bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cells of
Escherichia coli
Contain a Protein-Tyrosine Kinase, Wzc, and a Phosphotyrosine-Protein Phosphatase, Wzb
Authors: Vincent Carole , Doublet Patricia , Grangeasse Christophe , Vaganay Elisabeth , Cozzone Alain J. , Duclos Bertrand 
Published: December 2019

ABSTRACT

Two proteins of
Escherichia coli
, termed Wzc and Wzb, were analyzed for their capacity to participate in the reversible phosphorylation of proteins on tyrosine. First, Wzc was overproduced from its specific gene and purified to homogeneity by affinity chromatography. Upon incubation in the presence of radioactive ATP, it was found to effectively autophosphorylate. Two-dimensional analysis of its phosphoamino acid content revealed that it was modified exclusively at tyrosine. Second, Wzb was also overproduced from the corresponding gene and purified to homogeneity by affinity chromatography. It was shown to contain a phosphatase activity capable of cleaving the synthetic substrate
p
-nitrophenyl phosphate into
p
-nitrophenol and free phosphate. In addition, it was assayed on individual phosphorylated amino acids and appeared to dephosphorylate specifically phosphotyrosine, with no effect on phosphoserine or phosphothreonine. Such specificity for phosphotyrosine was confirmed by the observation that Wzb was able to dephosphorylate previously autophosphorylated Wzc. Together, these data demonstrate, for the first time, that
E. coli
cells contain both a protein-tyrosine kinase and a phosphotyrosine-protein phosphatase. They also provide evidence that this phosphatase can utilize the kinase as an endogenous substrate, which suggests the occurrence of a regulatory mechanism connected with reversible protein phosphorylation on tyrosine. From comparative analysis of amino acid sequences, Wzc was found to be similar to a number of proteins present in other bacterial species which are all involved in the synthesis or export of exopolysaccharides. Since these polymers are considered important virulence factors, we suggest that reversible protein phosphorylation on tyrosine may be part of the cascade of reactions that determine the pathogenicity of bacteria.
Control of Acid Resistance in
Escherichia coli
Authors: Castanie-Cornet Marie-Pierre , Penfound Thomas A. , Smith Dean , Elliott John F. , Foster John W. 
Published: December 2019

ABSTRACT

Acid resistance (AR) in
Escherichia coli
is defined as the ability to withstand an acid challenge of pH 2.5 or less and is a trait generally restricted to stationary-phase cells. Earlier reports described three AR systems in
E. coli
. In the present study, the genetics and control of these three systems have been more clearly defined. Expression of the first AR system (designated the oxidative or glucose-repressed AR system) was previously shown to require the alternative sigma factor RpoS. Consistent with glucose repression, this system also proved to be dependent in many situations on the cyclic AMP receptor protein. The second AR system required the addition of arginine during pH 2.5 acid challenge, the structural gene for arginine decarboxylase (
adiA
), and the regulator
cysB
, confirming earlier reports. The third AR system required glutamate for protection at pH 2.5, one of two genes encoding glutamate decarboxylase (
gadA
or
gadB
), and the gene encoding the putative glutamate:γ-aminobutyric acid antiporter (
gadC
). Only one of the two glutamate decarboxylases was needed for protection at pH 2.5. However, survival at pH 2 required both glutamate decarboxylase isozymes. Stationary phase and acid pH regulation of the
gad
genes proved separable. Stationary-phase induction of
gadA
and
gadB
required the alternative sigma factor ς
S
encoded by
rpoS
. However, acid induction of these enzymes, which was demonstrated to occur in exponential- and stationary-phase cells, proved to be ς
S
independent. Neither
gad
gene required the presence of volatile fatty acids for induction. The data also indicate that AR via the amino acid decarboxylase systems requires more than an inducible decarboxylase and antiporter. Another surprising finding was that the ς
S
-dependent oxidative system, originally thought to be acid induced, actually proved to be induced following entry into stationary phase regardless of the pH. However, an inhibitor produced at pH 8 somehow interferes with the activity of this system, giving the illusion of acid induction. The results also revealed that the AR system affording the most effective protection at pH 2 in complex medium (either Luria-Bertani broth or brain heart infusion broth plus 0.4% glucose) is the glutamate-dependent GAD system. Thus,
E. coli
possesses three overlapping acid survival systems whose various levels of control and differing requirements for activity ensure that at least one system will be available to protect the stationary-phase cell under naturally occurring acidic environments.
Role of Region C in Regulation of the Heat Shock Gene-Specific Sigma Factor of
Escherichia coli
, ς
32
Authors: Arsène Florence , Tomoyasu Toshifumi , Mogk Axel , Schirra Christiane , Schulze-Specking Agnes , Bukau Bernd 
Published: December 2019

ABSTRACT

Expression of heat shock genes is controlled in
Escherichia coli
by the antagonistic action of the ς
32
subunit of RNA polymerase and the DnaK chaperone system, which inactivates ς
32
by stress-dependent association and mediates ς
32
degradation by the FtsH protease. A stretch of 23 residues (R122 to Q144) conserved among ς
32
homologs, termed region C, was proposed to play a role in ς
32
degradation, and peptide analysis identified two potential DnaK binding sites central and peripheral to region C. Region C is thus a prime candidate for mediating stress control of ς
32
, a hypothesis that we tested in the present study. A peptide comprising the central DnaK binding site was an excellent substrate for FtsH, while a peptide comprising the peripheral DnaK binding site was a poor substrate. Replacement of a single hydrophobic residue in each DnaK binding site by negatively charged residues (I123D and F137E) strongly decreased the binding of the peptides to DnaK and the degradation by FtsH. However, introduction of these and additional region C alterations into the ς
32
protein did not affect ς
32
degradation in vivo and in vitro or DnaK binding in vitro. These findings do not support a role for region C in ς
32
control by DnaK and FtsH. Instead, the ς
32
mutants had reduced affinities for RNA polymerase and decreased transcriptional activities in vitro and in vivo. Furthermore, cysteines inserted into region C allowed cysteine-specific cross-linking of ς
32
to RNA polymerase. Region C thus confers on ς
32
a competitive advantage over other ς factors to bind RNA polymerase and thereby contributes to the rapidity of the heat shock response.
The
mdoC
Gene of
Escherichia coli
Encodes a Membrane Protein That Is Required for Succinylation of Osmoregulated Periplasmic Glucans
Authors: Lacroix Jean-Marie , Lanfroy Eric , Cogez Virginie , Lequette Yannick , Bohin Anne , Bohin Jean-Pierre 
Published: December 2019

ABSTRACT

Osmoregulated periplasmic glucans (OPGs) of
Escherichia coli
are anionic oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. Their anionic character is provided by the substitution of the glucosidic backbone by phosphoglycerol originating from the membrane phospholipids and by succinyl residues from unknown origin. A phosphoglycerol-transferase-deficient
mdoB
mutant was subjected to Tn
5
transposon mutagenesis, and putative mutant clones were screened for changes in the anionic character of OPGs by thin-layer chromatography. One mutant deficient in succinylation of OPGs was obtained, and the gene inactivated in this mutant was characterized and named
mdoC
.
mdoC
, which encodes a membrane-bound protein, is closely linked to the
mdoGH
operon necessary for the synthesis of the OPG backbone.
relA
Is Required for Actinomycin Production in
Streptomyces antibioticus
Authors: Hoyt Shannan , Jones George H. 
Published: December 2019

ABSTRACT

The
relA
gene from
Streptomyces antibioticus
has been cloned and sequenced. The gene encodes a protein with an
M
r
of 93,653, which is 91% identical to the corresponding protein from
Streptomyces coelicolor
. Disruption of
S. antibioticus relA
produces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact
relA
gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted
relA
gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the
relA
disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.
The Exopolygalacturonate Lyase PelW and the Oligogalacturonate Lyase Ogl, Two Cytoplasmic Enzymes of Pectin Catabolism in
Erwinia chrysanthemi
3937
Authors: Shevchik Vladimir E. , Condemine Guy , Robert-Baudouy Janine , Hugouvieux-Cotte-Pattat Nicole 
Published: December 2019

ABSTRACT

Erwinia chrysanthemi
3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of
E. chrysanthemi
. The
E. chrysanthemi
3937 gene
kdgC
has been shown to exhibit a high degree of similarity to the genes
pelY
of
Yersinia pseudotuberculosis
and
pelB
of
Erwinia carotovora
, which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the
kdgC
gene was renamed
pelW
. PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite −2 to +1, while it is probably two for Ogl, extending from subsite −1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca
2+
is unable to restore the activity of PelW or Ogl, while several other cations, including Co
2+
, Mn
2+
, and Ni
2+
, can activate both cytoplasmic lyases.
Elements Involved in Catabolite Repression and Substrate Induction of the Lactose Operon in
Lactobacillus casei
Authors: Gosalbes María José , Monedero Vicente , Pérez-Martínez Gaspar 
Published: December 2019

ABSTRACT

In
Lactobacillus casei
ATCC 393, the chromosomally encoded lactose operon,
lacTEGF
, encodes an antiterminator protein (LacT), lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) elements (LacE and LacF), and a phospho-β-galactosidase.
lacT
,
lacE
, and
lacF
mutant strains were constructed by double crossover. The
lacT
strain displayed constitutive termination at a ribonucleic antiterminator (RAT) site, whereas
lacE
and
lacF
mutants showed an inducer-independent antiterminator activity, as shown analysis of enzyme activity obtained from transcriptional fusions of
lac
promoter (
lacp
) and
lacpΔRAT
with the
Escherichia coli gusA
gene in the different
lac
mutants. These results strongly suggest that in vivo under noninducing conditions, the lactose-specific PTS elements negatively modulate LacT activity. Northern blot analysis detected a 100-nucleotide transcript starting at the transcription start site and ending a consensus RAT sequence and terminator region. In a
ccpA
mutant, transcription initiation was derepressed but no elongation through the terminator was observed in the presence of glucose and the inducing sugar, lactose. Full expression of
lacTEGF
was found only in a
man ccpA
double mutant, indicating that PTS elements are involved in the CcpA-independent catabolite repression mechanism probably via LacT.
The Extracellular Xylan Degradative System in
Clostridium cellulolyticum
Cultivated on Xylan: Evidence for Cell-Free Cellulosome Production
Authors: Mohand-Oussaid O. , Payot S. , Guedon E. , Gelhaye E. , Youyou A. , Petitdemange H. 
Published: December 2019

ABSTRACT

In this study, we demonstrate that the cellulosome of
Clostridium cellulolyticum
grown on xylan is not associated with the bacterial cell. Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan. Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC. The same repartition is observed with carboxymethyl cellulase activity. The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions. In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate. We propose that, depending on the growth substrate,
C. cellulolyticum
is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.
Identification of Site-Specific Recombination Genes
int
and
xis
of the
Rhizobium
Temperate Phage
16-3
Authors: Semsey Szabolcs , Papp IstvAn , Buzas Zsuzsanna , Patthy Andras , Orosz Laszlo , Papp Peter P. 
Published: December 2019

ABSTRACT

Phage
16-3
is a temperate phage of
Rhizobium meliloti
41 which integrates its genome with high efficiency into the host chromosome by site-specific recombination through DNA sequences of
attB
and
attP
. Here we report the identification of two phage-encoded genes required for recombinations at these sites:
int
(phage integration) and
xis
(prophage excision). We concluded that Int protein of phage
16-3
belongs to the integrase family of tyrosine recombinases. Despite similarities to the cognate systems of the lambdoid phages, the
16-3 int xis att
system is not active in
Escherichia coli
, probably due to requirements for host factors that differ in
Rhizobium meliloti
and
E. coli
. The application of the
16-3
site-specific recombination system in biotechnology is discussed.
Identification and Characterization of a New Lipoprotein, NlpI, inEscherichia coliK-12
Authors: Ohara Masaru , Wu Henry C. , Sankaran Krishnan , Rick Paul D. 
Published: December 2019

ABSTRACTWe report here the identification of a new lipoprotein, NlpI, inEscherichia coliK-12. The NlpI structural gene (nlpI) is located between the genespnp(polynucleotide phosphorylase) anddeaD(RNA helicase) at 71 min on theE. colichromosome. ThenlpIgene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. AnnlpI::cmmutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation innlpI, since it was complemented by the wild-typenlpIgene alone. Overexpression of the unalterednlpIgene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.
Regulated Expression of a Highly Conserved Regulatory Gene Cluster Is Necessary for Controlling Photosynthesis Gene Expression in Response to Anaerobiosis in
Rhodobacter capsulatus
Authors: Du Shouying , Kouadio Jean-Louis K. , Bauer Carl E. 
Published: December 2019

ABSTRACT

We utilized primer extension analysis to demonstrate that the divergently transcribed
regB
and
senC-regA-hvrA
transcripts contain stable 5′ ends 43 nucleotides apart within the
regB-senC
intergenic region. DNA sequence analysis indicates that this region contains two divergent promoters with overlapping ς
70
type −35 and −10 promoter recognition sequences. In vivo analysis of expression patterns of
regB
::
lacZ
and
senC-regA-hvrA
::
lacZ
reporter gene fusions demonstrates that the
regB
and
senC-regA-hvrA
transcripts are both negatively regulated by the phosphorylated form of the global response regulator RegA. DNase I protection assays with a constitutively active variant of RegA indicate that RegA binds between
regB
and
senC
overlapping −10 and −35 promoter recognition sequences. Two mutations were also isolated in a
regB
-deficient background that increased expression of the
senC-regA-hvrA
operon 10- and 5-fold, respectively. As a consequence of increased RegA expression, these mutants exhibited elevated aerobic and anaerobic photosynthesis (
puf
) gene expression, even in the absence of the sensor kinase RegB. These results indicate that autoregulation by RegA is a factor contributing to the maintenance of an optimal low level of RegA expression that allows responsiveness to activation by phosphorylation.
Transcriptional Activation of
Agrobacterium tumefaciens
Virulence Gene Promoters in
Escherichia coli
Requires the
A. tumefaciens rpoA
Gene, Encoding the Alpha Subunit of RNA Polymerase
Authors: Lohrke S. M. , Nechaev S. , Yang H. , Severinov K. , Jin S. J. 
Published: December 2019

ABSTRACT

The two-component regulatory system, composed of
virA
and
virG
, is indispensable for transcription of virulence genes within
Agrobacterium tumefaciens
. However,
virA
and
virG
are insufficient to activate transcription from virulence gene promoters within
Escherichia coli
cells, indicating a requirement for additional
A. tumefaciens
genes. In a search for these additional genes, we have identified the
rpoA
gene, encoding the α subunit of RNA polymerase (RNAP), which confers significant expression of a
virB
promoter (
virBp
)::
lacZ
fusion in
E. coli
in the presence of an active transcriptional regulator
virG
gene. We conducted in vitro transcription assays using either reconstituted
E. coli
RNAP or hybrid RNAP in which the α subunit was derived from
A. tumefaciens
. The two forms of RNAP were equally efficient in transcription from a ς
70
-dependent
E. coli gal
P1 promoter; however, only the hybrid RNAP was able to transcribe
virBp
in a
virG
-dependent manner. In addition, we provide evidence that the α subunit from
A. tumefaciens
, but not from
E. coli
, is able to interact with the VirG protein. These data suggest that transcription of virulence genes requires specific interaction between VirG and the α subunit of
A. tumefaciens
and that the α subunit from
E. coli
is unable to effectively interact with the VirG protein. This work provides the basis for future studies designed to examine
vir
gene expression as well as the T-DNA transfer process in
E. coli.
Mutational Analysis and Membrane Topology of ComP, a Quorum-Sensing Histidine Kinase of
Bacillus subtilis
Controlling Competence Development
Authors: Piazza Flavia , Tortosa Pablo , Dubnau David 
Published: December 2019

ABSTRACT

ComP is a sensor histidine kinase of
Bacillus subtilis
required for the signal transduction pathway that initiates the development of competence for genetic transformation. It is believed that ComP senses the presence of ComX, a modified extracellular peptide pheromone, and donates a phosphate to ComA, thereby activating this transcription factor for binding to the
srfA
promoter. In the present study, fusions to the
Escherichia coli
proteins PhoA and LacZ and analysis of its susceptibility to the protease kallikrein were used to probe the membrane topology of ComP. These data suggest that ComP contains six or eight membrane-spanning segments and two large extracytoplasmic loops in its N-terminal membrane-associated domain. Deletions were introduced involving the large extracellular loops to explore the role of the N-terminal domain of ComP in signal transduction. The absence of the second loop conferred a phenotype in which ComP was active in the absence of ComX. The implications of these data are discussed.
The
Bacillus subtilis yaaH
Gene Is Transcribed by SigE RNA Polymerase during Sporulation, and Its Product Is Involved in Germination of Spores
Authors: Kodama Takeko , Takamatsu Hiromu , Asai Kei , Kobayashi Kazuo , Ogasawara Naotake , Watabe Kazuhito 
Published: December 2019

ABSTRACT

The expression of 21 novel genes located in the region from
dnaA
to
abrB
of the
Bacillus subtilis
chromosome was analyzed. One of the genes,
yaaH
, had a predicted promoter sequence conserved among SigE-dependent genes. Northern blot analysis revealed that
yaaH
mRNA was first detected from 2 h after the cessation of logarithmic growth (
T
2
) of sporulation in wild-type cells and in
spoIIIG
(SigG
−
) and
spoIVCB
(SigK
−
) mutants but not in
spoIIAC
(SigF
−
) and
spoIIGAB
(SigE
−
) mutants. The transcription start point was determined by primer extension analysis; the −10 and −35 regions are very similar to the consensus sequences recognized by SigE-containing RNA polymerase. A YaaH-His tag fusion encoded by a plasmid with a predicted promoter for the
yaaH
gene was produced from
T
2
of sporulation in a
B. subtilis
transformant and extracted from mature spores, indicating that the
yaaH
gene product is a spore protein. Inactivation of the
yaaH
gene by insertion of an erythromycin resistance gene did not affect vegetative growth or spore resistance to heat, chloroform, and lysozyme. The germination of
yaaH
mutant spores in a mixture of
l
-asparagine,
d
-glucose,
d
-fructose, and potassium chloride was almost the same as that of wild-type spores, but the mutant spores were defective in
l
-alanine-stimulated germination. These results suggest that
yaaH
is a novel gene encoding a spore protein produced in the mother cell compartment from
T
2
of sporulation and that it is required for the
l
-alanine-stimulated germination pathway.