bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization and Transcriptional Analysis of Gene Clusters for a Type IV Secretion Machinery in Human Granulocytic and Monocytic Ehrlichiosis Agents
Authors: Ohashi Norio , Zhi Ning , Lin Quan , Rikihisa Yasuko 
Published: July 2002

ABSTRACT

Anaplasma
(
Ehrlichia
)
phagocytophila
and
Ehrlichia chaffeensis
, the etiologic agents of granulocytic and monocytic ehrlichioses, respectively, are obligatory intracellular bacteria that cause febrile systemic illness in humans. We identified and characterized clusters of genes for a type IV secretion machinery in these two bacteria, and analyzed their gene expression in cell culture and mammalian hosts. Eight
virB
and
virD
genes were found in each bacterial genome, and all of the genes were transcribed in cell culture. Although the gene order and orientation were similar to those found in other bacteria, the eight
virB
and
virD
genes were clustered at two separate loci in each genome. Five of the genes (
virB8
,
virB9
,
virB10
,
virB11
, and
virD4
) were located downstream from a
ribA
gene. These five genes in both
A. phagocytophila
and
E. chaffeensis
were polycistronically transcribed and controlled through at least two tandem promoters located upstream of the
virB8
gene in human leukemia cell lines. The
virB9
gene of
A. phagocytophila
was transcriptionally active in peripheral blood leukocytes from human ehrlichiosis patients and experimentally infected animals. Three of the remaining genes (
virB3
,
virB4
, and
virB6
) of both
A. phagocytophila
and
E. chaffeensis
were arranged downstream from a
sodB
gene and cotranscribed with the
sodB
gene through one or more
sodB
promoters in human leukocytes. This suggests that transcription of the three
virB
genes in these two
Anaplasma
and
Ehrlichia
spp. is regulated by factors that influence the
sodB
gene expression. This unique regulation of gene expression for the type IV secretion system may be associated with intracellular survival and replication of
Anaplasma
and
Ehrlichia
spp. in granulocytes or monocytes.
A Regulatory RNA (PrrB RNA) Modulates Expression of Secondary Metabolite Genes in
Pseudomonas fluorescens
F113
Authors: Aarons Simon , Abbas Abdelhamid , Adams Claire , Fenton Anne , O'Gara Fergal 
Published: July 2002

ABSTRACT

The GacS-GacA two-component signal transduction system, which is highly conserved in gram-negative bacteria, is required for the production of exoenzymes and secondary metabolites in
Pseudomonas
spp. Screening of a
Pseudomonas fluorescens
F113 gene bank led to the isolation of a previously undefined locus which could restore secondary metabolite production to both
gacS
and
gacA
mutants of F113. Sequence analysis of this locus demonstrated that it did not contain any obvious
Pseudomonas
protein-coding open reading frames or homologues within available databases. Northern analysis indicated that the locus encodes an RNA (PrrB RNA) which is able to phenotypically complement
gacS
and
gacA
mutants and is itself regulated by the GacS-GacA two-component signal transduction system. Primer extension analysis of the 132-base transcript identified the transcription start site located downstream of a ς
70
promoter sequence from positions −10 to −35. Inactivation of the
prrB
gene in F113 resulted in a significant reduction of 2,4-diacetylphloroglucinol (Phl) and hydrogen cyanide (HCN) production, while increased metabolite production was observed when
prrB
was overexpressed. The
prrB
gene sequence contains a number of imperfect repeats of the consensus sequence 5′-AGGA-3′, and sequence analysis predicted a complex secondary structure featuring multiple putative stem-loops with the consensus sequences predominantly positioned at the single-stranded regions at the ends of the stem-loops. This structure is similar to the CsrB and RsmB regulatory RNAs in
Escherichia coli
and
Erwinia carotovora
, respectively. Results suggest that a regulatory RNA molecule is involved in GacA-GacS-mediated regulation of Phl and HCN production in
P. fluorescens
F113.
Analysis of the Role of
recA
in Phenotypic Switching of
Pseudomonas tolaasii
Authors: Sinha Himanshu , Pain Arnab , Johnstone Keith 
Published: July 2002

ABSTRACT

Switching between the pathogenic smooth (1116S) and nonpathogenic rough (1116R) forms of
Pseudomonas tolaasii
occurs due to the reversible duplication of a 661-bp element within the
pheN
locus. Disruption of the chromosomal
recA
locus of 1116S and 1116R produced strains 1116S
recA
and 1116R
recA
, respectively, which showed typical loss of UV resistance. Switching from the smooth to the rough form was virtually eliminated in the 1116S
recA
strain, whereas the extent of switching from the rough to the smooth form was almost identical in 1116R and 1116R
recA
. It is concluded that phenotypic switching from 1116S to 1116R is
recA
dependent whereas that from 1116R to 1116S is
recA
independent.
Functionality of Purified ς
N
(ς
54
) and a NifA-Like Protein from the Hyperthermophile
Aquifex aeolicus
Authors: Studholme David J. , Wigneshwereraraj Siva R. , Gallegos María-Trinidad , Buck Martin 
Published: July 2002

ABSTRACT

The genome sequence of the extremely thermophilic bacterium
Aquifex aeolicus
encodes alternative sigma factor ς
N
(ς
54
, RpoN) and five potential ς
N
-dependent transcriptional activators. Although
A. aeolicus
possesses no recognizable nitrogenase genes, two of the activators have a high degree of sequence similarity to NifA proteins from nitrogen-fixing proteobacteria. We identified five putative ς
N
-dependent promoters upstream of operons implicated in functions including sulfur respiration, nitrogen assimilation, nitrate reductase, and nitrite reductase activity. We cloned, overexpressed (in
Escherichia coli
), and purified
A. aeolicus
ς
N
and the NifA homologue, AQ_218. Purified
A. aeolicus
ς
N
bound to
E. coli
core RNA polymerase and bound specifically to a DNA fragment containing
E. coli
promoter
glnHp2
and to several
A. aeolicus
DNA fragments containing putative ς
N
-dependent promoters. When combined with
E. coli
core RNA polymerase,
A. aeolicus
ς
N
supported
A. aeolicus
NifA-dependent transcription from the
glnHp2
promoter. The
E. coli
activator PspFΔHTH did not stimulate transcription. The NifA homologue, AQ_218, bound specifically to a DNA sequence centered about 100 bp upstream of the
A. aeolicus glnBA
operon and so is likely to be involved in the regulation of nitrogen assimilation in this organism. These results argue that the ς
N
enhancer-dependent transcription system operates in at least one extreme environment, and that the activator and ς
N
have coevolved.
Effect on Heterocyst Differentiation of Nitrogen Fixation in Vegetative Cells of the Cyanobacterium
Anabaena variabilis
ATCC 29413
Authors: Thiel Teresa , Pratte Brenda 
Published: July 2002

ABSTRACT

Heterocysts are terminally differentiated cells of some filamentous cyanobacteria that fix nitrogen for the entire filament under oxic growth conditions.
Anabaena variabilis
ATCC 29413 is unusual in that it has two Mo-dependent nitrogenases; one, called Nif1, functions in heterocysts, while the second, Nif2, functions under anoxic conditions in vegetative cells. Both nitrogenases depended on expression of the global regulatory protein NtcA. It has long been thought that a product of nitrogen fixation in heterocysts plays a role in maintenance of the spaced pattern of heterocyst differentiation. This model assumes that each cell in a filament senses its own environment in terms of nitrogen sufficiency and responds accordingly in terms of differentiation. Expression of the Nif2 nitrogenase under anoxic conditions in vegetative cells was sufficient to support long-term growth of a
nif1
mutant; however, that expression did not prevent differentiation of heterocysts and expression of the
nif1
nitrogenase in either the
nif1
mutant or the wild-type strain. This suggested that the nitrogen sufficiency of individual cells in the filament did not affect the signal that induces heterocyst differentiation. Perhaps there is a global mechanism by which the filament senses nitrogen sufficiency or insufficiency based on the external availability of fixed nitrogen. The filament would then respond by producing heterocyst differentiation signals that affect the entire filament. This does not preclude cell-to-cell signaling in the maintenance of heterocyst pattern but suggests that overall control of the process is not controlled by nitrogen insufficiency of individual cells.
phzO
, a Gene for Biosynthesis of 2-Hydroxylated Phenazine Compounds in
Pseudomonas aureofaciens
30-84
Authors: Delaney Shannon M. , Mavrodi Dmitri V. , Bonsall Robert F. , Thomashow Linda S. 
Published: July 2002

ABSTRACT

Certain strains of root-colonizing fluorescent
Pseudomonas
spp. produce phenazines, a class of antifungal metabolites that can provide protection against various soilborne root pathogens. Despite the fact that the phenazine biosynthetic locus is highly conserved among fluorescent
Pseudomonas
spp., individual strains differ in the range of phenazine compounds they produce. This study focuses on the ability of
Pseudomonas aureofaciens
30-84 to produce 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA) and 2-hydroxyphenazine from the common phenazine metabolite phenazine-1-carboxylic acid (PCA).
P. aureofaciens
30-84 contains a novel gene located downstream from the core phenazine operon that encodes a 55-kDa aromatic monooxygenase responsible for the hydroxylation of PCA to produce 2-OH-PCA. Knowledge of the genes responsible for phenazine product specificity could ultimately reveal ways to manipulate organisms to produce multiple phenazines or novel phenazines not previously described.
Aspartic Peptide Hydrolases in
Salmonella enterica
Serovar Typhimurium
Authors: Larsen Rachel A. , Knox Tina M. , Miller Charles G. 
Published: July 2002

ABSTRACT

Two well-characterized enzymes in
Salmonella enterica
serovar Typhimurium and
Escherichia coli
are able to hydrolyze N-terminal aspartyl (Asp) dipeptides: peptidase B, a broad-specificity aminopeptidase, and peptidase E, an Asp-specific dipeptidase. A serovar Typhimurium strain lacking both of these enzymes, however, can still utilize most N-terminal Asp dipeptides as sources of amino acids, and extracts of such a strain contain additional enzymatic activities able to hydrolyze Asp dipeptides. Here we report two such activities from extracts of
pepB pepE
mutant strains of serovar Typhimurium identified by their ability to hydrolyze Asp-Leu. Although each of these activities hydrolyzes Asp-Leu at a measurable rate, the preferred substrates for both are N-terminal isoAsp peptides. One of the activities is a previously characterized isoAsp dipeptidase from
E. coli
, the product of the
iadA
gene. The other is the product of the serovar Typhimurium homolog of
E. coli ybiK
, a gene of previously unknown function. This gene product is a member of the N-terminal nucleophile structural family of amidohydrolases. Like most other members of this family, the mature enzyme is generated from a precursor protein by proteolytic cleavage and the active enzyme is a heterotetramer. Based on its ability to hydrolyze an N-terminal isoAsp tripeptide as well as isoAsp dipeptides, the enzyme appears to be an isoAsp aminopeptidase, and we propose that the gene encoding it be designated
iaaA
(isoAsp aminopeptidase). A strain lacking both IadA and IaaA in addition to peptidase B and peptidase E has been constructed. This strain utilizes Asp-Leu as a leucine source, and extracts of this strain contain at least one additional, as-yet-uncharacterized, peptidase able to cleave Asp dipeptides.
Identification and Characterization of a Second Superoxide Dismutase Gene (
sodM
) from
Staphylococcus aureus
Authors: Valderas Michelle Wright , Hart Mark E. 
Published: July 2002

ABSTRACT

A gene encoding superoxide dismutase (SOD),
sodM
, from
S. aureus
was cloned and characterized. The deduced amino acid sequence specifies a 187-amino-acid protein with 75% identity to the
S. aureus
SodA protein. Amino acid sequence comparisons with known SODs and relative insensitivity to hydrogen peroxide and potassium cyanide indicate that SodM most likely uses manganese (Mn) as a cofactor. The
sodM
gene expressed from a plasmid rescued an
Escherichia coli
double mutant (
sodA sodB
) under conditions that are otherwise lethal. SOD activity gels of
S. aureus
RN6390 whole-cell lysates revealed three closely migrating bands of activity. The two upper bands were absent in a
sodM
mutant, while the two lower bands were absent in a
sodA
mutant. Thus, the middle band of activity most likely represents a SodM-SodA hybrid protein. All three bands of activity increased as highly aerated cultures entered the late exponential phase of growth, SodM more so than SodA. Viability of the
sodA
and
sodM sodA
mutants but not the
sodM
mutant was drastically reduced under oxidative stress conditions generated by methyl viologen (MV) added during the early exponential phase of growth. However, only the viability of the
sodM sodA
mutant was reduced when MV was added during the late exponential and stationary phases of growth. These data indicate that while SodA may be the major SOD activity in
S. aureus
throughout all stages of growth, SodM, under oxidative stress, becomes a major source of activity during the late exponential and stationary phases of growth such that viability and growth of an
S. aureus sodA
mutant are maintained.
Characterization and Analysis of the PikD Regulatory Factor in the Pikromycin Biosynthetic Pathway of
Streptomyces venezuelae
Authors: Wilson Daniel J. , Xue Yongquan , Reynolds Kevin A. , Sherman David H. 
Published: July 2002

ABSTRACT

The
Streptomyces venezuelae pikD
gene from the pikromycin biosynthetic cluster was analyzed, and its deduced product (PikD) was found to have amino acid sequence homology with a small family of bacterial regulatory proteins. Database comparisons revealed two hypothetical domains, including an N-terminal triphosphate-binding domain and a C-terminal helix-turn-helix DNA-binding motif. Analysis of PikD was initiated by deletion of the corresponding gene (
pikD
) from the chromosome of
S. venezuelae
, resulting in complete loss of antibiotic production. Complementation by a plasmid carrying
pikD
restored macrolide biosynthesis, demonstrating that PikD is a positive regulator. Mutations were made in the predicted nucleotide triphosphate-binding domain, confirming the active-site amino acid residues of the Walker A and B motifs. Feeding of macrolide intermediates was carried out to gauge the points of operon control by PikD. Although the
pikD
mutant strain was unable to convert macrolactones (10-deoxymethynolide and narbonolide) to glycosylated products, macrolide intermediates (YC-17 and narbomycin) were hydroxylated with high efficiency. To study further the control of biosynthesis, presumed promoter regions from
pik
cluster loci were linked to the
xylE
reporter and placed in
S. venezuelae
wild-type and
pikD
mutant strains. This analysis demonstrated that PikD-mediated transcriptional regulation occurs at promoters controlling expression of
pikRII
,
pikAI
, and
desI
but not those controlling
pikRI
or
pikC
.
Methionine Regeneration and Aspartate Aminotransferase in Parasitic Protozoa
Authors: Berger Louise C. , Wilson Judith , Wood Pamela , Berger Bradley J. 
Published: July 2002

ABSTRACT

Aspartate aminotransferases have been cloned and expressed from
Crithidia fasciculata
,
Trypanosoma brucei brucei
,
Giardia intestinalis
, and
Plasmodium falciparum
and have been found to play a role in the final step of methionine regeneration from methylthioadenosine. All five enzymes contain sequence motifs consistent with membership in the Ia subfamily of aminotransferases; the crithidial and giardial enzymes and one trypanosomal enzyme were identified as cytoplasmic aspartate aminotransferases, and the second trypanosomal enzyme was identified as a mitochondrial aspartate aminotransferase. The plasmodial enzyme contained unique sequence substitutions and appears to be highly divergent from the existing members of the Ia subfamily. In addition, the
P. falciparum
enzyme is the first aminotransferase found to lack the invariant residue G197 (P. K. Mehta, T. I. Hale, and P. Christen, Eur. J. Biochem
.
214:549–561, 1993), a feature shared by sequences discovered in
P. vivax
and
P. berghei
. All five enzymes were able to catalyze aspartate-ketoglutarate, tyrosine-ketoglutarate, and amino acid-ketomethiobutyrate aminotransfer reactions. In the latter, glutamate, phenylalanine, tyrosine, tryptophan, and histidine were all found to be effective amino donors. The crithidial and trypanosomal cytosolic aminotransferases were also able to catalyze alanine-ketoglutarate and glutamine-ketoglutarate aminotransfer reactions and, in common with the giardial aminotransferase, were able to catalyze the leucine-ketomethiobutyrate aminotransfer reaction. In all cases, the kinetic constants were broadly similar, with the exception of that of the plasmodial enzyme, which catalyzed the transamination of ketomethiobutyrate significantly more slowly than aspartate-ketoglutarate aminotransfer. This result obtained with the recombinant
P. falciparum
aminotransferase parallels the results seen for total ketomethiobutyrate transamination in malarial homogenates; activity in the latter was much lower than that in homogenates from other organisms. Total ketomethiobutyrate transamination in
Trichomonas vaginalis
and
G. intestinalis
homogenates was extensive and involved lysine-ketomethiobutyrate enzyme activity in addition to the aspartate aminotransferase activity. The methionine production in these two species could be inhibited by the amino-oxy compounds canaline and carboxymethoxylamine. Canaline was also found to be an uncompetitive inhibitor of the plasmodial aspartate aminotransferase, with a

K
i

of 27 μM.
DNA Inversion in the Tail Fiber Gene Alters the Host Range Specificity of Carotovoricin Er, a Phage-Tail-Like Bacteriocin of Phytopathogenic
Erwinia carotovora
subsp.
carotovora
Er
Authors: Nguyen Hoa Anh , Tomita Toshio , Hirota Morihiko , Kaneko Jun , Hayashi Tetsuya , Kamio Yoshiyuki 
Published: July 2002

ABSTRACT

Carotovoricin Er is a phage-tail-like bacteriocin produced by
Erwinia carotovora
subsp.
carotovora
strain Er, a causative agent for soft rot disease in plants. Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues. A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence. In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected. DNA inversion of the 790-bp fragment also occurred in
Escherichia coli
DH5α when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were cotransformed into the bacterium. Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin.
The Pyrimidine Operon
pyrRPB-carA
from
Lactococcus lactis
Authors: Martinussen Jan , Schallert Jette , Andersen Birgit , Hammer Karin 
Published: July 2002

ABSTRACT

The four genes
pyrR, pyrP, pyrB
, and
carA
were found to constitute an operon in
Lactococcus lactis
subsp.
lactis
MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene,
pyrR
, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation. The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil. The last two genes in the operon,
pyrB
and
carA
, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (
pyrB
) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (
carA
) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate. The
carA
gene product is shown to be required for both pyrimidine and arginine biosynthesis. The expression of the pyrimidine biosynthetic genes including the
pyrRPB-carA
operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein.
Expression of the Putative Siderophore Receptor Gene
bfrZ
Is Controlled by the Extracytoplasmic-Function Sigma Factor BupI in
Bordetella bronchiseptica
Authors: Pradel Elizabeth , Locht Camille 
Published: July 2002

ABSTRACT

A new gene from
Bordetella bronchiseptica, bfrZ
encoding a putative siderophore receptor, was identified in a Fur-repressor titration assay. A
bfrZ
null mutant was constructed by allelic exchange. The protein profile of this mutant is similar to that of the wild-type parent strain. The BfrZ
−
-BfrZ
+
isogenic pair was tested for utilization of 132 different siderophores as iron sources. None of these iron sources acted as a ligand for BfrZ. Translational
bfrZ
::
phoA
and transcriptional
bfrZ
::
lacZ
fusions were introduced into the
B. bronchiseptica bfrZ
locus. No alkaline phosphatase or β-galactosidase activity was detected. Sequence analysis of the
bfrZ
upstream region revealed the presence of two tightly linked genes,
bupI
and
bupR
. Both of these genes are located downstream from a Fur-binding sequence. BupI is homologous to
Escherichia coli
FecI and
Pseudomonas putida
PupI and belongs to the family of extracytoplasmic-function sigma factors involved in transcription of genes with extracytoplasmic functions. BupR is homologous to the FecR and PupR antisigma factors and is predicted to be localized in the inner membrane. Similar to the surface signaling receptors FecA and PupB, BfrZ bears an N-terminal extension. We found that
bfrZ
is not transcribed when
bupI
and
bupR
are expressed at the same level. However, overexpression of
bupI
from a multicopy plasmid triggers
bfrZ
transcription, and under these conditions BfrZ was detected in membrane fractions. By analogy with the FecI-FecR-FecA and PupI-PupR-PupB systems, our data suggest that
bfrZ
expression is inducible by binding of the cognate ligand to BfrZ and transduction of a signal through the envelope.