bluuebunny/crossref_metadata_2025_split huggingface dataset:
Roles of LcrG and LcrV during Type III Targeting of Effector Yops by
Yersinia enterocolitica
Authors: DeBord Kristin L. , Lee Vincent T. , Schneewind Olaf 
Published: July 2002

ABSTRACT

Yersinia enterocolitica
target effector Yop proteins into the cytosol of eukaryotic cells by a mechanism requiring the type III machinery. LcrG and LcrV have been suggested to fulfill essential functions during the type III targeting of effector Yops. It is reported here that knockout mutations of
lcrG
caused mutant yersiniae to prematurely secrete Yops into the extracellular medium without abolishing the type III targeting mechanism (Los phenotype [loss of type III targeting specificity]). Knockout mutations in
lcrV
reduced type III targeting of mutant yersiniae but did not promote secretion into the extracellular medium (Not [no type III targeting]). However, knockout mutations in both genes caused Δ
lcrGV
yersiniae to display a Los phenotype similar to that of strains carrying knockout mutations in
lcrG
alone. LcrG binding to LcrV resulted in the formation of soluble LcrGV complexes in the bacterial cytoplasm. Membrane-associated, bacterial-surface-displayed or -secreted LcrG could not be detected. Most of LcrV was located in the bacterial cytoplasm; however, small amounts were secreted into the extracellular medium. These data support a model whereby LcrG may act as a negative regulator of type III targeting in the bacterial cytoplasm, an activity that is modulated by LcrG binding to LcrV. No support could be gathered for the hypothesis whereby LcrG and LcrV may act as a bacterial surface receptor for host cells, allowing effector Yop translocation across the eukaryotic plasma membrane.
The HilA Box and Sequences outside It Determine the Magnitude of HilA-Dependent Activation of P

prgH

from
Salmonella
Pathogenicity Island 1
Authors: Lostroh C. Phoebe , Lee Catherine A. 
Published: July 2002

ABSTRACT

Salmonella
requires genes on the
Salmonella
pathogenicity island 1 (SPI1) for the intestinal phase of infection in several models of pathogenesis. In
Salmonella enterica
serovar Typhimurium, most SPI1 genes are arranged in operons that are coordinately regulated by the SPI1-encoded protein HilA. In the past, it has been shown that HilA directly activates two promoters on SPI1, P

invF-1

and P

prgH

. P

invF-1

contains a HilA binding site, termed a HilA box, that is necessary and sufficient for activation by HilA. The HilA box is 17 nucleotides long and contains a direct repeat comprised of two hexamers separated by 5 nucleotides, centered at −45 relative to the start site of transcription. P

prgH

also contains a HilA box, and here we investigate its role at P

prgH

. We have found that the HilA box is necessary, but not sufficient, for HilA-dependent activation of P

prgH

. Instead, half-site-like hexamers outside the HilA box appear to be required for HilA-dependent activation of P

prgH

, even though HilA binds to the HilA box in the absence of these hexamers. Thus, although HilA-dependent activation of P

invF-1

and P

prgH

coordinates the expression of the structural genes for a type III secretion apparatus and the effectors secreted by that apparatus, it is also possible that mechanisms not apparent under in vitro inducing conditions could separate the expression of
invFGEABC-spaMNOPQRS-sicA-sipBCDA-iacP-sicP-sptP
and
prgHIJK-orgABC
.
Quorum Sensing Is a Global Regulatory Mechanism in Enterohemorrhagic
Escherichia coli
O157:H7
Authors: Sperandio Vanessa , Torres Alfredo G. , Girón Jorge A. , Kaper James B. 
Published: July 2002

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the
luxS
gene. In this study we hybridized an
E. coli
gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic
luxS
mutant. We observed that 404 genes were regulated by
luxS
at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the
luxS
mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the
luxS
mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon::
lacZ
fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the
luxS
mutant. The average swimming speeds for the wild-type strain and the
luxS
mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of
E. coli
as well as for virulence factors.
Selection and Characterization of
Escherichia coli
Variants Capable of Growth on an Otherwise Toxic Tryptophan Analogue
Authors: Bacher Jamie M. , Ellington Andrew D. 
Published: July 2002

ABSTRACT

Escherichia coli
isolates that were tolerant of incorporation of high proportions of 4-fluorotryptophan were evolved by serial growth. The resultant strain still preferred tryptophan for growth but showed improved growth relative to the parental strain on other tryptophan analogues. Evolved clones fully substituted fluorotryptophan for tryptophan in their proteomes within the limits of mass spectral and amino acid analyses. Of the genes sequenced, many genes were found to be unaltered in the evolved strain; however, three genes encoding enzymes involved in tryptophan uptake and utilization were altered: the aromatic amino acid permease (
aroP
) and tryptophanyl-tRNA synthetase (
trpS
) contained several amino acid substitutions, and the tyrosine repressor (
tyrR
) had a nonsense mutation. While kinetic analysis of the tryptophanyl-tRNA synthetase suggests discrimination against 4-fluorotryptophan, an analysis of the incorporation and growth patterns of the evolved bacteria suggest that other mutations also aid in the adaptation to the tryptophan analogue. These results suggest that the incorporation of unnatural amino acids into organismal proteomes may be possible but that extensive evolution may be required to reoptimize proteins and metabolism to accommodate such analogues.
Identification of thenikGene Cluster ofBrucella suis: Regulation and Contribution to Urease Activity
Authors: Jubier-Maurin Véronique , Rodrigue Agnès , Ouahrani-Bettache Safia , Layssac Marion , Mandrand-Berthelot Marie-Andrée , Köhler Stephan , Liautard Jean-Pierre 
Published: July 2002

ABSTRACTAnalysis of aBrucella suis1330 gene fused to agfpreporter, and identified as being induced in J774 murine macrophage-like cells, allowed the isolation of a gene homologous tonikA, the first gene of theEscherichia colioperon encoding the specific transport system for nickel. DNA sequence analysis of the correspondingB. suis niklocus showed that it was highly similar to that ofE. coliexcept for localization of thenikRregulatory gene, which lies upstream from the structuralnikABCDEgenes and in the opposite orientation. Protein sequence comparisons suggested that the deducednikABCDEgene products belong to a periplasmic binding protein-dependent transport system. ThenikApromoter-gfpfusion was activated in vitro by low oxygen tension and metal ion deficiency and was repressed by NiCl2excess. Insertional inactivation ofnikAstrongly reduced the activity of the nickel metalloenzyme urease, which was restored by addition of a nickel excess. Moreover, thenikAmutant ofB. suiswas functionally complemented with theE. coli nikgene cluster, leading to the recovery of urease activity. Reciprocally, anE. colistrain harboring a deletednikoperon recovered hydrogenase activity by heterologous complementation with theB. suis niklocus. Taking into account these results, we propose that theniklocus ofB. suisencodes a nickel transport system. The results further suggest that nickel could enterB. suisvia other transport systems. Intracellular growth rates of theB. suiswild-type andnikAmutant strains in human monocytes were similar, indicating thatnikAwas not essential for this step of infection. We discuss a possible role of nickel transport in maintaining enzymatic activities which could be crucial for survival of the bacteria under the environmental conditions encountered within the host.
Growth Phase and Growth Rate Regulation of the
rapA
Gene, Encoding the RNA Polymerase-Associated Protein RapA in
Escherichia coli
Authors: Cabrera Julio E. , Jin Ding Jun 
Published: July 2002

ABSTRACT

The
Escherichia coli rapA
gene encodes the RNA polymerase (RNAP)-associated protein RapA, which is a bacterial member of the SWI/SNF helicase-like protein family. We have studied the
rapA
promoter and its regulation in vivo and determined the interaction between RNAP and the promoter in vitro. We have found that the expression of
rapA
is growth phase dependent, peaking at the early log phase. The growth phase control of
rapA
is determined at least by one particular feature of the promoter: it uses CTP as the transcription-initiating nucleotide instead of a purine, which is used for most
E. coli
promoters. We also found that the
rapA
promoter is subject to growth rate regulation in vivo and that it forms intrinsic unstable initiation complexes with RNAP in vitro. Furthermore, we have shown that a GC-rich or discriminator sequence between the −10 and +1 positions of the
rapA
promoter is responsible for its growth rate control and the instability of its initiation complexes with RNAP.
DNA as a Nutrient: Novel Role for Bacterial Competence Gene Homologs
Authors: Finkel Steven E. , Kolter Roberto 
Published: July 2002

ABSTRACT

The uptake and stable maintenance of extracellular DNA, genetic transformation, is universally recognized as a major force in microbial evolution. We show here that extracellular DNA, both homospecific and heterospecific, can also serve as the sole source of carbon and energy supporting microbial growth. Mutants unable to consume DNA suffer a significant loss of fitness during stationary-phase competition. In
Escherichia coli
, the use of DNA as a nutrient depends on homologs of proteins involved in natural genetic competence and transformation in
Haemophilus influenzae
and
Neisseria gonorrhoeae
. Homologs of these
E. coli
genes are present in many members of the γ subclass of
Proteobacteria
, suggesting that the mechanisms for consumption of DNA may have been widely conserved during evolution.
Precise Deletion of
tagD
and Controlled Depletion of Its Product, Glycerol 3-Phosphate Cytidylyltransferase, Leads to Irregular Morphology and Lysis of
Bacillus subtilis
Grown at Physiological Temperature
Authors: Bhavsar Amit P. , Beveridge Terry J. , Brown Eric D. 
Published: July 2002

ABSTRACT

Using a previously reported conditional expression system for use in
Bacillus subtilis
(A. P. Bhavsar, X. Zhao, and E. D. Brown, Appl. Environ. Microbiol. 67:403–410, 2001), we report the first precise deletion of a teichoic acid biosynthesis (
tag
) gene,
tagD,
in
B. subtilis
. This teichoic acid mutant showed a lethal phenotype when characterized at a physiological temperature and in a defined genetic background. This
tagD
mutant was subject to full phenotypic rescue upon expression of the complementing copy of
tagD.
Depletion of the
tagD
gene product (glycerol 3-phosphate cytidylyltransferase) via modulated expression of
tagD
from the
amyE
locus revealed structural defects centered on shape, septation, and division. Thickening of the wall and ultimately lysis followed these events.
Genetic Evidence for Parallel Pathways of Chaperone Activity in the Periplasm of
Escherichia coli
Authors: Rizzitello Amy E. , Harper Jill R. , Silhavy Thomas J. 
Published: July 2002

ABSTRACT

The periplasm of
Escherichia coli
contains many proteins proposed to have redundant functions in protein folding. Using depletion analysis, we directly demonstrated that null mutations in
skp
and
surA,
as well as in
degP
and
surA,
result in synthetic phenotypes, suggesting that Skp, SurA, and DegP are functionally redundant. The Δ
skp surA
::
kan
combination has a bacteriostatic effect and leads to filamentation, while the
degP
::Tn
10 surA
::
kan
combination is bactericidal. The steady-state levels of several envelope proteins are greatly reduced upon depletion of a wild-type copy of
surA
in both instances. We suggest that the functional redundancy of Skp, SurA, and DegP lies in the periplasmic chaperone activity. Taken together, our data support a model in which the periplasm of
E. coli
contains parallel pathways for chaperone activity. In particular, we propose that Skp and DegP are components of the same pathway and that SurA is a component of a separate pathway. The loss of either pathway has minimal effects on the cell, while the loss of both pathways results in the synthetic phenotypes observed.
Biochemical Analysis of Replication Factor C from the Hyperthermophilic ArchaeonPyrococcus furiosus
Authors: Cann Isaac K. O. , Ishino Sonoko , Yuasa Mihoko , Daiyasu Hiromi , Toh Hiroyuki , Ishino Yoshizumi 
Published: July 2002

ABSTRACTReplication factor C (RFC) and proliferating cell nuclear antigen (PCNA) are accessory proteins essential for processive DNA synthesis in the domainEucarya. The function of RFC is to load PCNA, a processivity factor of eukaryotic DNA polymerases δ and ɛ, onto primed DNA templates. RFC-like genes, arranged in tandem in thePyrococcus furiosusgenome, were cloned and expressed individually inEscherichia colicells to determine their roles in DNA synthesis. TheP. furiosusRFC (PfuRFC) consists of a small subunit (RFCS) and a large subunit (RFCL). Highly purified RFCS possesses an ATPase activity, which was stimulated up to twofold in the presence of both single-stranded DNA (ssDNA) andP. furiosusPCNA (PfuPCNA). The ATPase activity of PfuRFC itself was as strong as that of RFCS. However, in the presence of PfuPCNA and ssDNA, PfuRFC exhibited a 10-fold increase in ATPase activity under the same conditions. RFCL formed very large complexes by itself and had an extremely weak ATPase activity, which was not stimulated by PfuPCNA and DNA. The PfuRFC stimulated PfuPCNA-dependent DNA synthesis by both polymerase I and polymerase II fromP. furiosus. We propose that PfuRFC is required for efficient loading of PfuPCNA and that the role of RFC in processive DNA synthesis is conserved inArchaeaandEucarya.
Isogenic Strain Construction and Gene Targeting in
Candida dubliniensis
Authors: Staib Peter , Moran Gary P. , Sullivan Derek J. , Coleman David C. , Morschhäuser Joachim 
Published: July 2002

ABSTRACT

Candida dubliniensis
is a recently described opportunistic fungal pathogen that is closely related to
Candida albicans
but differs from it with respect to epidemiology, certain virulence characteristics, and the ability to develop fluconazole resistance in vitro. A comparison of
C. albicans
and
C. dubliniensis
at the molecular level should therefore provide clues about the mechanisms used by these two species to adapt to their human host. In contrast to
C. albicans
, no auxotrophic
C. dubliniensis
strains are available for genetic manipulations. Therefore, we constructed homozygous
ura3
mutants from a
C. dubliniensis
wild-type isolate by targeted gene deletion. The two
URA3
alleles were sequentially inactivated using the

MPA
R

-flipping strategy, which is based on the selection of integrative transformants carrying a mycophenolic acid resistance marker that is subsequently deleted again by site-specific, FLP-mediated recombination. The
URA3
gene from
C. albicans
(
CaURA3
) was then used as a selection marker for targeted integration of a fusion between the
C. dubliniensis MDR1
(
CdMDR1
) promoter and a
C. albicans
-adapted
GFP
reporter gene. Uridine-prototrophic transformants were obtained with high frequency, and all transformants of two independent
ura3
-negative parent strains had correctly integrated the reporter gene fusion into the
CdMDR1
locus, demonstrating that the
CaURA3
gene can be used for efficient and specific targeting of recombinant DNA into the
C. dubliniensis
genome. Transformants carrying the reporter gene fusion did not exhibit detectable fluorescence during growth in yeast extract-peptone-dextrose medium in vitro, suggesting that
CdMDR1
is not significantly expressed under these conditions. Fluconazole had no effect on
MDR1
expression, but the addition of the drug benomyl strongly activated the reporter gene fusion in a dose-dependent fashion, demonstrating that the
CdMDR1
gene, which encodes an efflux pump mediating resistance to toxic compounds, is induced by the presence of certain drugs.
ATP-Binding Cassette Transport System Involved in Regulation of Morphological Differentiation in Response to Glucose in
Streptomyces griseus
Authors: Seo Jeong-Woo , Ohnishi Yasuo , Hirata Aiko , Horinouchi Sueharu 
Published: July 2002

ABSTRACT

Streptomyces griseus
NP4, which was derived by UV mutagenesis from strain IFO13350, showed a bald and wrinkled colony morphology in response to glucose. Mutant NP4 formed ectopic septa at intervals along substrate hyphae, and each of the compartments developed into a spore which was indistinguishable from an aerial spore in size, shape, and thickness of the spore wall and in susceptibility to lysozyme and heat. The ectopic spores of NP4 formed in liquid medium differed from “submerged spores” in lysozyme sensitivity. Shotgun cloning experiments with a library of the chromosomal DNA of the parental strain and mutant NP4 as the host gave rise to DNA fragments giving two different phenotypes; one complementing the bald phenotype of the host, and the other causing much severe wrinkled morphology in the host. Subcloning identified a gene (
dasR
) encoding a transcriptional repressor belonging to the GntR family that was responsible for the reversal of the bald phenotype and a gene (
dasA
) encoding a lipoprotein probably serving as a substrate-binding protein in an ATP-binding cassette (ABC) transport system that was responsible for the severe wrinkled morphology. These genes were adjacent but divergently encoded. Two genes, named
dasB
and
dasC
, encoding a membrane-spanning protein were present downstream of
dasA
, which suggested that
dasRABC
comprises a gene cluster for an ABC transporter, probably for sugar import.
dasR
was transcribed actively during vegetative growth, and
dasA
was transcribed just after commencement of aerial hypha formation and during sporulation, indicating that both were developmentally regulated. Transcriptional analysis and direct sequencing of
dasRA
in mutant NP4 suggested a defect of this mutant in the regulatory system to control the expression of these genes. Introduction of multicopies of
dasA
into the wild-type strain caused ectopic septation in very young substrate hyphae after only 1 day of growth and subsequent sporulation in response to glucose. The ectopic spores of the wild type had a thinner wall than those of mutant NP4, in agreement with the observation that the former was sensitive to lysozyme and heat. Disruption of the chromosomal
dasA
or
dasR
in the wild-type strain resulted in growth as substrate mycelium, suggesting an additional role of these genes in aerial mycelium formation. The ectopic septation and sporulation in mutant NP4 and the wild-type strain carrying multicopies of
dasA
were independent of a microbial hormone, A-factor (2-isocapryloyl-3
R
-hydroxymethyl-γ-butyrolactone), that acts as a master switch of aerial mycelium formation and secondary metabolism.
The Molybdate-Responsive
Escherichia coli
ModE Transcriptional Regulator Coordinates Periplasmic Nitrate Reductase
(napFDAGHBC)
Operon Expression with Nitrate and Molybdate Availability
Authors: McNicholas Paul M. , Gunsalus Robert P. 
Published: July 2002

ABSTRACT

Expression of the
Escherichia coli napFDAGHBC
operon (also known as
aeg46.5
), which encodes the periplasmic molybdoenzyme for nitrate reduction, is increased in response to anaerobiosis and further stimulated by the addition of nitrate or to a lesser extent by nitrite to the cell culture medium. These changes are mediated by the transcription factors Fnr and NarP, respectively. Utilizing a
napF-lacZ
operon fusion, we demonstrate that
napF
gene expression is impaired in strain defective for the molybdate-responsive ModE transcription factor. This control abrogates nitrate- or nitrite-dependent induction during anaerobiosis. Gel shift and DNase I footprinting analyses establish that ModE binds to the
napF
promoter with an apparent

K
d

of about 35 nM at a position centered at −133.5 relative to the start of
napF
transcription. Although the ModE binding site sequence is similar to other
E. coli
ModE binding sites, the location is atypical, because it is not centered near the start of transcription. Introduction of point mutations in the ModE recognition site severely reduced or abolished ModE binding in vitro and conferred a
modE
phenotype (i.e., loss of molybdate-responsive gene expression) in vivo. In contrast, deletion of the upstream ModE region site rendered
napF
expression independent of
modE.
These findings indicate the involvement of an additional transcription factor to help coordinate nitrate- and molybdate-dependent
napF
expression by the Fnr, NarP, NarL, and ModE proteins. The upstream ModE regulatory site functions to override nitrate control of
napF
gene expression when the essential enzyme component, molybdate, is limiting in the cell environment.