bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Extracytoplasmic Function Sigma Subunits, ς
E
and ς
FecI
, of
Escherichia coli
: Promoter Selectivity and Intracellular Levels
Authors: Maeda Hiroto , Jishage Miki , Nomura Tasuku , Fujita Nobuyuki , Ishihama Akira 
Published: July 2002

ABSTRACT

The promoter selectivity of two extracytoplasmic function (ECF) subfamily ς subunits, ς
E
(ς
24
) and ς
FecI
(ς
18
), of
Escherichia coli
RNA polymerase was analyzed by using an in vitro transcription system and various promoters. The Eς
E
holoenzyme recognized only the known cognate promoters,
rpoE
P2,
rpo
HP3, and
degP
, and the Eς
FecI
recognized only one known cognate promoter,
fecA
. The strict promoter recognition properties of ς
E
and ς
FecI
are similar to those of other minor ς subunits. Transcription by Eς
E
and Eς
FecI
was enhanced by high concentrations of glutamate, as in the case of other minor ς subunits. The optimum temperature for transcription by Eς
FecI
was low, around 25°C, apparently in agreement with the high rate of iron sequestration by
E. coli
at low temperatures. By quantitative Western blot analysis, the intracellular levels of ς
E
and ς
FecI
in the uninduced steady-state culture of
E. coli
W3110 (type A) were determined to be 0.7 to 2.0 and 0.1 to 0.2 fmol per μg of total proteins (or 3 to 9 and 0.4 to 0.9 molecules per cell), respectively, and less than 1% of the level of the major ς
70
subunit.
Regulation of the
cnr
Cobalt and Nickel Resistance Determinant of
Ralstonia eutropha
(
Alcaligenes eutrophus
) CH34
Authors: Tibazarwa C. , Wuertz S. , Mergeay M. , Wyns L. , van der Lelie D. 
Published: July 2002

ABSTRACT

The linked resistance to nickel and cobalt of
Ralstonia eutropha
-like strain CH34 (
Alcaligenes eutrophus
CH34) is encoded by the
cnr
operon, which is localized on the megaplasmid pMOL28. The regulatory genes
cnrYXH
have been cloned, overexpressed, and purified in
Escherichia coli
. CnrY fractionated as a 10.7-kDa protein in in vitro translation assays. CnrX, a periplasmic protein of 16.5 kDa, was overproduced and purified as a histidine-tagged fusion protein in
E. coli
. His-CnrX was found to posses a secondary structure content rich in alpha-helical and beta-sheet structures. CnrH, a sigma factor of the extracytoplasmic function family, was purified as an N-terminally histidine-tagged fusion. In gel shift mobility assays, His-CnrH, in the presence of
E. coli
core RNA polymerase enzyme, could retard at least two different promoter DNA targets,
cnrYp
and
cnrHp
, localized within the
cnrYXH
locus. These promoters and their transcription start sites were confirmed by primer extension. Purified His-CnrX did not inhibit the DNA-binding activity of His-CnrH and is therefore unlikely to be an anti-sigma factor, as previously hypothesized (EMBL
M91650
description entry). To study the transcriptional response of the regulatory locus to metals and to probe promoter regions, transcriptional fusions were constructed between fragments of
cnrYXH
and the
luxCDABE
, luciferase reporter genes. Nickel and cobalt specifically induced the
cnrYXH-luxCDABE
fusion at optimal concentrations of 0.3 mM Ni
2+
and 2.0 mM Co
2+
in a noncomplexing medium for metals. The two promoter regions P
Y
(upstream
cnrY
) and P
H
(upstream
cnrH
) were probed and characterized using this vector and were found to control the nickel-inducible regulatory response of the
cnr
operon. The
cnrHp
promoter was responsible for full transcription of the
cnrCBA
structural resistance genes, while the
cnrYp
promoter was necessary to obtain metal-inducible transcription from the
cnrHp
promoter. The zinc resistance phenotype (ZinB) of a spontaneous
cnr
mutant strain, AE963, was investigated and could be attributed to an insertion of IS
1087
, a member of the IS
2
family of insertion elements, within the
cnrY
gene.
RegA, Iron, and Growth Phase Regulate Expression of the
Pseudomonas aeruginosa tol-oprL
Gene Cluster
Authors: Duan Kangmin , Lafontaine Eric R. , Majumdar Sonali , Sokol Pamela A. 
Published: July 2002

ABSTRACT

The
tol-oprL
region in
Pseudomonas aeruginosa
appears to be involved in pyocin uptake and required for cell viability. The complete nucleotide sequences of the
tolQRA
and
oprL
genes as well as the incomplete sequences of
tolB
and
orf2
have been previously reported. In addition, the sequence of a
P. aeruginosa
iron-regulated gene (
pig6
) has been described and found to share homology with an open reading frame located upstream of the
Escherichia coli tolQRA
genes (U. A. Ochsner and M. L. Vasil, Proc. Natl. Acad. Sci. USA 93:4409–4414, 1996). In this study, we cloned the remainder of the
P. aeruginosa tol-oprL
gene cluster and determined its nucleotide sequence. This cluster was found to consist of seven genes in the order
orf1 tolQ tolR tolA tolB oprL orf2
. Transcriptional analysis of this gene cluster was performed by detecting the presence of mRNAs spanning adjacent genes as well as by using a promoterless
lacZ
reporter gene fused to each of the seven genes contained in the
tol-oprL
locus. The results show that there are three major transcriptional units or operons in this region,
orf1-tolQRA
,
tolB
, and
oprL-orf2
, in contrast to the
E. coli tol-pal
region, where there are only two operons,
orf1-tolQRA
and
tolB-pal-orf2
. Analysis of gene expression indicated that the
tol-oprL
genes of
P. aeruginosa
are both iron and growth phase modulated. The first operon,
orf1-tolQRA
, is iron regulated throughout growth, but iron-regulated expression of
tolB
and
oprL
fusions occurs only in late log phase. The expression of the three operons was significantly less repressed by iron in
fur
mutants than in the wild-type strain, suggesting the involvement of Fur in the iron regulation of all three operons. RegA is a positive yet nonessential regulator of
tol-oprL
expression.
Domain Structure, Oligomeric State, and Mutational Analysis of PpsR, the
Rhodobacter sphaeroides
Repressor of Photosystem Gene Expression
Authors: Gomelsky Mark , Horne Irene M. , Lee Hye-Joo , Pemberton John M. , McEwan Alastair G. , Kaplan Samuel 
Published: July 2002

ABSTRACT

The transcription factor PpsR from the facultative photoheterotroph
Rhodobacter sphaeroides
is involved in repression of photosystem gene expression under aerobic growth conditions. We have isolated a number of spontaneous mutations as well as constructed directed mutations and deletions in
ppsR
. Repressor activities and the oligomeric state of the wild-type and mutant proteins were assayed. Our results suggest that the wild-type PpsR exists in cell extracts as a tetramer. Analysis of the PpsR mutants confirmed that the carboxy-terminal region of PpsR (residues 400 to 464) is involved in DNA binding. The central region of the protein (residues 150 to 400) was found to contain two PAS domains (residues 161 to 259 and 279 to 367). PAS domains are ubiquitous protein modules involved in sensory transduction as well as in protein-protein interactions. All spontaneously isolated mutations, which significantly impaired repressor activity and which mapped outside the DNA binding region, were positioned in the PAS domains. None of these, however, affected the overall oligomeric state. This implies that the conformation of the PAS domains within the tetramer is critical for repressor activity. Upstream of the first PAS domain resides a putative glutamine-rich hinge (residues 127 to 136) that connects the first PAS domain to the amino-terminal region (residues 1 to 135). The role of the amino terminus of PpsR is not obvious; however, extended deletions within this region abolish repressor activity, thus suggesting that the amino terminus is essential for structural integrity of the protein. We present a model of the domain architecture of the PpsR protein according to which PpsR is comprised of three regions: the carboxy terminus responsible for DNA binding, the central region primarily involved in protein oligomerization and possibly signal sensing, and the amino terminus of unknown function. This model may prove useful for determining the mode of PpsR action.
Peptidase E, a Peptidase Specific for N-Terminal Aspartic Dipeptides, Is a Serine Hydrolase
Authors: Lassy Rachel A. L. , Miller Charles G. 
Published: July 2002

ABSTRACTSalmonella entericaserovar Typhimurium peptidase E (PepE) is an N-terminal Asp-specific dipeptidase. PepE is not inhibited by any of the classical peptidase inhibitors, and its amino acid sequence does not place it in any of the known peptidase structural classes. A comparison of the amino acid sequence of PepE with a number of related sequences has allowed us to define the amino acid residues that are strongly conserved in this family. To ensure the validity of this comparison, we have expressed one of the most distantly related relatives (Xenopus) inEscherichia coliand have shown that it is indeed an Asp-specific dipeptidase with properties very similar to those of serovar Typhimurium PepE. The sequence comparison suggests that PepE is a serine hydrolase. We have used site-directed mutagenesis to change all of the conserved Ser, His, and Asp residues and have found that Ser120, His157, and Asp135 are all required for activity. Conversion of Ser120 to Cys leads to severely reduced (104-fold) but still detectable activity, and this activity but not that of the parent is inhibited by thiol reagents; these results confirm that this residue is likely to be the catalytic nucleophile. These results suggest that PepE is the prototype of a new family of serine peptidases. The phylogenetic distribution of the family is unusual, since representatives are found in eubacteria, an insect (Drosophila), and a vertebrate (Xenopus) but not in theArchaeaor in any of the other eukaryotes for which genome sequences are available.
Characterization of
Escherichia coli
Type 1 Pilus Mutants with Altered Binding Specificities
Authors: Harris Sandra L. , Spears Patricia A. , Havell Edward A. , Hamrick Terri S. , Horton John R. , Orndorff Paul E. 
Published: July 2002

ABSTRACT

PCR mutagenesis and a unique enrichment scheme were used to obtain two mutants, each with a single lesion in
fimH
, the chromosomal gene that encodes the adhesin protein (FimH) of
Escherichia coli
type 1 pili. These mutants were noteworthy in part because both were altered in the normal range of cell types bound by FimH. One mutation altered an amino acid at a site previously shown to be involved in temperature-dependent binding, and the other altered an amino acid lining the predicted FimH binding pocket.
Insertional Mutagenesis in the
n
-Alkane-Assimilating Yeast
Yarrowia lipolytica
: Generation of Tagged Mutations in Genes Involved in Hydrophobic Substrate Utilization
Authors: Mauersberger Stephan , Wang Hui-Jie , Gaillardin Claude , Barth Gerold , Nicaud Jean-Marc 
Published: July 2002

ABSTRACT

Tagged mutants affected in the degradation of hydrophobic compounds (HC) were generated by insertion of a
zeta-URA3
mutagenesis cassette (MTC) into the genome of a
zeta
-free and
ura3
deletion-containing strain of
Yarrowia lipolytica
. MTC integration occurred predominantly at random by nonhomologous recombination. A total of 8,600 Ura
+
transformants were tested by replica plating for (i) growth on minimal media with alkanes of different chain lengths (decane, dodecane, and hexadecane), oleic acid, tributyrin, or ethanol as the C source and (ii) colonial defects on different glucose-containing media (YPD, YNBD, and YNBcas). A total of 257 mutants were obtained, of which about 70 were affected in HC degradation, representing different types of non-alkane-utilizing (Alk
−
) mutants (phenotypic classes alkA to alkE) and tributyrin degradation mutants. Among Alk
−
mutants, growth defects depending on the alkane chain length were observed (alkAa to alkAc). Furthermore, mutants defective in yeast-hypha transition and ethanol utilization and selected auxotrophic mutants were isolated. Flanking borders of the integrated MTC were sequenced to identify the disrupted genes. Sequence analysis indicated that the MTC was integrated in the
LEU1
locus in N083, a leucine-auxotrophic mutant, in the isocitrate dehydrogenase gene of N156 (alkE leaky), in the thioredoxin reductase gene in N040 (alkAc), and in a peroxine gene (
PEX14
) in N078 (alkD). This indicates that MTC integration is a powerful tool for generating and analyzing tagged mutants in
Y. lipolytica
.
Analysis of Catabolite Control Protein A-Dependent Repression in
Staphylococcus xylosus
by a Genomic Reporter Gene System
Authors: Jankovic Ivana , Egeter Oliver , Brückner Reinhold 
Published: July 2002

ABSTRACT

A single-copy reporter system for
Staphylococcus xylosus
has been developed, that uses a promoterless version of the endogenous β-galactosidase gene
lacH
as a reporter gene and that allows integration of promoters cloned in front of
lacH
into the lactose utilization gene cluster by homologous recombination. The system was applied to analyze carbon catabolite repression of
S. xylosus
promoters by the catabolite control protein CcpA. To test if
lacH
is a suitable reporter gene, β-galactosidase activities directed by two promoters known to be subject to CcpA regulation were measured. In these experiments, repression of the
malRA
maltose utilization operon promoter and autoregulation of the
ccpA
promoters were confirmed, proving the applicability of the system. Subsequently, putative CcpA operators, termed catabolite-responsive elements (
cre
s), from promoter regions of several
S. xylosus
genes were tested for their ability to confer CcpA regulation upon a constitutive promoter, P

veg
II

. For that purpose,
cre
sequences were placed at position +3 or +4 within the transcribed region of P

veg
II

. Measurements of β-galactosidase activities in the presence or absence of glucose yielded repression ratios between two- and eightfold. Inactivation of
ccpA
completely abolished glucose-dependent regulation. Therefore, the tested
cre
s functioned as operator sites for CcpA. With promoters exclusively regulated by CcpA, signal transduction leading to CcpA activation in
S. xylosus
was examined. Glucose-dependent regulation was measured in a set of isogenic mutants showing defects in genes encoding glucose kinase GlkA, glucose uptake protein GlcU, and HPr kinase HPrK. GlkA and GlcU deficiency diminished glucose-dependent CcpA-mediated repression, but loss of HPr kinase activity abolished regulation. These results clearly show that HPr kinase provides the essential signal to activate CcpA in
S. xylosus
. Glucose uptake protein GlcU and glucose kinase GlkA participate in activation, but they are not able to trigger CcpA-mediated regulation independently from HPr kinase.
Clonal Population Structure of
Pseudomonas stutzeri
, a Species with Exceptional Genetic Diversity
Authors: Rius Núria , Fusté M. Carme , Guasp Caterina , Lalucat Jorge , Lorén José G. 
Published: July 2002

ABSTRACT

Genetic diversity and genetic relationships among 42
Pseudomonas stutzeri
strains belonging to several genomovars and isolated from different sources were investigated in an examination of 20 metabolic enzymes by multilocus enzyme electrophoresis analysis. Forty-two distinct allele profiles were identified, indicating that all multilocus genotypes were represented by a single strain. All 20 loci were exceptionally polymorphic, with an average of 15.9 alleles per locus. To the best of our knowledge, this
P. stutzeri
sample exhibited the highest mean genetic diversity (
H
= 0.876) found to date in all bacterial species studied by multilocus enzyme electrophoresis. A high frequency of occurrence of null alleles was identified. The index of association (

I
A

) for the
P. stutzeri
strains analyzed was 1.10. The

I
A

values were always significantly different from zero for all subgroups studied, including clinical and environmental isolates and strains classified as genomovar 1. These results suggest that the population structure of
P. stutzeri
is strongly clonal, indicating that there is no significant level of assortative recombination that might destroy linkage disequilibrium.
Cloning, Sequencing, and Characterization of the Iturin A Operon
Authors: Tsuge Kenji , Akiyama Takanori , Shoda Makoto 
Published: July 2002

ABSTRACT

Bacillus subtilis
RB14 is a producer of the antifungal lipopeptide iturin A. Using a transposon, we identified and cloned the iturin A synthetase operon of RB14, and the sequence of this operon was also determined. The iturin A operon spans a region that is more than 38 kb long and is composed of four open reading frames,
ituD, ituA, ituB
, and
ituC
. The
ituD
gene encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production. The second gene,
ituA
, encodes a 449-kDa protein that has three functional modules homologous to fatty acid synthetase, amino acid transferase, and peptide synthetase. The third gene,
ituB
, and the fourth gene,
ituC
, encode 609- and 297-kDa peptide synthetases that harbor four and two amino acid modules, respectively. Mycosubtilin, which is produced by
B. subtilis
ATCC 6633, has almost the same structure as iturin A, but the amino acids at positions 6 and 7 in the mycosubtilin sequence are
d
-Ser→
l
-Asn, while in iturin A these amino acids are inverted (i.e.,
d
-Asn→
l
-Ser). Comparison of the amino acid sequences encoded by the iturin A operon and the mycosubtilin operon revealed that
ituD, ituA
, and
ituB
have high levels of homology to the counterpart genes
fenF
(79%),
mycA
(79%), and
mycB
(79%), respectively. Although the overall level of homology of the amino acid sequences encoded by
ituC
and
mycC
, the counterpart of
ituC
, is relatively low (64%), which indicates that there is a difference in the amino acid sequences of the two lipopeptides, the levels of homology between the putative serine adenylation domains and between the asparagine adenylation domains in the two synthetases are high (79 and 80%, respectively), implying that there is an intragenic domain change in the synthetases. The fact that the flanking sequence of the iturin A synthetase coding region was highly homologous to the flanking sequence that of
xynD
of
B. subtilis
168 and the fact that the promoter of the iturin A operon which we identified was also conserved in an upstream sequence of
xynD
imply that horizontal transfer of this operon occurred. When the promoter was replaced by the
repU
promoter of the plasmid pUB110 replication protein, production of iturin A increased threefold.
A 12-Amino-Acid Segment, Present in Type s2 but Not Type s1
Helicobacter pylori
VacA Proteins, Abolishes Cytotoxin Activity and Alters Membrane Channel Formation
Authors: McClain Mark S. , Cao Ping , Iwamoto Hideki , Vinion-Dubiel Arlene D. , Szabo Gabor , Shao Zhifeng , Cover Timothy L. 
Published: July 2002

ABSTRACT

Helicobacter pylori
, a gram-negative bacterium associated with gastritis, peptic ulceration, and gastric adenocarcinoma in humans, secretes a protein toxin, VacA, that causes vacuolar degeneration of epithelial cells. Several different families of
H. pylori vacA
alleles can be distinguished based on sequence diversity in the “middle” region (i.e., m1 and m2) and in the 5′ end of the gene (i.e., s1 and s2). Type s2 VacA toxins contain a 12-amino-acid amino-terminal hydrophilic segment, which is absent from type s1 toxins. To examine the functional properties of VacA toxins containing this 12-amino-acid segment, we analyzed a wild-type s1/m1 VacA and a chimeric s2/m1 VacA protein. Purified s1/m1 VacA from
H. pylori
strain 60190 induced vacuolation in HeLa and Vero cells, whereas the chimeric s2/m1 toxin (in which the s1 sequence of VacA from strain 60190 was replaced with the s2 sequence from strain Tx30a) lacked detectable cytotoxic activity. Type s1/m1 VacA from strain 60190 formed membrane channels in a planar lipid bilayer assay at a significantly higher rate than did s2/m1 VacA. However, membrane channels formed by type s1 VacA and type s2 VacA proteins exhibited similar anion selectivities (permeability ratio, P
Cl
/P
Na
= 5). When an equimolar mixture of the chimeric s2/m1 toxin and the wild-type s1/m1 toxin was added to HeLa cells, the chimeric toxin completely inhibited the activity of the s1/m1 toxin. Thus, the s2/m1 toxin exhibited a dominant-negative phenotype similar to that of a previously described mutant toxin, VacA-(Δ6–27). Immunoprecipitation experiments indicated that both s2/m1 VacA and VacA-(Δ6–27) could physically interact with a c-myc epitope-tagged s1/m1 VacA, which suggests that the dominant-negative phenotype results from the formation of heterooligomeric VacA complexes with defective functional activity. Despite detectable differences in the channel-forming activities and cytotoxic properties of type s1 and type s2 VacA proteins, the conservation of type s2 sequences in many
H. pylori
isolates suggests that type s2 VacA proteins retain an important biological activity.
Transcript Analysis of Multiple Copies of
amo
(Encoding Ammonia Monooxygenase) and
hao
(Encoding Hydroxylamine Oxidoreductase) in
Nitrosomonas europaea
Authors: Hommes Norman G. , Sayavedra-Soto Luis A. , Arp Daniel J. 
Published: July 2002

ABSTRACT

The genes encoding ammonia monooxygenase (
amoCAB
), hydroxylamine oxidoreductase (
hao
), and the
c
-type cytochrome
c
-554 (
hcy
) are present in multiple copies in the genome of
Nitrosomonas europaea.
The upstream regions of the two copies of
amoC
, the three copies of
hao
, and one copy of
hcy
were cloned and sequenced. Primer extension reactions were done to identify transcription start sites for these genes, as well as for
amoA
. Putative ς
70
promoter sequences were found associated with all but one of the mapped transcription start sites. Primer extensions were done with
amoC
primers using RNA harvested from cells incubated with and without ammonium. The experiments suggested that
N. europaea
cells may be able to use different promoters in the presence and absence of ammonium.
Initiation of Biofilm Formation by
Pseudomonas aeruginosa
57RP Correlates with Emergence of Hyperpiliated and Highly Adherent Phenotypic Variants Deficient in Swimming, Swarming, and Twitching Motilities
Authors: Déziel Eric , Comeau Yves , Villemur Richard 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
is a ubiquitous environmental bacterium capable of forming biofilms on surfaces as a survival strategy. It exhibits a large variety of competition/virulence factors, such as three types of motilities: flagellum-mediated swimming, flagellum-mediated swarming, and type IV pilus-mediated twitching. A strategy frequently used by bacteria to survive changing environmental conditions is to create a phenotypically heterogeneous population by a mechanism called phase variation. In this report, we describe the characterization of phenotypic variants forming small, rough colonies that spontaneously emerged when
P. aeruginosa
57RP was cultivated as a biofilm or in static liquid cultures. These small-colony (S) variants produced abundant type IV fimbriae, displayed defective swimming, swarming, and twitching motilities, and were impaired in chemotaxis. They also autoaggregated in liquid cultures and rapidly initiated the formation of strongly adherent biofilms. In contrast, the large-colony variant (parent form) was poorly adherent, homogeneously dispersed in liquid cultures, and produced scant polar fimbriae. Further analysis of the S variants demonstrated differences in a variety of other phenotypic traits, including increased production of pyocyanin and pyoverdine and reduced elastase activity. Under appropriate growth conditions, cells of each phenotype switched to the other phenotype at a fairly high frequency. We conclude that these S variants resulted from phase variation and were selectively enriched when
P. aeruginosa
57RP was grown as a biofilm or in static liquid cultures. We propose that phase variation ensures the prior presence of phenotypic forms well adapted to initiate the formation of a biofilm as soon as environmental conditions are favorable.