bluuebunny/crossref_metadata_2025_split huggingface dataset:
Redox-Dependent Gene Regulation in
Rhodobacter sphaeroides
2.4.1
T
: Effects on Dimethyl Sulfoxide Reductase (
dor
) Gene Expression
Authors: Mouncey Nigel J. , Kaplan Samuel 
Published: December 2019

ABSTRACT

The ability of
Rhodobacter sphaeroides
2.4.1
T
to respire anaerobically with the alternative electron acceptor dimethyl sulfoxide (DMSO) or trimethylamine
N
-oxide (TMAO) is manifested by the molybdoenzyme DMSO reductase, which is encoded by genes of the
dor
locus. Previously, we have demonstrated that
dor
expression is regulated in response to lowered oxygen tensions and the presence of DMSO or TMAO in the growth medium. Several regulatory proteins have been identified as key players in this regulatory cascade: FnrL, DorS-DorR, and DorX-DorY. To further examine the role of redox potentiation in the regulation of
dor
expression, we measured DMSO reductase synthesis and β-galactosidase activity from
dor
::
lacZ
fusions in strains containing mutations in the redox-active proteins CcoP and RdxB, which have previously been implicated in the generation of a redox signal affecting photosynthesis gene expression. Unlike the wild-type strain, both mutants were able to synthesize DMSO reductase under strictly aerobic conditions, even in the absence of DMSO. When cells were grown photoheterotrophically,
dorC
::
lacZ
expression was stimulated by increasing light intensity in the CcoP mutant, whereas it is normally repressed in the wild-type strain under such conditions. Furthermore, the expression of genes encoding the DorS sensor kinase and DorR response regulator proteins was also affected by the
ccoP
mutation. By using CcoP-DorR and CcoP-DorY double mutants, it was shown that the DorR protein is strictly required for altered
dor
expression in CcoP mutants. These results further demonstrate a role for redox-generated responses in the expression of genes encoding DMSO reductase in
R. sphaeroides
and identify the DorS-DorR proteins as a redox-dependent regulatory system controlling
dor
expression.
Identification of an N-Terminal Region of Sigma 54 Required for Enhancer Responsiveness
Authors: Syed Adeela , Gralla Jay D. 
Published: December 2019

ABSTRACT
Sigma 54 associates with bacterial core RNA polymerase and converts it into an enhancer-responsive enzyme. Deletion of the N-terminal 40 amino acids is known to result in loss of the ability to respond to enhancer binding proteins. In this work PCR mutagenesis and genetic screens were used to identify a small patch, from amino acids 33 to 37, that is required for proper response to activator in vivo. Site-directed single point mutants within this segment were constructed and studied. Two of these were defective in responding to the enhancer binding protein in vitro. The mutants could still direct the polymerase to bind to DNA and initiate transient melting. However, they failed in directing activator-dependent formation of a heparin-stable open complex. Thus, amino acid region 33 to 37 includes critical activation response determinants. This region overlaps the larger leucine patch negative-control region, suggesting that anti-inhibition and positive activation are closely coupled events.
The Phenolic Recognition Profiles of the
Agrobacterium tumefaciens
VirA Protein Are Broadened by a High Level of the Sugar Binding Protein ChvE
Authors: Peng Wen-Tao , Lee Yong-Woog , Nester Eugene W. 
Published: December 2019

ABSTRACT

The formation of crown gall tumors by
Agrobacterium tumefaciens
requires that the virulence (
vir
) genes be induced by chemical signals which consist of specific phenolic compounds and monosaccharides, synthesized at plant wound sites. Signal transduction in the activation of these genes is mediated by the VirA-VirG two-component regulatory system, together with ChvE, a glucose-galactose binding protein which interacts with VirA. We have previously presented genetic evidence that
virA
senses phenolic compounds directly (Y.-W. Lee, S. Jin, W.-S. Sim, and E. W. Nester, Proc. Natl. Acad. Sci. USA 92:12245–12249, 1995). The
vir
genes of strain KU12 can be induced by 4-hydroxyacetophenone,
p
-coumaric acid, and phenol, whereas these same phenolic compounds are weak inducers of the
vir
genes of strain A6. In this report, we show that a specific inducing sugar can broaden the specificity of the phenolic compound which VirA senses. 4-Hydroxyacetophenone and other related phenolic compounds function as inducing phenolic compounds with the
virA
gene of A6 if arabinose replaces glucose as the inducing sugar. We further demonstrate that this broadened specificity for phenolic inducers results from the increased level of ChvE through induction by arabinose via the regulatory protein GbpR. If high levels of ChvE are present, then poorly inducing phenolic compounds can induce the
vir
genes to high levels in combination with glucose. Comparing the induction response of the wild type and that of a VirA mutant with a mutation in its receiver domain revealed that the activity of the receiver domain is controlled by the periplasmic domain. We discuss these observations in terms of how VirA senses and transduces signals elicited by the two classes of plant signal molecules.
Induction of
ermAMR
from a Clinical Strain of
Enterococcus faecalis
by 16-Membered-Ring Macrolide Antibiotics
Authors: Oh Tae-Gwon , Kwon Ae-Ran , Choi Eung-Chil 
Published: December 2019

ABSTRACT

We cloned the MLS
B
resistance determinant by PCR from a clinical isolate of
Enterococcus faecalis
373, which is induced more strongly by a 16-membered-ring macrolide, tylosin, than by erythromycin. To elucidate the molecular basis of resistance of
E. faecalis
373, we analyzed the cloned gene, designated
ermAMR
, by site-directed mutagenesis and reporter gene assay. Our results showed that an arginine-to-cysteine change in the seventh codon of the putative leader peptide endowed tylosin with resistance inducibility and that TAAA duplication enabled the control region to express the downstream methylase gene at a drastically increased level.
Identification of a Zinc-Specific Metalloregulatory Protein, Zur, Controlling Zinc Transport Operons in
Bacillus subtilis
Authors: Gaballa Ahmed , Helmann John D. 
Published: December 2019

ABSTRACT

Zinc is an essential nutrient for all cells, but remarkably little is known regarding bacterial zinc transport and its regulation. We have identified three of the key components acting to maintain zinc homeostasis in
Bacillus subtilis
. Zur is a metalloregulatory protein related to the ferric uptake repressor (Fur) family of regulators and is required for the zinc-specific repression of two operons implicated in zinc uptake,
yciC
and
ycdHIyceA
. A
zur
mutant overexpresses the 45-kDa YciC membrane protein, and purified Zur binds specifically, and in a zinc-responsive manner, to an operator site overlapping the
yciC
control region. A similar operator precedes the
ycdH
-containing operon, which encodes an ABC transporter. Two lines of evidence suggest that the
ycdH
operon encodes a high-affinity zinc transporter whereas YciC may function as part of a lower-affinity pathway. First, a
ycdH
mutant is impaired in growth in low-zinc medium, and this growth defect is exacerbated by the additional presence of a
yciC
mutation. Second, mutation of
ycdH
, but not
yciC
, alters the regulation of both the
yciC
and
ycdH
operons such that much higher levels of exogenous zinc are required for repression. We conclude that Zur is a Fur-like repressor that controls the expression of two zinc homeostasis operons in response to zinc. Thus, Fur-like regulators control zinc homeostasis in addition to their previously characterized roles in regulating iron homeostasis, acid tolerance responses, and oxidative stress functions.
Targeted Mutagenesis of ς
54
Activator Proteins in
Myxococcus xanthus
Authors: Gorski Lisa , Kaiser Dale 
Published: December 2019

ABSTRACT

Myxococcus xanthus
DNA segments related to the highly conserved central sequence of ς
54
activator proteins have been investigated. A genetic technique designed to inactivate a gene that encodes such an activator by inserting a plasmid-borne internal fragment of the putative gene has been tested. When the internal fragment inserted by homologous recombination into the corresponding chromosomal locus, the expected duplication of the gene was observed by Southern hybridization. The single restriction fragment characteristic of each segment was replaced in the insertion strains by two hybridizing fragments, and one of these fragments hybridized with the kanamycin resistance gene of the plasmid vector. The combined molecular weights of the two fragments from the insertion strains were equal to the molecular weight of the original fragment plus the expected molecular weight contributed by the vector. In the duplication, one copy is expected to have an N-terminal deletion and the other copy is expected to have a C-terminal deletion. In most cases, the net result should be loss of activator function. If an activator is essential for vegetative growth, then it should not be possible to obtain the insertion strain by plasmid integration. Indeed, integrants for three of the segments were not obtained in repeated trials; however, a plausible explanation for these results other than lethality can be offered. Of the seven insertions validated by Southern hybridization, four strains exhibited defects in the development of fruiting bodies. One of these failed to develop in submerged culture, though it developed normally on agar. The other three showed arrested development of fruiting bodies, each at a morphologically different stage of aggregation. One of the mutants may be defective in the reception pathway of A-signal.
Characterization of the Lysogenic Bacteriophage MAV1 from
Mycoplasma arthritidis
Authors: Voelker LeRoy L. , Dybvig Kevin 
Published: December 2019

ABSTRACT

The lysogenic bacteriophage MAV1, which is associated with the arthritogenicity of
Mycoplasma arthritidis
, was characterized. Several strains of
M. arthritidis
were examined for their ability to support growth of MAV1. A PFU assay was developed, and the sensitivity of phage to various chemical treatments was assayed. The most notable result was the resistance of MAV1 to proteinase K. The MAV1 genome is a double-stranded, linear DNA molecule of about 16 kb. The site of MAV1 DNA integration in the host chromosome was investigated. The ends of MAV1 DNA were cloned from three independent lysogens shown to have MAV1 DNA inserted at different sites in the host. The nucleotide sequences of the ends of the MAV1 genome and of the MAV1 DNA-chromosomal DNA junctions from each of three lysogens were determined. Sequences flanking the integrated prophage and the ends of native MAV1 DNA were determined, allowing the identification of the phage DNA (
attP
) and bacterial DNA (
attB
) recombination sites. Analysis of the left MAV1 DNA-chromosomal DNA junction sites showed a single-base heterogeneity located within MAV1 DNA sequences immediately adjacent to the
attB
sequence. A model for MAV1 integration-excision is proposed.
Corynebacterium glutamicum
Is Equipped with Four Secondary Carriers for Compatible Solutes: Identification, Sequencing, and Characterization of the Proline/Ectoine Uptake System, ProP, and the Ectoine/Proline/Glycine Betaine Carrier, EctP
Authors: Peter Heidi , Weil Brita , Burkovski Andreas , Krämer Reinhard , Morbach Susanne 
Published: December 2019

ABSTRACT

Gram-positive soil bacterium
Corynebacterium glutamicum
uses the compatible solutes glycine betaine, proline, and ectoine for protection against hyperosmotic shock. Osmoregulated glycine betaine carrier BetP and proline permease PutP have been previously characterized; we have identified and characterized two additional osmoregulated secondary transporters for compatible solutes in
C. glutamicum
, namely, the proline/ectoine carrier, ProP, and the ectoine/glycine betaine/proline carrier, EctP. A Δ
betP ΔputP ΔproP ΔectP
mutant was unable to respond to hyperosmotic stress, indicating that no additional uptake system for these compatible solutes is present. Osmoregulated ProP consists of 504 residues and preferred proline (

K
m

, 48 μM) to ectoine (

K
m

, 132 μM). The
proP
gene could not be expressed from its own promoter in
C. glutamicum
; however, expression was observed in
Escherichia coli
. ProP belongs to the major facilitator superfamily, whereas EctP, together with the betaine carrier, BetP, is a member of a newly established subfamily of the sodium/solute symporter superfamily. The constitutively expressed
ectP
codes for a 615-residue transporter. EctP preferred ectoine (

K
m

, 63 μM) to betaine (

K
m

, 333 μM) and proline (

K
m

, 1,200 μM). Its activity was regulated by the external osmolality. The related betaine transporter, BetP, could be activated directly by altering the membrane state with local anesthetics, but this was not the case for EctP. Furthermore, the onset of osmotic activation was virtually instantaneous for BetP, whereas it took about 10 s for EctP.
Transport of Intact Porphyrin by HpuAB, the Hemoglobin-Haptoglobin Utilization System ofNeisseria meningitidis
Authors: Lewis L. A. , Sung M.-H. , Gipson M. , Hartman K. , Dyer D. W. 
Published: December 2019

ABSTRACTThe meningococcalhemAgene was cloned and used to construct a porphyrin biosynthesis mutant. An analysis of thehemAmutant indicated that meningococci can transport intact porphyrin from heme (Hm), hemoglobin (Hb), and Hb-haptoglobin (Hp). By constructing a HemA−HpuAB−double mutant, we demonstrated that HpuAB is required for the transport of porphyrin from Hb and Hb-Hp.
An Upstream Sequence Element Required for NucC-Dependent Expression of the
Serratia marcescens
Extracellular Nuclease
Authors: Winslow Robert H. , Julien Bryan , Calendar Richard , Christie Gail E. 
Published: December 2019

ABSTRACT

The
Serratia marcescens
extracellular nuclease gene,
nucA
, is positively regulated by the product of the
nucC
gene. In this study, the upstream region required for NucC-dependent nuclease expression was defined by using fusions to the gene encoding chloramphenicol acetyltransferase (
cat
). This sequence includes an element of hyphenated dyad symmetry identified previously as the binding site for the P2 Ogr family of activators. Footprint analysis confirmed that members of this family of activator proteins bind to this site, protecting a region between −76 and −59 relative to the start of transcription. The activator binding site in the
nucA
promoter lies one turn of the helix upstream from the corresponding sites in the P2 and P4 late promoters. The effects of deletions between the downstream end of the activator binding site and the putative −35 region are consistent with a strict helical phasing requirement for activation.
Sequence Diversity of the
oprI
Gene, Coding for Major Outer Membrane Lipoprotein I, among rRNA Group I Pseudomonads
Authors: De Vos Daniel , Bouton Christiane , Sarniguet Alain , De Vos Paul , Vauterin Marc , Cornelis Pierre 
Published: December 2019

ABSTRACT

The sequence of
oprI
, the gene coding for the major outer membrane lipoprotein I, was determined by PCR sequencing for representatives of 17 species of rRNA group I pseudomonads, with a special emphasis on
Pseudomonas aeruginosa
and
Pseudomonas fluorescens
. Within the
P. aeruginosa
species,
oprI
sequences for 25 independent isolates were found to be identical, except for one silent substitution at position 96. The
oprI
sequences diverged more for the other rRNA group I pseudomonads (85 to 91% similarity with
P. aeruginosa oprI
). An accumulation of silent and also (but to a much lesser extent) nonsilent substitutions in the different sequences was found. A clustering according to the respective presence and/or positions of the
Hae
III,
Pvu
II, and
Sph
I sites could also be obtained. A sequence cluster analysis showed a rather widespread distribution of
P. fluorescens
isolates. All other rRNA group I pseudomonads clustered in a manner that was in agreement with other studies, showing that the
oprI
gene can be useful as a complementary phylogenetic marker for classification of rRNA group I pseudomonads.
Gene Organization and Transcription Analysis of the
Agrobacterium tumefaciens
Glycogen (
glg
) Operon: Two Transcripts for the Single Phosphoglucomutase Gene
Authors: Ugalde Juan E. , Lepek Viviana , Uttaro Antonio , Estrella Julia , Iglesias Alberto , Ugalde Rodolfo A. 
Published: December 2019

ABSTRACT

The gene organization and transcription of the
Agrobacterium glg
operon differ from those in other bacteria.
Agrobacterium tumefaciens
A348 contains a 9.1-kb gene cluster harboring genes for glycogen metabolism. The nucleotide sequence and gene organization of a region containing ADP-glucose pyrophosphorylase (
glgC
), glycogen synthetase (
glgA
), and phosphoglucomutase (
pgm
) genes have been previously described (A. Uttaro and R. A. Ugalde, Gene 150:117–122, 1994). In this work we report that the glycogen phosphorylase (
glgP
) and branching enzyme (
glgB
) genes are located immediately upstream of this region. The complete nucleotide sequences of the
glgP
and
glgB
genes were obtained, and mutants were constructed by targeted insertional mutagenesis with a kanamycin cassette. Enzymatic assays and reverse transcription PCR carried out with the wild type and with
glgP
and
glgB
mutants, as well as primer extension experiments and β-galactosidase fusions, revealed that this region containing five open reading frames (
glgPBCA
and
pgm
) is transcribed unidirectionally as a single operon under the control of a promoter located upstream of the glycogen phosphorylase gene (
glgP
). An alternative transcript was identified starting 168 bp upstream of an internal ATG start codon of the
pgm
gene, which is translated as a 71-amino-acid-shorter Pgm protein which complements in vivo a
pgm
mutant. This alternative transcript has a promoter with the motif TATCAAN
5
G, identified in octopine Ti plasmid as an autoinducible TraR promoter. This promoter is >200 times more efficient in
A. tumefaciens
than in
Escherichia coli
, as judged by the level of enzymatic activity of a
lacZ-pgm
fusion.
Stability of the
Agrobacterium tumefaciens
VirB10 Protein Is Modulated by Growth Temperature and Periplasmic Osmoadaption
Authors: Banta Lois M. , Bohne Jutta , Lovejoy S. Dawn , Dostal Kathleen 
Published: December 2019

ABSTRACT

Export of oncogenic T-DNA from the phytopathogen
Agrobacterium tumefaciens
is mediated by the products of the
virB
operon. It has recently been reported (K. J. Fullner and E. W. Nester, J. Bacteriol. 178:1498–1504, 1996) that DNA transfer does not occur at elevated temperatures; these observations correlate well with much earlier studies on the temperature sensitivity of crown gall tumor development on plants. In testing the hypothesis that this loss of DNA movement reflects a defect in assembly or maintenance of a stable DNA transfer machinery at high temperature, we have found that steady-state levels of VirB10 are sensitive to growth temperature while levels of several other VirB proteins are considerably less affected. This temperature-dependent failure to accumulate VirB10 is exacerbated in an attachment-deficient mutant strain (
chvB
) which exhibits pleiotropic defects in periplasmic osmoadaption, and virulence of a
chvB
mutant can be partially restored by lowering the temperature at which the bacteria and the plant tissue are cocultivated. Furthermore, the stability of VirB10 is diminished in cells lacking functional VirB9, but only under conditions of low osmolarity. We propose that newly synthesized VirB10 is inherently labile in the presence of a large osmotic gradient across the inner membrane and is rapidly degraded unless it is stabilized by VirB9-dependent assembly into oligomeric complexes. The possibility that VirB10-containing complexes are not assembled properly at elevated temperatures suggests an explanation for the decades-old observation that tumor formation is exquisitely sensitive to ambient temperature.
The Two-Component Regulators GacS and GacA Influence Accumulation of the Stationary-Phase Sigma Factor ς
S
and the Stress Response in
Pseudomonas fluorescens
Pf-5
Authors: Whistler Cheryl A. , Corbell Nathan A. , Sarniguet Alain , Ream Walter , Loper Joyce E. 
Published: December 2019

ABSTRACT

Three global regulators are known to control antibiotic production by
Pseudomonas fluorescens
. A two-component regulatory system comprised of the sensor kinase GacS (previously called ApdA or LemA) and GacA, a member of the FixJ family of response regulators, is required for antibiotic production. A mutation in
rpoS
, which encodes the stationary-phase sigma factor ς
S
, differentially affects antibiotic production and reduces the capacity of stationary-phase cells of
P. fluorescens
to survive exposure to oxidative stress. The
gacA
gene of
P. fluorescens
Pf-5 was isolated, and the influence of
gacS
and
gacA
on
rpoS
transcription, ς
S
levels, and oxidative stress response of Pf-5 was determined. We selected a
gacA
mutant of Pf-5 that contained a single nucleotide substitution within a predicted α-helical region, which is highly conserved among the FixJ family of response regulators. At the entrance to stationary phase, ς
S
content in
gacS
and
gacA
mutants of Pf-5 was less than 20% of the wild-type level. Transcription of
rpoS
, assessed with an
rpoS-lacZ
transcriptional fusion, was positively influenced by GacS and GacA, an effect that was most evident at the transition between exponential growth and stationary phase. Mutations in
gacS
and
gacA
compromised the capacity of stationary-phase cells of Pf-5 to survive exposure to oxidative stress. The results of this study provide evidence for the predominant roles of GacS and GacA in the regulatory cascade controlling stress response and antifungal metabolite production in
P. fluorescens
.
Genome Size, Complexity, and Ploidy of the Pathogenic Fungus
Histoplasma capsulatum
Authors: Carr Jeanne , Shearer Glenmore 
Published: December 2019

ABSTRACT

The genome size, complexity, and ploidy of the dimorphic pathogenic fungus
Histoplasma capsulatum
was determined by using DNA renaturation kinetics, genomic reconstruction, and flow cytometry. Nuclear DNA was isolated from two strains, G186AS and Downs, and analyzed by renaturation kinetics and genomic reconstruction with three putative single-copy genes (calmodulin, α-tubulin, and β-tubulin). G186AS was found to have a genome of approximately 2.3 × 10
7
bp with less than 0.5% repetitive sequences. The Downs strain, however, was found to have a genome approximately 40% larger with more than 16 times more repetitive DNA. The Downs genome was determined to be 3.2 × 10
7
bp with approximately 8% repetitive DNA. To determine ploidy, the DNA mass per cell measured by flow cytometry was compared with the 1
n
genome estimate to yield a DNA index (DNA per cell/1
n
genome size). Strain G186AS was found to have a DNA index of 0.96, and Downs had a DNA index of 0.94, indicating that both strains are haploid. Genomic reconstruction and Southern blot data obtained with α- and β-tubulin probes indicated that some genetic duplication has occurred in the Downs strain, which may be aneuploid or partially diploid.