bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Dual-Signaling Mechanism Mediated by the ArcB Hybrid Sensor Kinase Containing the Histidine-Containing Phosphotransfer Domain in
Escherichia coli
Authors: Matsushika Akinori , Mizuno Takeshi 
Published: December 2019

ABSTRACT

The two components ArcB and ArcA play a crucial role in the signal transduction implicated in the complex transcriptional regulatory network that allows
Escherichia coli
to sense various respiratory growth conditions. ArcB is a hybrid sensor kinase having multiple phosphorylation sites in its primary amino acid sequence, including a transmitter, a receiver, and a histidine-containing phosphotransfer (HPt) domain. ArcA is a DNA-binding transcriptional regulator with a receiver domain. Results of recent in vitro studies revealed multistep His-to-Asp phosphotransfer circuitry in the ArcB-ArcA signaling system. For this report we conducted a series of in vivo experiments using a set of crucial ArcB mutants to evaluate the regulation of the
sdh
operon. The results suggested that the phosphorylated His-717 site in the HPt domain of ArcB is essential for anaerobic repression of
sdh
. Nonetheless, the ArcB mutant lacking this crucial His-717 site does not necessarily exhibit a null phenotype with respect to ArcB-ArcA signaling. The HPt mutant appears to maintain an ability to signal ArcA, particularly under aerobic conditions, which results in a significant repression of
sdh
. Based on these and other in vivo results, we propose a model in which ArcB functions in its own right as a dual-signaling sensor that is capable of propagating two types of stimuli through two distinct phosphotransfer pathways.
Multicellular Stalk-Like Structures in
Saccharomyces cerevisiae
Authors: Engelberg David , Mimran Avishai , Martinetto Horacio , Otto Joel , Simchen Giora , Karin Michael , Fink Gerald R. 
Published: December 2019

ABSTRACT

Stalk formation is a novel pattern of multicellular organization. Yeast cells which survive UV irradiation form colonies that grow vertically to form very long (0.5 to 3.0 cm) and thin (0.5 to 4 mm in diameter) multicellular structures. We describe the conditions required to obtain these stalk-like structures reproducibly in large numbers. Yeast mutants, mutated for control of cell polarity, developmental processes, UV response, and signal transduction cascades were tested and found capable of forming stalk-like structures. We suggest a model that explains the mechanism of stalk formation by mechanical environmental forces. We show that other microorganisms (
Candida albicans
,
Schizosaccharomyces pombe
, and
Escherichia coli
) also form stalks, suggesting that the ability to produce stalks may be a general property of microorganisms. Diploid yeast stalks sporulate at an elevated frequency, raising the possibility that the physiological role of stalks might be disseminating spores.
Role of PhoP∼P in Transcriptional Regulation of Genes Involved in Cell Wall Anionic Polymer Biosynthesis in
Bacillus subtilis
Authors: Qi Ying , Hulett F. Marion 
Published: December 2019

ABSTRACT

tagA
,
tagD
, and
tuaA
operons are responsible for the synthesis of cell wall anionic polymer, teichoic acid, and teichuronic acid, respectively, in
Bacillus subtilis
. Under phosphate starvation conditions, teichuronic acid is synthesized while teichoic acid synthesis is inhibited. Expression of these genes is controlled by PhoP-PhoR, a two-component system. It has been proposed that PhoP∼P plays a key role in the activation of
tuaA
and the repression of
tagA
and
tagD
. In this study, we demonstrated the role of PhoP∼P in the switch process from teichoic acid synthesis to teichuronic acid synthesis, by using an in vitro transcription system. The results indicate that PhoP∼P is sufficient to repress the transcription of the
tagA
and
tagD
promoters and also to activate the transcription of the
tuaA
promoter.
Molecular Characterization of a Chromosomal Determinant Conferring Resistance to Zinc and Cobalt Ions in
Staphylococcus aureus
Authors: Xiong Anming , Jayaswal Radheshyam K. 
Published: December 2019

ABSTRACT

A DNA fragment conferring resistance to zinc and cobalt ions was isolated from a genomic DNA library of
Staphylococcus aureus
RN450. The DNA sequence analysis revealed two consecutive open reading frames, designated
zntR
and
zntA
. The predicted ZntR and ZntA showed significant homology to members of ArsR and cation diffusion families, respectively. A mutant strain containing the null allele of
zntA
was more sensitive to zinc and cobalt ions than was the parent strain. The metal-sensitive phenotype of the mutant was complemented by a 2.9-kb DNA fragment containing
zntR
and
zntA
. An
S. aureus
strain harboring multiple copies of
zntR
and
zntA
showed an increased resistance to zinc. The resistance to zinc in the wild-type strain was inducible. Transcriptional analysis indicated that
zntR
and
zntA
genes were cotranscribed. The zinc uptake studies suggested that the
zntA
product was involved in the export of zinc ions out of cells.
A Redox-Responsive Pathway for Aerobic Regulation of Photosynthesis Gene Expression in
Rhodobacter sphaeroides
2.4.1
Authors: O’Gara James P. , Eraso Jesus M. , Kaplan Samuel 
Published: December 2019

ABSTRACT

To further understand the proposed signal transduction pathway involving the presumed redox proteins RdxBH and
cbb
3
cytochrome oxidase in
Rhodobacter sphaeroides
2.4.1, a series of mutants lacking components of both the Prr two-component activation system and the
cbb
3
-type cytochrome oxidase or RdxBH were constructed. We report that under highly aerobic conditions, aberrant photosynthesis gene expression and spectral complex formation typical of
cbb
3
- or RdxBH-deficient mutants were no longer observed when either
prrA
(encoding the response regulator of the Prr system) or
prrB
(encoding the presumed sensor kinase) was also deleted. These double-mutant strains are phenotypically identical to single-mutant PrrA and PrrB strains, suggesting that the signal(s) originating from the
cbb
3
terminal oxidase affects downstream
puc
and
puf
operon expression by acting exclusively through the Prr system. When the same double-mutant strains were examined under anaerobic dark dimethyl sulfoxide growth conditions, photosynthesis gene expression was obligatorily linked to the two-component activation system. However, photosynthesis gene expression under the same growth conditions was significantly higher in the
cbb
3
mutant strain when compared to that in the wild type. Similarly, under anaerobic photosynthetic conditions the high levels of the oxidized carotenoid, spheroidenone, which accumulate in
cbb
3
-deficient mutants were nearly restored to normal in a PrrB
−
CcoP
−
double mutant. This observation, together with previously published results, suggests that the regulation of the CrtA-catalyzed reaction possesses both transcriptional and posttranscriptional regulatory effectors. We propose that the
cbb
3
cytochrome oxidase, which by definition can interact with external oxygen, serves to control the activity of the Prr two-component activation system under both aerobic and anaerobic conditions. Although independent from the
cbb
3
oxidase, the RdxBH proteins are also required for normal functioning of the Prr two-component activation system and are therefore believed to lie between the
cbb
3
oxidase in this oxygen-sensing, redox signaling pathway and the Prr activation system.
Branched-Chain Amino Acid Biosynthesis in
Salmonella typhimurium
: a Quantitative Analysis
Authors: Epelbaum Sabine , LaRossa Robert A. , VanDyk Tina K. , Elkayam T. , Chipman David M. , Barak Ze’ev 
Published: December 2019

ABSTRACT

We report here the first quantitative study of the branched-chain amino acid biosynthetic pathway in
Salmonella typhimurium
LT2. The intracellular levels of the enzymes of the pathway and of the 2-keto acid intermediates were determined under various physiological conditions and used for estimation of several of the fluxes in the cells. The results led to a revision of previous ideas concerning the way in which multiple acetohydroxy acid synthase (AHAS) isozymes contribute to the fitness of enterobacteria. In wild-type LT2, AHAS isozyme I provides most of the flux to valine, leucine, and pantothenate, while isozyme II provides most of the flux to isoleucine. With acetate as a carbon source, a strain expressing AHAS II only is limited in growth because of the low enzyme activity in the presence of elevated levels of the inhibitor glyoxylate. A strain with AHAS I only is limited during growth on glucose by the low tendency of this enzyme to utilize 2-ketobutyrate as a substrate; isoleucine limitation then leads to elevated threonine deaminase activity and an increased 2-ketobutyrate/2-ketoisovalerate ratio, which in turn interferes with the synthesis of coenzyme A and methionine. The regulation of threonine deaminase is also crucial in this regard. It is conceivable that, because of fundamental limitations on the specificity of enzymes, no single AHAS could possibly be adequate for the varied conditions that enterobacteria successfully encounter.
Structural and Functional Analysis of the Gene Cluster Encoding the Enzymes of the Arginine Deiminase Pathway of
Lactobacillus sake
Authors: Zúñiga Manuel , Champomier-Verges Marie , Zagorec Monique , Pérez-Martínez Gaspar 
Published: December 2019

ABSTRACT

Lactobacillus sake
can use arginine via the arginine deiminase (ADI) pathway. We designed degenerate primers based on an alignment of known sequences of ornithine transcarbamoylase (OTC)-encoding genes in order to amplify the
L. sake
counterpart sequences by PCR. Screening a genomic library of
L. sake
in λEMBL3 allowed us to isolate a clone containing a 10-kb
L. sake
genomic DNA insert. Sequence analysis revealed that the genes involved in arginine catabolism were clustered and encoded ADI (
arcA
), OTC (
arcB
), carbamate kinase (
arcC
), and a putative carrier with high similarity to the arginine/ornithine antiporter of
Pseudomonas aeruginosa
(
arcD
). Additionally, a putative transaminase-encoding gene (
arcT
) was located in this region. The genes followed the order
arcA arcB arcC arcT arcD
, which differs from that found in other microorganisms.
arcA
,
arcB
,
arcC
, and
arcD
mutants were constructed, and the ADI pathway was impaired in all of them. Transcriptional studies indicated that
arcA
gene is subject to catabolite repression, and under the conditions used, several transcripts could be detected, suggesting the existence of different initiation sites or processing of a larger mRNA.
Substitution of an Alanine Residue for Glycine 146 in TMP Kinase from
Escherichia coli
Is Responsible for Bacterial Hypersensitivity to Bromodeoxyuridine
Authors: Tourneux Lise , Bucurenci Nadia , Lascu Ioan , Sakamoto Hiroshi , Briand Gilbert , Gilles Anne-Marie 
Published: December 2019

ABSTRACT

The wild-type TMP kinases from
Escherichia coli
and from a strain hypersensitive to 5-bromo-2′-deoxyuridine were characterized comparatively. The mutation at codon 146 causes the substitution of an alanine residue for glycine in the enzyme, which is accompanied by changes in the relative affinities for 5-Br-UMP and TMP compared to those of the wild-type TMP kinase. Plasmids carrying the wild-type
tmk
gene from
Escherichia coli
or
Bacillus subtilis
, but not the defective
tmk
gene, restored the resistance to bromodeoxyuridine of an
E. coli
mutant strain.
Identification and Expression of the
Bacillus subtilis
Fructose-1,6-Bisphosphatase Gene (
fbp
)
Authors: Fujita Yasutaro , Yoshida Ken-Ichi , Miwa Yasuhiko , Yanai Nobuo , Nagakawa Eishi , Kasahara Yasuhiro 
Published: December 2019

ABSTRACT

The
Bacillus subtilis fbp
gene encoding fructose-1,6-bisphosphatase (FBPase) was originally identified as
yydE
. The
fbp
gene was expressed at a fairly constant level in cells undergoing glycolysis or gluconeogenesis.
fbp
transcription was initiated 94 bp upstream of the translation initiation codon, resulting in a 2.4-kb monocistronic transcript. Interestingly,
B. subtilis
FBPase exhibited no significant similarity to other FBPases in protein sequence databases.
Efficiency and Frequency of Translational Coupling between the Bacteriophage T4 Clamp Loader Genes
Authors: Torgov Michael Y. , Janzen Deanna M. , Reddy Michael K. 
Published: December 2019

ABSTRACT

The bacteriophage T4 DNA polymerase holoenzyme is composed of the core polymerase, gene product 43 (gp43), in association with the “sliding clamp” of the T4 system, gp45. Sliding clamps are the processivity factors of DNA replication systems. The T4 sliding clamp comes to encircle DNA via the “clamp loader” activity inherent in two other T4 proteins: 44 and 62. These proteins assemble into a pentameric complex with a precise 4:1 stoichiometry of proteins 44 and 62. Previous work established that T4 genes
44
and
62
, which are directly adjacent on polycistronic mRNA molecules, are—to some degree—translationally coupled. In the present study, measurement of the levels (monomers/cell) of the clamp loader subunits during the course of various T4 infections in different host cell backgrounds was accomplished by quantitative immunoblotting. The efficiency of translational coupling was obtained by determining the in vivo levels of gp62 that were synthesized when its translation was either coupled to or uncoupled from the upstream translation of gene
44
. Levels of gp44 were also measured to determine the relative stoichiometry of synthesis and the percentage of gp44 translation that was transmitted across the intercistronic junction (coupling frequency). The results indicated a coupling efficiency of ∼85% and a coupling frequency of ∼25% between the
44-62
gene pair during the course of infection. Thus, translational coupling is the major factor in maintaining the 4:1 stoichiometry of synthesis of the clamp loader subunits. However, coupling does not appear to be an absolute requirement for the synthesis of gp62.
Chitinolytic Activity in
Chromobacterium violaceum
: Substrate Analysis and Regulation by Quorum Sensing
Authors: Chernin Leonid S. , Winson Michael K. , Thompson Jacquelyn M. , Haran Shoshan , Bycroft Barrie W. , Chet Ilan , Williams Paul , Stewart Gordon S. A. B. 
Published: December 2019

ABSTRACT

Quorum sensing control mediated by
N
-acyl homoserine lactone (AHL) signaling molecules has been established as a key feature of the regulation of exoenzyme production in many gram-negative bacteria. In
Chromobacterium violaceum
ATCC 31532 a number of phenotypic characteristics, including production of the purple pigment violacein, hydrogen cyanide, antibiotics, and exoproteases are known to be regulated by the endogenous AHL
N
-hexanoyl-
l
-homoserine lactone (HHL). In this study we show that
C. violaceum
produces a set of chitinolytic enzymes whose production is regulated by HHL. The chitinolytic activity was induced in strains grown in the presence of chitin as the sole carbon source and quantitated in the secreted proteins by using
p
-nitrophenol analogs of disaccharide, trisaccharide, and tetrasaccharide oligomers of
N
-acetylglucosamine. By using 4-methylumbelliferyl analogs of the same oligomers of
N
-acetylglucosamine as substrates for proteins separated and renatured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, at least six enzymes were detected: a chitobiase with high specificity to a dimeric substrate of 87 kDa, two
N
-acetylglucosaminidases with apparent molecular masses of 162 and 133 kDa, two endochitinases of 108 and 67 kDa, and a chitobiosidase of 56 kDa. In addition, two unidentified bands of >205 kDa were found where a tetrameric chitin derivative was used as a substrate. A pleiotropic mini-Tn
5
mutant of
C. violaceum
(CV026) that is defective in HHL production and other quorum-sensing-regulated factors was also found to be completely deficient in chitinolytic activity. Growth of this mutant on minimal medium with chitin supplemented with culture supernatant from the
C. violaceum
wild-type strain or 10 μM synthetic HHL restored chitinase production to the level shown by the parental strain. These results constitute the most complete evidence so far for regulation of chitinolytic activity by AHL signaling in a gram-negative bacterium.
Similarities between the
antABC
-Encoded Anthranilate Dioxygenase and the
benABC
-Encoded Benzoate Dioxygenase of
Acinetobacter
sp. Strain ADP1
Authors: Bundy Becky M. , Campbell Alan L. , Neidle Ellen L. 
Published: December 2019

ABSTRACT

Acinetobacter
sp. strain ADP1 can use benzoate or anthranilate as a sole carbon source. These structurally similar compounds are independently converted to catechol, allowing further degradation to proceed via the β-ketoadipate pathway. In this study, the first step in anthranilate catabolism was characterized. A mutant unable to grow on anthranilate, ACN26, was selected. The sequence of a wild-type DNA fragment that restored growth revealed the
antABC
genes, encoding 54-, 19-, and 39-kDa proteins, respectively. The deduced AntABC sequences were homologous to those of class IB multicomponent aromatic ring-dihydroxylating enzymes, including the dioxygenase that initiates benzoate catabolism. Expression of
antABC
in
Escherichia coli
, a bacterium that normally does not degrade anthranilate, enabled the conversion of anthranilate to catechol. Unlike benzoate dioxygenase (BenABC), anthranilate dioxygenase (AntABC) catalyzed catechol formation without requiring a dehydrogenase. In
Acinetobacter
mutants,
benC
substituted for
antC
during growth on anthranilate, suggesting relatively broad substrate specificity of the BenC reductase, which transfers electrons from NADH to the terminal oxygenase. In contrast, the
benAB
genes did not substitute for
antAB
. An
antA
point mutation in ACN26 prevented anthranilate degradation, and this mutation was independent of a
mucK
mutation in the same strain that prevented exogenous muconate degradation. Anthranilate induced expression of
antA
, although no associated transcriptional regulators were identified. Disruption of three open reading frames in the immediate vicinity of
antABC
did not prevent the use of anthranilate as a sole carbon source. The
antABC
genes were mapped on the ADP1 chromosome and were not linked to the two known supraoperonic gene clusters involved in aromatic compound degradation.
Molecular Characterization and Sequence of a Methionine Biosynthetic Locus from
Pseudomonas syringae
Authors: Andersen Gary L. , Beattie Gwyn A. , Lindow Steven E. 
Published: December 2019

ABSTRACT

Two methionine biosynthetic genes in
Pseudomonas syringae
pv.
syringae
,
metX
and
metW
, were isolated, sequenced, and evaluated for their roles in methionine biosynthesis and bacterial fitness on leaf surfaces. The
metXW
locus was isolated on a 1.8-kb DNA fragment that was required for both methionine prototrophy and wild-type epiphytic fitness. Sequence analysis identified two consecutive open reading frames (ORFs), and in vitro transcription-translation experiments provided strong evidence that the ORFs encode proteins with the predicted molecular masses of 39 and 22.5 kDa. The predicted amino acid sequence of MetX (39 kDa) showed homology to several known and putative homoserine
O
-acetyltransferases. This enzyme is the first enzyme in the methionine biosynthetic pathway of fungi, gram-negative bacteria of the genus
Leptospira
, and several gram-positive bacterial genera. Both
metX
and
metW
were required for methionine biosynthesis, and transcription from both genes was not repressed by methionine. MetW (22.5 kDa) did not show significant homology to any known protein, including prokaryotic and eukaryotic methionine biosynthetic enzymes. Several classes of methionine auxotrophs, including
metX
and
metW
mutants, exhibit reduced fitness on leaf surfaces, indicating a requirement for methionine prototrophy in wild-type epiphytic fitness. This requirement is enhanced under environmentally stressful conditions, suggesting a role for methionine prototrophy in bacterial stress tolerance.
Vibrio cholerae
O139 Bengal: Combined Physical and Genetic Map and Comparative Analysis with the Genome of
V. cholerae
O1
Authors: Khetawat Gopal , Bhadra Rupak K. , Kar Sujata , Das Jyotirmoy 
Published: December 2019

ABSTRACT

A combined physical and genetic map of the genome of strain SG24 of
Vibrio cholerae
O139 Bengal, a novel non-O1 strain having epidemic potential, has been constructed by using the enzymes
Not
I,
Sfi
I, and
Ceu
I. The genome of SG24 is circular, and the genome size is about 3.57 Mb. The linkages between 47
Not
I and 32
Sfi
I fragments of
V. cholerae
SG24 genomic DNA were determined by combining two approaches: (i) identification of fragments produced by enzyme I in fragments produced by enzyme II by the method of fragment excision, redigestion, and end labeling and (ii) use of the linking clone libraries generated from the genome of classical O1 strain 569B. The linkages between nine
Ceu
I fragments were determined primarily by analyses of partial fragments of the
Ceu
I-digested genome. More than 80 cloned homologous and heterologous genes, including several operons, have been positioned on the physical map. The map of the SG24 genome represents the second map of a
V. cholerae
genome, and a comparison of this map with that of classical O1 strain 569B revealed considerable diversity in DNA restriction sites and allowed identification of hypervariable regions. Several genetic markers, including virulence determinant genes, are in different positions in the SG24 and 569B genomes.
Mutational Analysis of Plasmid R64 Thin Pilus Prepilin: the Entire Prepilin Sequence Is Required for Processing by Type IV Prepilin Peptidase
Authors: Horiuchi Takayuki , Komano Teruya 
Published: December 2019

ABSTRACT

The thin pili of IncI1 plasmid R64, which is required for conjugation in liquid media, belong to the type IV pilus family. They consist of a major subunit, the
pilS
product, and a minor component, one of the seven
pilV
products. The
pilS
product is first synthesized as a 22-kDa prepilin, processed to a 19-kDa mature pilin by the function of the
pilU
product, and then secreted outside the cell. The mature pilin is assembled to form a thin pilus with the
pilV
product. To reveal the relationship between the structure and function of the
pilS
product, 27 missense mutations, three N-terminal deletions, and two C-terminal deletions were constructed by PCR and site-directed mutagenesis. The characteristics of 32 mutant
pilS
products were analyzed. Four
pilS
mutant phenotype classes were identified. The products of 10 class I mutants were not processed by prepilin peptidase; the extracellular secretion of the products of two class II mutants was inhibited; from 11 class III mutants, thin pili with reduced activities in liquid mating were formed; from 9 class IV mutants, thin pili with mating activity similar to that of the wild-type
pilS
gene were formed. The point mutations of the class I mutants were distributed throughout the prepilin sequence, suggesting that processing of the
pilS
product requires the entire prepilin sequence.
Novel Pathway for Conversion of Chlorohydroxyquinol to Maleylacetate in
Burkholderia cepacia
AC1100
Authors: Zaborina Olga , Daubaras Dayna L. , Zago Anna , Xun Luying , Saido Katsuhiko , Klem Thomas , Nikolic Dejan , Chakrabarty A. M. 
Published: December 2019

ABSTRACT

Burkholderia cepacia
AC1100 metabolizes 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) via formation of 5-chlorohydroxyquinol (5-CHQ), hydroxyquinol (HQ), maleylacetate, and β-oxoadipate. The step(s) leading to the dechlorination of 5-CHQ to HQ has remained unidentified. We demonstrate that a dechlorinating enzyme, TftG, catalyzes the conversion of 5-CHQ to hydroxybenzoquinone, which is then reduced to HQ by a hydroxybenzoquinone reductase (HBQ reductase). HQ is subsequently converted to maleylacetate by hydroxyquinol 1,2-dioxygenase (HQDO). All three enzymes were purified. We demonstrate specific product formation by colorimetric assay and mass spectrometry when 5-CHQ is treated successively with the three enzymes: TftG, TftG plus HBQ reductase, and TftG plus HBQ reductase plus HQDO. This study delineates the complete enzymatic pathway for the degradation of 5-CHQ to maleylacetate.