bluuebunny/crossref_metadata_2025_split huggingface dataset:
Intracellular α-Amylase of
Streptococcus mutans
Authors: Simpson Christine L. , Russell Roy R. B. 
Published: December 2019

ABSTRACT

Sequencing upstream of the
Streptococcus mutans
gene for a CcpA gene homolog,
regM
, revealed an open reading frame, named
amy
, with homology to genes encoding α-amylases. The deduced amino acid sequence showed a strong similarity (60% amino acid identity) to the intracellular α-amylase of
Streptococcus bovis
and, in common with this enzyme, lacked a signal sequence. Amylase activity was found only in
S. mutans
cell extracts, with no activity detected in culture supernatants. Inactivation of
amy
by insertion of an antibiotic resistance marker confirmed that
S. mutans
has a single α-amylase activity. The amylase activity was induced by maltose but not by starch, and no acid was produced from starch.
S. mutans
can, however, transport limit dextrins and maltooligosaccharides generated by salivary amylase, but inactivation of
amy
did not affect growth on these substrates or acid production. The amylase digested the glycogen-like intracellular polysaccharide (IPS) purified from
S. mutans
, but the
amy
mutant was able to digest and produce acid from IPS; thus, amylase does not appear to be essential for IPS breakdown. However, when grown on excess maltose, the
amy
mutant produced nearly threefold the amount of IPS produced by the parent strain. The role of Amy has not been established, but Amy appears to be important in the accumulation of IPS in
S. mutans
grown on maltose.
Mutations in
Salmonella
Pathogenicity Island 2 (SPI2) Genes Affecting Transcription of SPI1 Genes and Resistance to Antimicrobial Agents
Authors: Deiwick Jörg , Nikolaus Thomas , Shea Jaqueline E. , Gleeson Colin , Holden David W. , Hensel Michael 
Published: December 2019

ABSTRACT

The
Salmonella typhimurium
genome contains two pathogenicity islands (SPI) with genes encoding type III secretion systems for virulence proteins. SPI1 is required for the penetration of the epithelial layer of the intestine. SPI2 is important for the subsequent proliferation of bacteria in the spleens of infected hosts. Although most mutations in SPI2 lead to a strong reduction of virulence, they have different effects in vitro, with some mutants having significantly increased sensitivity to gentamicin and the antibacterial peptide polymyxin B. Previously we showed that certain mutations in SPI2 affect the ability of
S. typhimurium
to secrete SPI1 effector proteins and to invade cultured eukaryotic cells. In this study, we show that these SPI2 mutations affect the expression of the SPI1 invasion genes. Analysis of reporter fusions to various SPI1 genes reveals highly reduced expression of
sipC
,
prgK
, and
hilA
, the transcriptional activator of SPI1 genes. These observations indicate that the expression of one type III secretion system can be influenced dramatically by mutations in genes encoding a second type III secretion system in the same cell.
Probing the Role of Cysteine Residues in Glucosamine-1-Phosphate Acetyltransferase Activity of the Bifunctional GlmU Protein from
Escherichia coli
: Site-Directed Mutagenesis and Characterization of the Mutant Enzymes
Authors: Pompeo Frédérique , van Heijenoort Jean , Mengin-Lecreulx Dominique 
Published: December 2019

ABSTRACT

The glucosamine-1-phosphate acetyltransferase activity but not the uridyltransferase activity of the bifunctional GlmU enzyme from
Escherichia coli
was lost when GlmU was stored in the absence of β-mercaptoethanol or incubated with thiol-specific reagents. The enzyme was protected from inactivation in the presence of its substrate acetyl coenzyme A (acetyl-CoA), suggesting the presence of an essential cysteine residue in or near the active site of the acetyltransferase domain. To ascertain the role of cysteines in the structure and function of the enzyme, site-directed mutagenesis was performed to change each of the four cysteines to alanine, and plasmids were constructed for high-level overproduction and one-step purification of histidine-tagged proteins. Whereas the kinetic parameters of the bifunctional enzyme appeared unaffected by the C296A and C385A mutations, 1,350- and 8-fold decreases of acetyltransferase activity resulted from the C307A and C324A mutations, respectively. The

K
m

values for acetyl-CoA and GlcN-1-P of mutant proteins were not modified, suggesting that none of the cysteines was involved in substrate binding. The uridyltransferase activities of wild-type and mutant GlmU proteins were similar. From these studies, the two cysteines Cys307 and Cys324 appeared important for acetyltransferase activity and seemed to be located in or near the active site.
The
Bacteroides fragilis
BtgA Mobilization Protein Binds to the
oriT
Region of pBFTM10
Authors: Sitailo Leonid A. , Zagariya Alexander M. , Arnold Patrick J. , Vedantam Gayatri , Hecht David W. 
Published: December 2019

ABSTRACT

The
Bacteroides fragilis
conjugal plasmid pBFTM10 contains two genes,
btgA
and
btgB
, and a putative
oriT
region necessary for transfer in
Bacteroides fragilis
and
Escherichia coli
. The BtgA protein was predicted to contain a helix-turn-helix motif, indicating possible DNA binding activity. DNA sequence analysis of the region immediately upstream of
btgA
revealed three sets of inverted repeats, potentially locating the
oriT
region. A 304-bp DNA fragment comprising this putative
oriT
region was cloned and confirmed to be the functional pBFTM10
oriT
by bacterial conjugation experiments using
E. coli
and
B. fragilis. btgA
was cloned and overexpressed in
E. coli
, and the purified protein was used in electrophoretic mobility shift assays, demonstrating specific binding of BtgA protein to its cognate
oriT
. DNase I footprint analysis demonstrated that BtgA binds apparently in a single-stranded fashion to the
oriT
-containing fragment, overlapping inverted repeats I, II, and III and the putative nick site.
Characterization of
dacC
, Which Encodes a New Low-Molecular-Weight Penicillin-Binding Protein in
Bacillus subtilis
Authors: Pedersen Lotte B. , Murray Thomas , Popham David L. , Setlow Peter 
Published: December 2019

ABSTRACT

The
pbp
gene (renamed
dacC
), identified by the
Bacillus subtilis
genome sequencing project, encodes a putative 491-residue protein with sequence homology to low-molecular-weight penicillin-binding proteins. Use of a transcriptional
dacC-lacZ
fusion revealed that
dacC
expression (i) is initiated at the end of stationary phase; (ii) depends strongly on transcription factor ς
H
; and (iii) appears to be initiated from a promoter located immediately upstream of
yoxA
, a gene of unknown function located upstream of
dacC
on the
B. subtilis
chromosome. A
B. subtilis dacC
insertional mutant grew and sporulated identically to wild-type cells, and
dacC
and wild-type spores had the same heat resistance, cortex structure, and germination and outgrowth kinetics. Expression of
dacC
in
Escherichia coli
showed that this gene encodes an ∼59-kDa membrane-associated penicillin-binding protein which is highly toxic when overexpressed.
Heterologous Expression of the
Desulfovibrio gigas
[NiFe] Hydrogenase in
Desulfovibrio fructosovorans
MR400
Authors: Rousset Marc , Magro Valérie , Forget Nicole , Guigliarelli Bruno , Belaich Jean-Pierre , Hatchikian E. Claude 
Published: December 2019

ABSTRACT

The ability of
Desulfovibrio fructosovorans
MR400 Δ
hynABC
to express the heterologous cloned [NiFe] hydrogenase of
Desulfovibrio gigas
was investigated. The [NiFe] hydrogenase operon from
D. gigas
,
hynABCD
, was cloned, sequenced, and introduced into
D. fructosovorans
MR400. A portion of the recombinant heterologous [NiFe] hydrogenase was totally matured, exhibiting catalytic and spectroscopic properties identical to those of the native
D. gigas
protein. A chimeric operon containing
hynAB
from
D. gigas
and
hynC
from
D. fructosovorans
placed under the control of the
D. fructosovorans hynAp
promoter was constructed and expressed in
D. fructosovorans
MR400. Under these conditions, the same level of activity was obtained as with the
D. gigas
hydrogenase operon.
A New Class of
Caulobacter crescentus
Flagellar Genes
Authors: Leclerc Guy , Wang Shui Ping , Ely Bert 
Published: December 2019

ABSTRACT

Eight
Caulobacter crescentus
flagellar genes,
flmA
,
flmB
,
flmC
,
flmD
,
flmE
,
flmF
,
flmG
, and
flmH
, have been cloned and characterized. These eight genes are clustered in pairs (
flmAB
,
flmCD
,
flmEF
, and
flmGH
) that appear to be structurally organized as operons. Homology comparisons suggest that the proteins encoded by the
flm
genes may be involved in posttranslational modification of flagellins or proteins that interact with flagellin monomers prior to their assembly into a flagellar filament. Expression of the
flmAB
,
flmEF
, and
flmGH
operons was shown to occur primarily in predivisional cells. In contrast, the
flmCD
operon was expressed throughout the cell cycle, with only a twofold increase in predivisional cells. The expression of the three temporally regulated operons was subject to positive regulation by the CtrA response regulator protein. Mutations in class II and III flagellar genes had no significant effect on the expression of the
flm
genes. Furthermore, the
flm
genes did not affect the expression of class II or class III flagellar genes. However, mutations in the
flm
genes did result in reduced synthesis of the class IV flagellin proteins. Taken together, these data indicate that the
flm
operons belong to a new class of flagellar genes.
Rare Homologous Gene Targeting in
Histoplasma capsulatum
: Disruption of the
URA5
Hc
Gene by Allelic Replacement
Authors: Woods Jon P. , Retallack Diane M. , Heinecke Elizabeth L. , Goldman William E. 
Published: December 2019

ABSTRACT

URA5
genes encode orotidine-5′-monophosphate pyrophosphorylase (OMPpase), an enzyme involved in pyrimidine biosynthesis. We cloned the
Histoplasma capsulatum URA5
gene (
URA5
Hc
) by using a probe generated by PCR with inosine-rich primers based on relatively conserved sequences in OMPpases from other organisms. Transformation with this gene restored uracil prototrophy and OMPpase activity to UV-mutagenized
ura5
strains of
H. capsulatum
. We attempted to target the genomic
URA5
locus in this haploid organism to demonstrate homologous allelic replacement with transforming DNA, which has not been previously done in
H. capsulatum
and has been challenging in some other pathogenic fungi. Several strategies commonly used in
Saccharomyces cerevisiae
and other eukaryotes were unsuccessful, due to the frequent occurrence of ectopic integration, linear plasmid formation, and spontaneous resistance to 5-fluoroorotic acid, which is a selective agent for
URA5
gene inactivation. Recent development of an efficient electrotransformation system and of a second selectable marker (
hph
, conferring hygromycin B resistance) for this fungus enabled us to achieve allelic replacement by using transformation with an insertionally inactivated Δ
ura5
Hc
::
hph
plasmid, followed by dual selection with hygromycin B and 5-fluoroorotic acid, or by screening hygromycin B-resistant transformants for uracil auxotrophy. The relative frequency of homologous gene targeting was approximately one allelic replacement event per thousand transformants. This work demonstrates the feasibility but also the potential challenge of gene disruption in this organism. To our knowledge, it represents the first example of experimentally directed allelic replacement in
H. capsulatum
, or in any dimorphic systemic fungal pathogen of humans.
A Complex Control System for Transcriptional Activation from the
sid
Promoter of Bacteriophage P4
Authors: Reiter Kaye , Lam Hugh , Young Edward , Julien Bryan , Calendar Richard 
Published: December 2019

ABSTRACT

The
sid
gene promoter (P

sid

), which controls expression of the late genes from satellite phage P4, is activated by a unique class of small DNA-binding proteins. The activators from both satellite and helper phages stimulate transcription from P

sid

. These activators bind to sites centered at position −55 in all the helper and satellite phage late promoters. P4 P

sid

is unique in that it has an additional activator binding site centered at position −18 (site II). We have constructed a mutant of site II that no longer binds activators. Transcription under the control of satellite phage activators is increased by the site II mutation. In contrast, helper phage activators do not show this increase in transcription from P

sid

mutated at site II. Competition gel shift analysis reveals that the P4 satellite phage activator, Delta, binds eightfold better to site II than to site I. The products of the
sid
transcription unit are needed only when a helper phage is present; thus, the satellite phage activators repress transcription until the helper is present to supply a nonrepressing activator.
Bradyrhizobium japonicum
FixK
2
, a Crucial Distributor in the FixLJ-Dependent Regulatory Cascade for Control of Genes Inducible by Low Oxygen Levels
Authors: Nellen-Anthamatten D. , Rossi P. , Preisig O. , Kullik I. , Babst M. , Fischer H. M. , Hennecke H. 
Published: December 2019

ABSTRACT

Bradyrhizobium japonicum
possesses a second
fixK
-like gene,
fixK
2
, in addition to the previously identified
fixK
1
gene. The expression of both genes depends in a hierarchical fashion on the low-oxygen-responsive two-component regulatory system FixLJ, whereby FixJ first activates
fixK
2
, whose product then activates
fixK
1
. While the target genes for control by FixK
1
are unknown, there is evidence for activation of the
fixNOQP
,
fixGHIS
, and
rpoN
1
genes and some heme biosynthesis and nitrate respiration genes by FixK
2
. FixK
2
also regulates its own structural gene, directly or indirectly, in a negative way.
Extracellular Signal Molecule(s) Involved in the Carbon Starvation Response of Marine
Vibrio
sp. Strain S14
Authors: Srinivasan Sujatha , Östling Jörgen , Charlton Timothy , de Nys Rocky , Takayama Kathy , Kjelleberg Staffan 
Published: December 2019

ABSTRACT

The role of exogenous metabolites as putative signal molecules mediating and/or regulating the carbon starvation adaptation program in
Vibrio
sp. strain S14 was investigated. Addition of the stationary-phase supernatant extract (SSE) of
Vibrio
sp. strain S14 to logarithmic-phase cells resulted in a significant number of carbon starvation-induced proteins being up-regulated. Halogenated furanones, putative antagonists of acylated homoserine lactones (AHLs), inhibited the synthesis of proteins specifically induced upon carbon starvation. The effect of the furanone was the opposite of that caused by SSE with respect to the up- and down-regulation of protein expression, indicating that both the furanone and the putative signalling molecules were acting on the same regulatory pathway. Culturability was rapidly lost when
Vibrio
sp. strain S14 was starved in the presence of the furanone at a low concentration. The furanone also had a negative effect on the ability of carbon-starved cells to mount resistance against UV irradiation and hydrogen peroxide exposure. The SSE of
Vibrio
sp. strain S14 had the ability to provide cross-protection against the loss in viability caused by the furanone. We have further demonstrated that the SSE taken from low- as well as high-cell-density cultures of
Vibrio
sp. strain S14 induced luminescence in
Vibrio harveyi
. Taken together, the results in this report provide evidence that
Vibrio
sp. strain S14 produces extracellular signalling metabolites during carbon and energy starvation and that these molecules play an important role in the expression of proteins crucial to the development of starvation- and stress-resistant phenotypes.
Cell Reproduction and Morphological Changes in
Mycoplasma capricolum
Authors: Seto Shintaro , Miyata Makoto 
Published: December 2019

ABSTRACT

The cell reproduction of
Mycoplasma capricolum
was studied. The velocity of DNA replication fork progression was about 6 kb/min, which is 10 times slower than that of
Escherichia coli
. The time required for one round of DNA replication accorded with the doubling time. The origin/terminus ratio was 2.0.
M. capricolum
cell morphology was classified into two types, rod and branched. In the ordinary-growth phase, the rod cells accounted for about 90% of the total population, with branched cells comprising the remaining 10%. The proportion of branched cells increased to 90% following inhibition of DNA replication by nucleoside starvation. An increase in the proportion of branched cells was induced by transfer of a temperature-sensitive mutant deficient in DNA replication to the restrictive temperature. The rod cells had a regular structure, a fixed cell length, and constrictions in the center. The DNA contents of individual rod cells were distributed with a standard deviation of 0.40 of average. The branched cells had irregular structures and a wide distribution of DNA contents. Counting of viable cells revealed that the cells ceased division upon cell type conversion; however, branched cells maintained a reproductive capacity. A model for the reproduction process is proposed.
Initiation and Velocity of Chromosome Replication in
Escherichia coli
B/r and K-12
Authors: Bipatnath Meeshel , Dennis Patrick P. , Bremer Hans 
Published: December 2019

ABSTRACT

The macromolecular composition and a number of parameters affecting chromosome replication were examined over a range of exponential growth rates in two common
Escherichia coli
strains, B/r and K-12 AB1157. Based on improved measurements of DNA after treatment of exponential cultures with rifampin, the cell mass per chromosomal replication origin (initiation mass) and the time required to replicate the chromosome from origin to terminus (
C
period) were determined. For these two strains, the initiation mass approached values of 8 × 10
−10
and 10 × 10
−10
units of optical density (at 460 nm) of culture mass per
oriC
, respectively, at growth rates above 1 doubling/h (at 37°C). The amount of protein per
oriC
decreased with increasing growth rate for AB1157 and remained nearly constant for the B/r strain. The
C
period decreased for both strains in an essentially identical manner from about 70 min at 0.6 doublings/h to about 33 min at 3 doublings/h. From the initiation mass and
C
period, relative or absolute copy numbers for genes with known map locations can be accurately determined at different growth rates. At growth rates above 2 doublings/h, when chromosomes are highly branched, genes near the origin are about threefold more prevalent than genes near the terminus. At a growth rate of 0.6 doubling/h, this ratio is only about 1.7, which reflects the lower degree of chromosome branching.
Properties of the P-Type ATPases Encoded by the
copAP
Operons of
Helicobacter pylori
and
Helicobacter felis
Authors: Bayle Denis , Wängler Sabine , Weitzenegger Thomas , Steinhilber Wolfram , Volz Jürgen , Przybylski Michael , Schäfer Klaus P. , Sachs George , Melchers Klaus 
Published: December 2019

ABSTRACT

The
cop
operons of
Helicobacter pylori
and
Helicobacter felis
were cloned by gene library screening. Both operons contain open reading frames for a P-type ion pump (CopA) with homology to Cd
2+
and Cu
2+
ATPases and a putative ion binding protein (CopP), the latter representing a CopZ homolog of the
copYZAB
operon of
Enterococcus hirae
. The predicted CopA ATPases contained an N-terminal GMXCXXC ion binding motif and a membrane-associated CPC sequence. A synthetic N-terminal peptide of the
H. pylori
CopA ATPase bound to Cu
2+
specifically, and gene disruption mutagenesis of CopA resulted in an enhanced growth sensitivity of
H. pylori
to Cu
2+
but not to other divalent cations. As determined experimentally,
H. pylori
CopA contains four pairs of transmembrane segments (H1 to H8), with the ATP binding and phosphorylation domains lying between H6 and H7, as found for another putative transition metal pump of
H. pylori
(K. Melchers, T. Weitzenegger, A. Buhmann, W. Steinhilber, G. Sachs, and K. P. Schäfer, J. Biol. Chem. 271:446–457, 1996). The corresponding transmembrane segments of the
H. felis
CopA pump were identified by hydrophobicity analysis and via sequence similarity. To define functional domains, similarly oriented regions of the two enzymes were examined for sequence identity. Regions with high degrees of identity included the N-terminal Cu
2+
binding domain, the regions of ATP binding and phosphorylation in the energy transduction domain, and a transport domain consisting of the last six transmembrane segments with conserved cysteines in H4, H6, and H7. The data suggest that
H. pylori
and
H. felis
employ conserved mechanisms of ATPase-dependent copper resistance.
MinCD Proteins Control the Septation Process during Sporulation of
Bacillus subtilis
Authors: Barák Imrich , Prepiak Peter , Schmeisser Falko 
Published: December 2019

ABSTRACT

Mutation of the
divIVB
locus in
Bacillus subtilis
causes misplacement of the septum during cell division and allows the formation of anucleate minicells. The
divIVB
locus contains five open reading frames (ORFs). The last two ORFs (
minCD
) are homologous to
minC
and
minD
of
Escherichia coli
but a
minE
homolog is lacking in
B. subtilis
. There is some similarity between minicell formation and the asymmetric septation that normally occurs during sporulation in terms of polar septum localization. However, it has been proposed that MinCD has no essential role in sporulation septum formation. We have used electron microscopic studies to show septation events during sporulation in some
minD
strains. We have observed an unusually thin septum at the midcell position in
minD
and also in
minD spoIIE71
mutant cells. Fluorescence microscopy also localized a SpoIIE-green fluorescent protein fusion protein at the midcell site in
minD
cells. We propose that the MinCD complex plays an important role in asymmetric septum formation during sporulation of
B. subtilis
cells.
Escherichia coli
Promoters with UP Elements of Different Strengths: Modular Structure of Bacterial Promoters
Authors: Ross Wilma , Aiyar Sarah E. , Salomon Julia , Gourse Richard L. 
Published: December 2019

ABSTRACT

The α subunit of
Escherichia coli
RNA polymerase (RNAP) participates in promoter recognition through specific interactions with UP element DNA, a region upstream of the recognition hexamers for the ς subunit (the −10 and −35 hexamers). UP elements have been described in only a small number of promoters, including the rRNA promoter
rrnB
P1, where the sequence has a very large (30- to 70-fold) effect on promoter activity. Here, we analyzed the effects of upstream sequences from several additional
E. coli
promoters (
rrnD
P1,
rrnB
P2, λ
p
R
,
lac
,
merT
, and RNA II). The relative effects of different upstream sequences were compared in the context of their own core promoters or as hybrids to the
lac
core promoter. Different upstream sequences had different effects, increasing transcription from 1.5- to ∼90-fold, and several had the properties of UP elements: they increased transcription in vitro in the absence of accessory protein factors, and transcription stimulation required the C-terminal domain of the RNAP α subunit. The effects of the upstream sequences correlated generally with their degree of similarity to an UP element consensus sequence derived previously. Protection of upstream sequences by RNAP in footprinting experiments occurred in all cases and was thus not a reliable indicator of UP element strength. These data support a modular view of bacterial promoters in which activity reflects the composite effects of RNAP interactions with appropriately spaced recognition elements (−10, −35, and UP elements), each of which contributes to activity depending on its similarity to the consensus.