria , Ratouchniak Jeanine , Bonnefoy Violaine , Varela Patricia , Jedlicki Eugenia
Published: July 2002

A 1.3-kb insertion sequence, termed ISAfe1 (U66426), fromAcidithiobacillus ferrooxidansATCC 19859 is described. ISAfe1 exhibits the features of a typical bacterial insertion sequence. It has 26-bp, imperfectly matched, terminal inverted repeats and an open reading frame (ORF) that potentially encodes a transposase (TPase) of 404 amino acids (AAB07489) with significant similarity to members of the ISL3 family of insertion sequences. A potential ribosome-binding site and potential −10 and −35 promoter sites for the TPase ORF were identified, and a +1 transcriptional start site was detected experimentally. A potential outwardly directed −35 site was identified in the right inverted repeat of ISAfe1. A second ORF (ORF B), of unknown function, was found on the complementary strand with significant similarity to ORF 2 of ISAe1 fromRalstonia eutropha. Southern blot analyses demonstrated that ISAfe1-like elements can be found in multiple copies in a variety ofA. ferrooxidansstrains and that they exhibit transposition. A codon adaptation index (CAI) analysis of the TPase of ISAfe1 indicates that is has a CAI of 0.726 and can be considered well adapted to its host, suggesting that ISAfe1 might be an ancient resident ofA. ferrooxidans. Analysis of six of its target sites of insertion in the genome ofA. ferrooxidansATCC 19859 indicates a preference for 8-bp pseudopalindromic sequences, one of which resembles the termini of its inverted repeats. Evidence is presented here that is consistent with the possibility that ISAfe1 can promote both plasmid cointegrate formation and resolution inE. coli.
Novel Genes of the
sox
Gene Cluster, Mutagenesis of the Flavoprotein SoxF, and Evidence for a General Sulfur-Oxidizing System in
Paracoccus pantotrophus
GB17
Authors: Rother Dagmar , Henrich Hans-Jürgen , Quentmeier Armin , Bardischewsky Frank , Friedrich Cornelius G.
Published: July 2002

The novel genes
soxFGH
were identified, completing the
sox
gene cluster of
Paracoccus pantotrophus
coding for enzymes involved in lithotrophic sulfur oxidation. The periplasmic SoxF, SoxG, and SoxH proteins were induced by thiosulfate and purified to homogeneity from the soluble fraction.
soxF
coded for a protein of 420 amino acids with a signal peptide containing a twin-arginine motif. SoxF was 37% identical to the flavoprotein FccB of flavocytochrome
c
sulfide dehydrogenase of
Allochromatium vinosum
. The mature SoxF (42,832 Da) contained 0.74 mol of flavin adenine dinucleotide per mol.
soxG
coded for a novel protein of 303 amino acids with a signal peptide containing a twin-arginine motif. The mature SoxG (29,657 Da) contained two zinc binding motifs and 0.90 atom of zinc per subunit of the homodimer.
soxH
coded for a periplasmic protein of 317 amino acids with a double-arginine signal peptide. The mature SoxH (32,317 Da) contained two metal binding motifs and 0.29 atom of zinc and 0.20 atom of copper per subunit of the homodimer. SoxXA, SoxYZ, SoxB, and SoxCD (C. G. Friedrich, A. Quentmeier, F. Bardischewsky, D. Rother, R. Kraft, S. Kostka, and H. Prinz, J. Bacteriol. 182:4476–4487, 2000) reconstitute a system able to perform thiosulfate-, sulfite-, sulfur-, and hydrogen sulfide-dependent cytochrome
c
reduction, and this system is the first described for oxidizing different inorganic sulfur compounds. SoxF slightly inhibited the rate of hydrogen sulfide oxidation but not the rate of sulfite or thiosulfate oxidation. From use of a homogenote mutant with an in-frame deletion in
soxF
and complementation analysis, it was evident that the
soxFGH
gene products were not required for lithotrophic growth with thiosulfate.
Rhamnose Biosynthesis Pathway Supplies Precursors for Primary and Secondary Metabolism in
Saccharopolyspora spinosa
Authors: Madduri Krishnamurthy , Waldron Clive , Merlo Donald J.
Published: July 2002

Rhamnose is an essential component of the insect control agent spinosad. However, the genes coding for the four enzymes involved in rhamnose biosynthesis in
Saccharopolyspora spinosa
are located in three different regions of the genome, all unlinked to the cluster of other genes that are required for spinosyn biosynthesis. Disruption of any of the rhamnose genes resulted in mutants with highly fragmented mycelia that could survive only in media supplemented with an osmotic stabilizer. It appears that this single set of genes provides rhamnose for cell wall synthesis as well as for secondary metabolite production. Duplicating the first two genes of the pathway caused a significant improvement in the yield of spinosyn fermentation products.
Catalytic Function of an α/β Hydrolase Is Required for Energy Stress Activation of the ς
B
Transcription Factor in
Bacillus subtilis
Authors: Brody Margaret S. , Vijay Kamni , W. Price Chester
Published: July 2002

The general stress response of
Bacillus subtilis
is controlled by the ς
B
transcription factor, which is activated in response to diverse energy and environmental stresses. These two classes of stress are transmitted by separate signaling pathways which converge on the direct regulators of ς
B
, the RsbV anti-anti-ς factor and the RsbW anti-ς factor. The energy signaling branch involves the RsbP phosphatase, which dephosphorylates RsbV in order to trigger the general stress response. The
rsbP
structural gene lies downstream from
rsbQ
in a two-gene operon. Here we identify the RsbQ protein as a required positive regulator inferred to act in concert with the RsbP phosphatase. RsbQ bound RsbP in the yeast two-hybrid system, and a large in-frame deletion in
rsbQ
had the same phenotype as a null allele of
rsbP
—an inability to activate ς
B
in response to energy stress. Genetic complementation studies indicated that this phenotype was not due to a polar effect of the
rsbQ
alteration on
rsbP
. The predicted
rsbQ
product is a hydrolase or acyltransferase of the α/β fold superfamily, members of which catalyze a wide variety of reactions. Notably, substitutions in the presumed catalytic triad of RsbQ also abolished the energy stress response but had no detectable effect on RsbQ structure, synthesis, or stability. We conclude that the catalytic activity of RsbQ is an essential constituent of the energy stress signaling pathway.
The Staphylococcal QacR Multidrug Regulator Binds a Correctly Spaced Operator as a Pair of Dimers
Authors: Grkovic Steve , Brown Melissa H. , Schumacher Maria A. , Brennan Richard G. , Skurray Ronald A.
Published: July 2002

Expression of the
Staphylococcus aureus
plasmid-encoded QacA multidrug transporter is regulated by the divergently encoded QacR repressor protein. To circumvent the formation of disulfide-bonded degradation products, site-directed mutagenesis to replace the two cysteine residues in wild-type QacR was undertaken. Analysis of a resultant cysteineless QacR derivative indicated that it retained full DNA-binding activities in vivo and in vitro and continued to be fully proficient for the mediation of induction of
qacA
expression in response to a range of structurally dissimilar multidrug transporter substrates. The cysteineless QacR protein was used in cross-linking and dynamic light-scattering experiments to show that its native form was a dimer, whereas gel filtration indicated that four QacR molecules bound per DNA operator site. The addition of inducing compounds led to the dissociation of the four operator-bound QacR molecules from the DNA as dimers. Binding of QacR dimers to DNA was found to be dependent on the correct spacing of the operator half-sites. A revised model proposed for the regulation of
qacA
expression by QacR features the unusual characteristic of one dimer of the regulatory protein binding to each operator half-site by a process that does not appear to require the prior self-assembly of QacR into tetramers.
Complex Regulatory Network Controls Initial Adhesion and Biofilm Formation in
Escherichia coli
via Regulation of the
csgD
Gene
Authors: Prigent-Combaret Claire , Brombacher Eva , Vidal Olivier , Ambert Arnaud , Lejeune Philippe , Landini Paolo , Dorel Corinne
Published: July 2002

The
Escherichia coli
OmpR/EnvZ two-component regulatory system, which senses environmental osmolarity, also regulates biofilm formation. Up mutations in the
ompR
gene, such as the
ompR234
mutation, stimulate laboratory strains of
E. coli
to grow as a biofilm community rather than in a planktonic state. In this report, we show that the OmpR234 protein promotes biofilm formation by binding the
csgD
promoter region and stimulating its transcription. The
csgD
gene encodes the transcription regulator CsgD, which in turn activates transcription of the
csgBA
operon encoding curli, extracellular structures involved in bacterial adhesion. Consistent with the role of the
ompR
gene as part of an osmolarity-sensing regulatory system, we also show that the formation of biofilm by
E. coli
is inhibited by increasing osmolarity in the growth medium. The
ompR234
mutation counteracts adhesion inhibition by high medium osmolarity; we provide evidence that the
ompR234
mutation promotes biofilm formation by strongly increasing the initial adhesion of bacteria to an abiotic surface. This increase in initial adhesion is stationary phase dependent, but it is negatively regulated by the stationary-phase-specific sigma factor RpoS. We propose that this negative regulation takes place via
rpoS
-dependent transcription of the transcription regulator
cpxR
;
cpxR
-mediated repression of
csgB
and
csgD
promoters is also triggered by osmolarity and by curli overproduction, in a feedback regulation loop.
Introduction of a Carboxyl Group in the First Transmembrane Helix ofEscherichia coliF1FoATPase Subunitcand Cytoplasmic pH Regulation
Authors: Jones Phil C.
Published: July 2002

The multicopy subunitcof the H+-transporting F1FoATP synthase ofEscherichia colifolds across the membrane as a hairpin of two hydrophobic α helices. The subunits interact in a front-to-back fashion, forming an oligomeric ring with helix 1 packing in the interior and helix 2 at the periphery. A conserved carboxyl, Asp61inE. coli, centered in the second transmembrane helix is essential for H+transport. A second carboxylic acid in the first transmembrane helix is found at a position equivalent to Ile28in several bacteria, some the cause of serious infectious disease. This side chain has been predicted to pack proximal to the essential carboxyl in helix 2. It appears that in some of these bacteria the primary function of the enzyme is H+pumping for cytoplasmic pH regulation. In this study, Ile28was changed to Asp and Glu. Both mutants were functional. However, unlike the wild type, the mutants showed pH-dependent ATPase-coupled H+pumping and passive H+transport through Fo. The results indicate that the presence of a second carboxylate enables regulation of enzyme function in response to cytoplasmic pH and that the ion binding pocket is aqueous accessible. The presence of a single carboxyl at position 28, in mutants I28D/D61G and I28E/D61G, did not support growth on a succinate carbon source. However, I28E/D61G was functional in ATPase-coupled H+transport. This result indicates that the side chain at position 28 is part of the ion binding pocket.
Tn
5
Transposase with an Altered Specificity for Transposon Ends
Authors: Naumann Todd A. , Reznikoff William S.
Published: July 2002

Tn
5
is a composite bacterial transposon that encodes a protein, transposase (Tnp), required for movement of the transposon. The initial step in the transposition pathway involves specific binding of Tnp to 19-bp end recognition sequences. Tn
5
contains two different specific end sequences, termed outside end (OE) and inside end (IE). In
Escherichia coli,
IE is methylated by Dam methylase (IE
ME
). This methylation greatly inhibits recognition by Tnp and greatly reduces the ability of transposase to facilitate movement of IE defined transposons. Through use of a combinatorial random mutagenesis technique (DNA shuffling), we have isolated an IE
ME
-specific hyperactive form of Tnp, Tnp sC7v.2.0, that is able to promote high levels of transposition of IE
ME
defined transposons in vivo and in vitro while functioning at wild-type levels with OE transposons. This protein contains a critical glutamate-to-valine mutation at amino acid 58 that is responsible for this change in end specificity.
Molecular Evolution of the Intimin Gene in O111 Clones of PathogenicEscherichia coli
Authors: Tarr Cheryl L. , Whittam Thomas S.
Published: July 2002

Intimin is an important virulence factor in two groups of enteric pathogens: enteropathogenicEscherichia coli(EPEC), which is a major cause of infant diarrhea in the developing world, and enterohemorrhagicE. coli(EHEC), which has caused large food-borne outbreaks of hemorrhagic colitis in the United States and other developed countries. Intimin is encoded on a 35-kb pathogenicity island called the locus of enterocyte effacement (LEE). At least five antigenic types have been described for the highly variable gene, and each type is generally characteristic of particular evolutionary lineages. We determined the nucleotide sequences of intimin and other LEE genes in two O111 clones that have not been amenable to typing. The sequences from both O111:H8 and O111:H9 differed from the Int-β that is typical of other clones in the same evolutionary lineage. The sequence from the O111:H8 strains was a mosaic of divergent segments that alternately clustered with Int-α, Int-β, or Int-γ. The sequence from the O111:H9 clone consistently showed a close relationship with that from E2348/69, a distantly related strain that expresses Int-α. The results suggest that there have been multiple acquisitions of the LEE in the EHEC 2/EPEC 2 clonal lineage, with a recent turnover in either O111:H8 or its close relatives. Amino acid substitutions that alter residue charge occurred more frequently than would be expected under random substitution in the extracellular domains of intimin, suggesting that diversifying selection has promoted divergence in this region of the protein. An N-terminal domain that presumably functions in the periplasm may also be under positive selection.
The Active Partition GeneincCof IncP Plasmids Is Required for Stable Maintenance in a Broad Range of Hosts
Authors: Siddique Azeem , Figurski David H.
Published: July 2002

Plasmids of incompatibility group P (IncP) are capable of replication and stable inheritance in a wide variety of gram-negative bacteria. Three determinants of IncP plasmids are components of an active partition locus that is predicted to function in the segregation of plasmid copies to daughter cells. These determinants areincC, which codes for a member of the ParA family of partition ATPases;korB, which specifies a DNA-binding protein that also functions as a global transcriptional repressor; and OB, the DNA target for KorB, which occurs at multiple locations on IncP plasmids. To determine the importance and host range of the IncC/KorB partition system in the maintenance of IncP plasmids, we constructed an in-frame deletion ofincCin the otherwise intact 60-kb IncPα plasmid R995. R995ΔincCwas found to be highly unstable inEscherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, Agrobacterium tumefaciens, andAcinetobacter calcoaceticus, whereas wild-type R995 is stable in all these hosts. In addition, R995ΔincCcould not be established inActinobacillus actinomycetemcomitans.trans-Complementation analysis showed that the coding region for IncC2 polypeptide, which is expressed from an internal translational start within theincCgene, was sufficient to restore stable maintenance to wild-type levels. The results show that the IncC/KorB active partition system of IncP plasmids is remarkably proficient for stable maintenance in diverse bacteria.
The Pyrimidine Nucleotide Reductase Step in Riboflavin and F
420
Biosynthesis in
Archaea
Proceeds by the Eukaryotic Route to Riboflavin
Authors: Graupner Marion , Xu Huimin , White Robert H.
Published: July 2002

The
Methanococcus jannaschii
gene MJ0671 was cloned and overexpressed in
Escherichia coli
, and its gene product was tested for its ability to catalyze the pyridine nucleotide-dependent reduction of either 2,5-diamino-6-ribosylamino-4(3
H
)-pyrimidinone 5′-phosphate (compound 3) to 2,5-diamino-6-ribitylamino-4(3
H
)-pyrimidinone 5′-phosphate (compound 4) or 5-amino-6-ribosylamino-2,4(1
H
,3
H
)-pyrimidinedione 5′-phosphate (compound 7) to 5-amino-6-ribitylamino-2,4(1
H
,3
H
)-pyrimidinedione 5′-phosphate (compound 5). Only compound 3 was found to serve as a substrate for the enzyme. NADPH and NADH functioned equally well as the reductants. This specificity for the reduction of compound 3 was also confirmed by using cell extracts of
M. jannaschii
and
Methanosarcina thermophila
. Thus, this step in riboflavin biosynthesis in these archaea is the same as that found in yeasts. The absence of the other genes in the biosynthesis of riboflavin in
Archaea
is discussed.
Effects of Pyrazinamide on Fatty Acid Synthesis by Whole Mycobacterial Cells and Purified Fatty Acid Synthase I
Authors: Boshoff Helena I. , Mizrahi Valerie , Barry Clifton E.
Published: July 2002

The effects of low extracellular pH and intracellular accumulation of weak organic acids were compared with respect to fatty acid synthesis by whole cells of
Mycobacterium tuberculosis
and
Mycobacterium smegmatis
. The profile of fatty acids synthesized during exposure to benzoic, nicotinic, or pyrazinoic acids, as well as that observed during intracellular hydrolysis of the corresponding amides, was not a direct consequence of modulation of fatty acid synthesis by these compounds but reflected the response to inorganic acid stress. Analysis of fatty acid synthesis in crude mycobacterial cell extracts demonstrated that pyrazinoic acid failed to directly modulate the fatty acid synthase activity catalyzed by fatty acid synthase I (FAS-I). However, fatty acid synthesis was irreversibly inhibited by 5-chloro-pyrazinamide in a time-dependent fashion. Moreover, we demonstrate that pyrazinoic acid does not inhibit purified mycobacterial FAS-I, suggesting that this enzyme is not the immediate target of pyrazinamide.