bluuebunny/crossref_metadata_2025_split huggingface dataset:
CtaA ofStaphylococcus aureusIs Required for Starvation Survival, Recovery, and Cytochrome Biosynthesis
Authors: Clements Mark O. , Watson Sean P. , Poole Robert K. , Foster Simon J. 
Published: December 2019

ABSTRACTAStaphylococcus aureusmutant (SPW3) apparently unable to survive long-term starvation was shown to have a transposon insertion within a gene homologous toctaAofBacillus subtiliswhich encodes a heme A synthase. Analysis of the cytochrome profiles of SPW3 revealed the absence of heme A-containing cytochromes compared to the parental 8325-4 strain. SPW3 demonstrated a 100-fold reduction in the ability to survive starvation induced by glucose limitation, under aerated conditions, compared to 8325-4. Analysis of starved cultures revealed that greater than 90% of the cells which demonstrated metabolism (as shown by rhodamine 123 accumulation) were unable to recover and form colonies on agar. Analysis of the lag phase and initial growth kinetics of those cells which could recover also showed a defect. This recovery defect could be partially alleviated by the inclusion of catalase in the recovery medium, indicating the probable involvement of oxidative stress. SPW3 also exhibited reduced colony size similar to that of a small-colony variant, increased resistance to aminoglycoside antibiotics, and reduced hemolysin and toxic shock syndrome toxin 1 production, but no alteration in the ability to form lesions in a subcutaneous mouse infection model.
cis
- and
trans
-Acting Elements Involved in Regulation of
aniA
, the Gene Encoding the Major Anaerobically Induced Outer Membrane Protein in
Neisseria gonorrhoeae
Authors: Householder Tracey C. , Belli Wesley A. , Lissenden Sarah , Cole Jeffrey A. , Clark Virginia L. 
Published: December 2019

ABSTRACT

AniA (formerly Pan1) is the major anaerobically induced outer membrane protein in
Neisseria gonorrhoeae
. AniA has been shown to be a major antigen in patients with gonococcal disease, and we have been studying its regulation in order to understand the gonococcal response to anaerobiosis and its potential role in virulence. This study presents a genetic analysis of
aniA
regulation. Through deletion analysis of the upstream region, we have determined the minimal promoter region necessary for
aniA
expression. This 130-bp region contains a sigma 70-type promoter and an FNR (fumarate and nitrate reductase regulator protein) binding site, both of which are absolutely required for anaerobic expression. Also located in the minimal promoter region are three T-rich direct repeats and several potential NarP binding sites. This 80-bp region is required for induction by nitrite. By site-directed mutagenesis of promoter sequences, we have determined that the transcription of
aniA
is initiated only from the sigma 70-type promoter. The gearbox promoter, previously believed to be the major promoter, does not appear to be active during anaerobiosis. The gonococcal FNR and NarP homologs are involved in the regulation of
aniA
, and we demonstrate that placing
aniA
under the control of the
tac
promoter compensates for the inability of a gonococcal
fnr
mutant to grow anaerobically.
The
ompB
Operon Partially Determines Differential Expression of OmpC in
Salmonella typhi
and
Escherichia coli
Authors: Martínez-Flores Irma , Cano Roxana , Bustamante Víctor H. , Calva Edmundo , Puente JoséLuis 
Published: December 2019

ABSTRACT

Expression of the
Escherichia coli
OmpC and OmpF outer membrane proteins is regulated by the osmolarity of the culture media. In contrast, expression of OmpC in
Salmonella typhi
is not influenced by osmolarity, while OmpF is regulated as in
E. coli
. To better understand the lack of osmoregulation of OmpC expression in
S. typhi
, we compared the expression of the
ompC
gene in
S. typhi
and
E. coli
, using
ompC-lacZ
fusions and outer membrane protein (OMP) electrophoretic profiles.
S. typhi ompC
expression levels in
S. typhi
were similar at low and high osmolarity along the growth curve, whereas osmoregulation of
E. coli ompC
in
E. coli
was observed during the exponential phase. Both genes were highly expressed at high and low osmolarity when present in
S. typhi
, while expression of both was regulated by osmolarity in
E. coli
. Complementation experiments with either the
S. typhi
or
E. coli ompB
operon in an
S. typhi ΔompB
strain carrying the
ompC-lacZ
fusions showed that both
S. typhi
and
E. coli ompC
were not regulated by osmolarity when they were under the control of
S. typhi ompB
. Interestingly, in the same strain, both genes were osmoregulated under
E. coli ompB
. Surprisingly, in
E. coli ΔompB
, they were both osmoregulated under
S. typhi
or
E. coli ompB
. Thus, the lack of osmoregulation of OmpC expression in
S. typhi
is determined in part by the
ompB
operon, as well as by other unknown
trans
-acting elements present in
S. typhi
.
The LysR Homolog LrhA Promotes RpoS Degradation by Modulating Activity of the Response Regulator SprE
Authors: Gibson Katherine E. , Silhavy Thomas J. 
Published: December 2019

ABSTRACT

Synthesis of the OmpF porin of
Escherichia coli
is regulated in response to environmental and growth phase signals. In order to identify constituents of the various regulatory pathways involved in modulating
ompF
transcriptional expression, transposon insertion mutagenesis was performed and mutations that increased
ompF′-lacZ
activity were identified as previously described. Mutations mapping to a previously identified gene of unknown function,
lrhA
, were obtained. We found that LrhA, a LysR homolog, functions as a regulatory component in the RpoS-dependent growth phase repression of
ompF
. In addition to altered growth phase regulation of
ompF
, these
lrhA
mutants have pleiotropic stationary-phase defects as a result of decreased RpoS levels. We provide evidence that LrhA promotes degradation of RpoS by functioning within a genetic pathway that includes the response regulator SprE and the ClpXP protease. LrhA functions upstream of the other components in the pathway and appears to modulate the activity of SprE.
Phosphorylation and Functional Properties of the IIA Domain of the Lactose Transport Protein of
Streptococcus thermophilus
Authors: Gunnewijk Marga G. W. , Postma Pieter W. , Poolman Bert 
Published: December 2019

ABSTRACT

The lactose-H
+
symport protein (LacS) of
Streptococcus thermophilus
has a carboxyl-terminal regulatory domain (IIA
LacS
) that is homologous to a family of proteins and protein domains of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in various organisms, of which IIA
Glc
of
Escherichia coli
is the best-characterized member. On the basis of these similarities, it was anticipated that IIA
LacS
would be able to perform one or more functions associated with IIA
Glc
, i.e., carry out phosphoryl transfer and/or affect other catabolic functions. The gene fragment encoding IIA
LacS
was overexpressed in
Escherichia coli
, and the protein was purified in two steps by metal affinity and anion-exchange chromatography. IIA
LacS
was unable to restore glucose uptake in a IIA
Glc
-deficient strain, which is consistent with a very low rate of phosphorylation of IIA
LacS
by phosphorylated HPr (HPr∼P) from
E. coli
. With HPr∼P from
S. thermophilus
, the rate was more than 10-fold higher, but the rate constants for the phosphorylation of IIA
LacS
(
k
1
= 4.3 × 10
2
M
−1
s
−1
) and dephosphorylation of IIA
LacS
∼P by HPr (
k
−1
= 1.1 × 10
3
M
−1
s
−1
) are still at least 4 orders of magnitude lower than for the phosphoryltransfer between IIA
Glc
and HPr from
E. coli
. This finding suggests that IIA
LacS
has evolved into a protein domain whose main function is not to transfer phosphoryl groups rapidly. On the basis of sequence alignment of IIA proteins with and without putative phosphoryl transfer functions and the known structure of IIA
Glc
, we constructed a double mutant [IIA
LacS
(I548E/G556D)] that was predicted to have increased phosphoryl transfer activity. Indeed, the phosphorylation rate of IIA
LacS
(I548E/G556D) by HPr∼P increased (
k
1
= 4.0 × 10
3
M
−1
s
−1
) and became nearly independent of the source of HPr∼P (
S. thermophilus
,
Bacillus subtilis
, or
E. coli
). The increased phosphoryl transfer rate of IIA
LacS
(I548E/G556D) was insufficient to complement IIA
Glc
in PTS-mediated glucose transport in
E. coli
. Both IIA
LacS
and IIA
LacS
(I548E/G556D) could replace IIA
Glc
, but in another function: they inhibited glycerol kinase (inducer exclusion) when present in the unphosphorylated form.
The Mithramycin Gene Cluster of
Streptomyces argillaceus
Contains a Positive Regulatory Gene and Two Repeated DNA Sequences That Are Located at Both Ends of the Cluster
Authors: Lombó Felipe , Braña Alfredo F. , Méndez Carmen , Salas José A. 
Published: December 2019

ABSTRACT

Sequencing of a 4.3-kb DNA region from the chromosome of
Streptomyces argillaceus
, a mithramycin producer, revealed the presence of two open reading frames (ORFs). The first one (
orfA
) codes for a protein that resembles several transport proteins. The second one (
mtmR
) codes for a protein similar to positive regulators involved in antibiotic biosynthesis (DnrI, SnoA, ActII-orf4, CcaR, and RedD) belonging to the
Streptomyces
antibiotic regulatory protein (SARP) family. Both ORFs are separated by a 1.9-kb, apparently noncoding region. Replacement of the
mtmR
region by an antibiotic resistance cassette completely abolished mithramycin biosynthesis. Expression of
mtmR
in a high-copy-number vector in
S. argillaceus
caused a 16-fold increase in mithramycin production. The
mtmR
gene restored actinorhodin production in
Streptomyces coelicolor
JF1 mutant, in which the actinorhodin-specific activator ActII-orf4 is inactive, and also stimulated actinorhodin production by
Streptomyces lividans
TK21. A 241-bp region located 1.9 kb upstream of
mtmR
was found to be repeated approximately 50 kb downstream of
mtmR
at the other end of the mithramycin gene cluster. A model to explain a possible route for the acquisition of the mithramycin gene cluster by
S. argillaceus
is proposed.
Cyclic AMP Receptor Protein and TyrR Are Required for Acid pH and Anaerobic Induction of
hyaB
and
aniC
in
Salmonella typhimurium
Authors: Park Kyeong R. , Giard Jean-Christophe , Eom Juno H. , Bearson Shawn , Foster John W. 
Published: December 2019

ABSTRACT

Two acid-inducible genes,
aniC
and
aciK
, that require anaerobiosis and tyrosine for expression were identified as
orf326a
encoding a potential amino acid/polyamine antiporter and
hyaB
encoding hydrogenase I, respectively. Cyclic AMP (cAMP) receptor protein, cAMP, and TyrR, regulator of aromatic amino acid metabolism, were strong positive regulators of both genes.
Genetic and Biochemical Characterization of a High-Affinity Betaine Uptake System (BusA) in
Lactococcus lactis
Reveals a New Functional Organization within Bacterial ABC Transporters
Authors: Obis David , Guillot Alain , Gripon Jean-Claude , Renault Pierre , Bolotin Alexander , Mistou Michel-Yves 
Published: December 2019

ABSTRACT

The cytoplasmic accumulation of exogenous betaine stimulates the growth of
Lactococcus lactis
cultivated under hyperosmotic conditions. We report that
L. lactis
possesses a single betaine transport system that belongs to the ATP-binding cassette (ABC) superfamily of transporters. Through transposon mutagenesis, a mutant deficient in betaine transport was isolated. We identified two genes,
busAA
and
busAB
, grouped in an operon,
busA
(betaine uptake system). The transcription of
busA
is strongly regulated by the external osmolality of the medium. The
busAA
gene codes for the ATP-binding protein.
busAB
encodes a 573-residue polypeptide which presents two striking features: (i) a fusion between the regions encoding the transmembrane domain (TMD) and the substrate-binding domain (SBD) and (ii) a swapping of the SBD subdomains when compared to the
Bacillus subtilis
betaine-binding protein, OpuAC. BusA of
L. lactis
displays a high affinity towards betaine (

K
m

= 1.7 μM) and is an osmosensor whose activity is tightly regulated by external osmolality, leading the betaine uptake capacity of
L. lactis
to be under dual control at the biochemical and genetic levels. A protein presenting the characteristics predicted for BusAB was detected in the membrane fraction of
L. lactis
. The fusion between the TMD and the SBD is the first example of a new organization within prokaryotic ABC transporters.
Cloning and Sequence Analysis of Two
Pseudomonas
Flavoprotein Xenobiotic Reductases
Authors: Blehert David S. , Fox Brian G. , Chambliss Glenn H. 
Published: December 2019

ABSTRACT

The genes encoding flavin mononucleotide-containing oxidoreductases, designated xenobiotic reductases, from
Pseudomonas putida
II-B and
P. fluorescens
I-C that removed nitrite from nitroglycerin (NG) by cleavage of the nitroester bond were cloned, sequenced, and characterized. The
P. putida
gene,
xenA
, encodes a 39,702-Da monomeric, NAD(P)H-dependent flavoprotein that removes either the terminal or central nitro groups from NG and that reduces 2-cyclohexen-1-one but did not readily reduce 2,4,6-trinitrotoluene (TNT). The
P. fluorescens
gene,
xenB
, encodes a 37,441-Da monomeric, NAD(P)H-dependent flavoprotein that exhibits fivefold regioselectivity for removal of the central nitro group from NG and that transforms TNT but did not readily react with 2-cyclohexen-1-one. Heterologous expression of
xenA
and
xenB
was demonstrated in
Escherichia coli
DH5α. The transcription initiation sites of both
xenA
and
xenB
were identified by primer extension analysis. BLAST analyses conducted with the
P. putida xenA
and the
P. fluorescens xenB
sequences demonstrated that these genes are similar to several other bacterial genes that encode broad-specificity flavoprotein reductases. The prokaryotic flavoprotein reductases described herein likely shared a common ancestor with old yellow enzyme of yeast, a broad-specificity enzyme which may serve a detoxification role in antioxidant defense systems.
rpoS
Function Is Essential for
bgl
Silencing Caused by C-Terminally Truncated H-NS in
Escherichia coli
Authors: Ohta Tomoko , Ueguchi Chiharu , Mizuno Takeshi 
Published: December 2019

ABSTRACT

From evolutionary and physiological viewpoints, the
Escherichia coli bgl
operon is intriguing because its expression is silent (Bgl
−
phenotype), at least under several laboratory conditions. H-NS, a nucleoid protein, is known as a DNA-binding protein involved in
bgl
silencing. However, we previously found that
bgl
expression is still silent in a certain subset of
hns
mutations, each of which results in a defect in its DNA-binding ability. Based on this fact, we proposed a model in which a postulated DNA-binding protein(s) has an adapter function by interacting with both the
cis
-acting element of the
bgl
promoter and the mutated H-NS. To identify such a presumed adapter molecule, we attempted to isolate mutants exhibiting the Bgl
+
phenotype in the background of
hns60
, encoding the mutant H-NS protein lacking the DNA-binding domain by random insertion mutagenesis with the mini-Tn
10cam
transposon. These isolated mutations were mapped to five loci on the chromosome. Among these loci, three appeared to be
leuO
,
hns
, and
bglJ
, which were previously characterized, while the other two were novel. Genetic analysis revealed that the two insertions are within the
rpoS
gene and in front of the
lrhA
gene, respectively. The former encodes the stationary-phase-specific sigma factor, ς
S
, and the latter encodes a LysR-like DNA-binding protein. It was found that ς
S
is defective in both types of mutant cells. These results showed that the
rpoS
function is involved in the mechanism underlying
bgl
silencing, at least in the
hns60
background used in this study. We also examined whether the H-NS homolog StpA has such an adapter function, as was previously proposed. Our results did not support the idea that StpA has an adapter function in the genetic background used.
Campylobacter jejuni
Contains Two Fur Homologs: Characterization of Iron-Responsive Regulation of Peroxide Stress Defense Genes by the PerR Repressor
Authors: van Vliet Arnoud H. M. , Baillon Marie-Louise A. , Penn Charles W. , Ketley Julian M. 
Published: December 2019

ABSTRACT

Expression of the peroxide stress genes alkyl hydroperoxide reductase (
ahpC
) and catalase (
katA
) of the microaerophile
Campylobacter jejuni
is repressed by iron. Whereas iron repression in gram-negative bacteria is usually carried out by the Fur protein, previous work showed that this is not the case in
C. jejuni
, as these genes are still iron repressed in a
C. jejuni fur
mutant. An open reading frame encoding a Fur homolog (designated PerR for “peroxide stress regulator”) was identified in the genome sequence of
C. jejuni
. The
perR
gene was disrupted by a kanamycin resistance cassette in
C. jejuni
wild-type and
fur
mutant strains. Subsequent characterization of the
C. jejuni perR
mutants showed derepressed expression of both AhpC and KatA at a much higher level than that obtained by iron limitation, suggesting that expression of these genes is controlled by other regulatory factors in addition to the iron level. Other iron-regulated proteins were not affected by the
perR
mutation. The
fur perR
double mutant showed derepressed expression of known iron-repressed genes. Further phenotypic analysis of the
perR
mutant,
fur
mutant, and the
fur perR
double mutant showed that the
perR
mutation made
C. jejuni
hyperresistant to peroxide stress caused by hydrogen peroxide and cumene hydroperoxide, a finding consistent with the high levels of KatA and AhpC expression, and showed that these enzymes were functional. Quantitative analysis of KatA expression showed that both the
perR
mutation and the
fur
mutation had profound effects on catalase activity, suggesting additional non-iron-dependent regulation of KatA and, by inference, AhpC. The PerR protein is a functional but nonhomologous substitution for the OxyR protein, which regulates peroxide stress genes in other gram-negative bacteria. Regulation of peroxide stress genes by a Fur homolog has recently been described for the gram-positive bacterium
Bacillus subtilis. C. jejuni
is the first gram-negative bacterium where non-OxyR regulation of peroxide stress genes has been described and characterized.
A Novel Role for
Escherichia coli
Endonuclease VIII in Prevention of Spontaneous G→T Transversions
Authors: Blaisdell Jeffrey O. , Hatahet Zafer , Wallace Susan S. 
Published: December 2019

ABSTRACT

In the bacterium
Escherichia coli
, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the
nth
and
nei
genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C→A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C→T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in
fpg mutY
double mutants was significantly enhanced by the addition of the
nei
mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C→T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.
Mechanism of Repression of the
aroP
P2 Promoter by the TyrR Protein of
Escherichia coli
Authors: Yang Ji , Wang Peixiang , Pittard A. J. 
Published: December 2019

ABSTRACT

Previously, we have shown that expression of the
Escherichia coli aroP
P2 promoter is partially repressed by the TyrR protein alone and strongly repressed by the TyrR protein in the presence of the coeffector tyrosine or phenylalanine (P. Wang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:4206–4212, 1997). Here we present in vitro results showing that the TyrR protein and RNA polymerase can bind simultaneously to the
aroP
P2 promoter. In the presence of tyrosine, the TyrR protein inhibits open complex formation at the P2 promoter, whereas in the absence of any coeffector or in the presence of phenylalanine, the TyrR protein inhibits a step(s) following the formation of open complexes. We also present mutational evidence which implicates the N-terminal domain of the TyrR protein in the repression of P2 expression. The TyrR binding site of
aroP
, which includes one weak and one strong TyrR box, is located 5 bp downstream of the transcription start site of P2. Results from a mutational analysis show that the strong box (which is located more closely to the P2 promoter), but not the weak box, plays a critical role in P2 repression.
Identification and Characterization of the Single-Stranded DNA-Binding Protein of Bacteriophage P1
Authors: Lehnherr Hansjörg , Bendtsen Jannick D. , Preuss Fabian , Ilyina Tatiana V. 
Published: December 2019

ABSTRACT

The genome of bacteriophage P1 harbors a gene coding for a 162-amino-acid protein which shows 66% amino acid sequence identity to the
Escherichia coli
single-stranded DNA-binding protein (SSB). The expression of the P1 gene is tightly regulated by P1 immunity proteins. It is completely repressed during lysogenic growth and only weakly expressed during lytic growth, as assayed by an
ssb
-P1/
lacZ
fusion construct. When cloned on an intermediate-copy-number plasmid, the P1 gene is able to suppress the temperature-sensitive defect of an
E. coli ssb
mutant, indicating that the two proteins are functionally interchangeable. Many bacteriophages and conjugative plasmids do not rely on the SSB protein provided by their host organism but code for their own SSB proteins. However, the close relationship between SSB-P1 and the SSB protein of the P1 host,
E. coli
, raises questions about the functional significance of the phage protein.
Expression of P
II
and Glutamine Synthetase Is Regulated by P
II
, the
ntrBC
Products, and Processing of the
glnBA
mRNA in
Rhodospirillum rubrum
Authors: Cheng Jiujun , Johansson Magnus , Nordlund Stefan 
Published: December 2019

ABSTRACT

We have studied the transcription of the
glnB
and
glnA
genes in
Rhodospirillum rubrum
with firefly luciferase as a reporter enzyme. Under NH
4
+
and N
2
conditions,
glnBA
was cotranscribed from a weak and a strong promoter. In nitrogen-fixing cultures, activity of the latter was highly enhanced by NtrC, but transcription from both promoters occurred under both conditions. There is no promoter controlling transcription of
glnA
alone, supporting our proposal that the
glnA
mRNA is produced by processing.
Contribution of the Cell Wall Component Teichuronopeptide to pH Homeostasis and Alkaliphily in the Alkaliphile
Bacillus lentus
C-125
Authors: Aono Rikizo , Ito Masahiro , Machida Takayoshi 
Published: December 2019

ABSTRACT

A teichuronopeptide (TUP) is one of major structural components of the cell wall of the facultative alkaliphilic strain
Bacillus lentus
C-125. A mutant defective in TUP synthesis grows slowly at alkaline pH. An upper limit of pH for growth of the mutant was 10.4, while that of the parental strain C-125 was 10.8. Gene
tupA
, directing synthesis of TUP, was cloned from C-125 chromosomal DNA. The primary translation product of this gene is likely a cytoplasmic protein (57.3 kDa) consisting of 489 amino acid residues. Introduction of the
tupA
gene into the TUP-defective mutant complemented the mutation responsible for the pleiotropic phenotypes of the mutant, leading to simultaneous disappearance of the defect in TUP synthesis, the diminished ability for cytoplasmic pH homeostasis, and the low tolerance for alkaline conditions. These results demonstrate that the acidic polymer TUP in the cell wall plays a role in pH homeostasis in this alkaliphile.