bluuebunny/crossref_metadata_2025_split huggingface dataset:
Genetic Diversity of the Streptococcal Competence (
com
) Gene Locus
Authors: Whatmore Adrian M. , Barcus Victoria A. , Dowson Christopher G. 
Published: December 2019

ABSTRACT

The
com
operon of naturally transformable streptococcal species contains three genes,
comC
,
comD
, and
comE
, involved in the regulation of competence. The
comC
gene encodes a competence-stimulating peptide (CSP) thought to induce competence in the bacterial population at a critical extracellular concentration. The
comD
and
comE
genes are believed to encode the transmembrane histidine kinase and response regulator proteins, respectively, of a two-component regulator, with the
comD
-encoded protein being a receptor for CSP. Here we report on the genetic variability of
comC
and
comD
within
Streptococcus pneumoniae
isolates. Comparative analysis of sequence variations of
comC
and
comD
shows that, despite evidence for horizontal gene transfer at this locus and the lack of transformability of many
S. pneumoniae
strains in the laboratory, there is a clear correlation between the presence of a particular
comC
allele and the cognate
comD
allele. These findings effectively rule out the possibility that the presence of noncognate
comC
and
comD
alleles may be responsible for the inability to induce competence in many isolates and indicate the importance of a functional
com
pathway in these isolates. In addition, we describe a number of novel CSPs from disease-associated strains of
S. mitis
and
S. oralis
. The CSPs from these isolates are much more closely related to those from
S. pneumoniae
than to most CSPs previously reported from
S. mitis
and
S. oralis
, suggesting that these particular organisms may be a potential source of DNA in recombination events generating the mosaic structures commonly reported in genes of
S. pneumoniae
that are under strong selective pressure.
Cell Cycle Expression and Transcriptional Regulation of DNA Topoisomerase IV Genes in
Caulobacter
Authors: Ward Doyle V. , Newton Austin 
Published: December 2019

ABSTRACT

DNA replication and differentiation are closely coupled during the
Caulobacter crescentus
cell cycle. We have previously shown that DNA topoisomerase IV (topo IV), which is encoded by the
parE
and
parC
genes, is required for chromosomal partitioning, cell division, and differentiation in this bacterium (D. Ward and A. Newton, Mol. Microbiol. 26:897–910, 1997). We have examined the cell cycle regulation of
parE
and
parC
and report here that transcription of these topo IV genes is induced during the swarmer-to-stalked-cell transition when cells prepare for initiation of DNA synthesis. The regulation of
parE
and
parC
expression is not strictly coordinated, however. The rate of
parE
transcription increases ca. 20-fold during the G
1
-to-S-phase transition and in this respect, its pattern of regulation is similar to those of several other genes required for chromosome duplication. Transcription from the
parC
promoter, by contrast, is induced only two- to threefold during this cell cycle period. Steady-state ParE levels are also regulated, increasing ca. twofold from low levels in swarmer cells to a maximum immediately prior to cell division, while differences in ParC levels during the cell cycle could not be detected. These results suggest that topo IV activity may be regulated primarily through
parE
expression. The presumptive promoters of the topo IV genes display striking similarities to, as well as differences from, the consensus promoter recognized by the major
Caulobacter
sigma factor ς
73
. We also present evidence that a conserved 8-mer sequence motif located in the spacers between the −10 and −35 elements of the
parE
and
parC
promoters is required for maximum levels of
parE
transcription, which raises the possibility that it may function as a positive regulatory element. The pattern of
parE
transcription and the
parE
and
parC
promoter architecture suggest that the topo IV genes belong to a specialized subset of cell cycle-regulated genes required for chromosome replication.
Saccharomyces cerevisiae
Mid2p Is a Potential Cell Wall Stress Sensor and Upstream Activator of the
PKC1-MPK1
Cell Integrity Pathway
Authors: Ketela Troy , Green Robin , Bussey Howard 
Published: December 2019

ABSTRACT

The
MID2
gene of
Saccharomyces cerevisiae
encodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor.
MID2
was isolated as a multicopy activator of the Skn7p transcription factor. Deletion of
MID2
causes resistance to calcofluor white, diminished production of stress-induced cell wall chitin under a variety of conditions, and changes in growth rate and viability in a number of different cell wall biosynthesis mutants. Overexpression of
MID2
causes hyperaccumulation of chitin and increased sensitivity to calcofluor white. α-Factor hypersensitivity of
mid2
Δ mutants can be suppressed by overexpression of upstream elements of the cell integrity pathway, including
PKC1
,
RHO1
,
WSC1
, and
WSC2
. Mid2p and Wsc1p appear to have overlapping roles in maintaining cell integrity since
mid2Δ wsc1
Δ mutants are inviable on medium that does not contain osmotic support. A role for
MID2
in the cell integrity pathway is further supported by the finding that
MID2
is required for induction of Mpk1p tyrosine phosphorylation during exposure to α-factor, calcofluor white, or high temperature. Our data are consistent with a role for Mid2p in sensing cell wall stress and in activation of a response that includes both increased chitin synthesis and the Mpk1p mitogen-activated protein kinase cell integrity pathway. In addition, we have identified an open reading frame,
MTL1
, which encodes a protein with both structural and functional similarity to Mid2p.
Redundant In Vivo Proteolytic Activities of
Escherichia coli
Lon and the ClpYQ (HslUV) Protease
Authors: Wu Whi-Fin , Zhou YanNing , Gottesman Susan 
Published: December 2019

ABSTRACT

The ClpYQ (HslUV) ATP-dependent protease of
Escherichia coli
consists of an ATPase subunit closely related to the Clp ATPases and a protease component related to those found in the eukaryotic proteasome. We found that this protease has a substrate specificity overlapping that of the Lon protease, another ATP-dependent protease in which a single subunit contains both the proteolytic active site and the ATPase. Lon is responsible for the degradation of the cell division inhibitor SulA;
lon
mutants are UV sensitive, due to the stabilization of SulA.
lon
mutants are also mucoid, due to the stabilization of another Lon substrate, the positive regulator of capsule transcription, RcsA. The overproduction of ClpYQ suppresses both of these phenotypes, and the suppression of UV sensitivity is accompanied by a restoration of the rapid degradation of SulA. Inactivation of the chromosomal copy of
clpY
or
clpQ
leads to further stabilization of SulA in a
lon
mutant but not in
lon
+
cells. While either
lon
,
lon clpY
, or
lon clpQ
mutants are UV sensitive at low temperatures, at elevated temperatures the
lon
mutant loses its UV sensitivity, while the double mutants do not. Therefore, the degradation of SulA by ClpYQ at elevated temperatures is sufficient to lead to UV resistance. Thus, a protease with a structure and an active site different from those of Lon is capable of recognizing and degrading two different Lon substrates and appears to act as a backup for Lon under certain conditions.
Structural and Functional Roles of the Surface-Exposed Loops of the β-Barrel Membrane Protein OmpA from
Escherichia coli
Authors: Koebnik Ralf 
Published: December 2019

ABSTRACT

The N-terminal domain of the OmpA protein from
Escherichia coli
, consisting of 170 amino acid residues, is embedded in the outer membrane, in the form of an antiparallel β-barrel whose eight transmembrane β-strands are connected by three short periplasmic turns and four relatively large surface-exposed hydrophilic loops. This protein domain serves as a paradigm for the study of membrane assembly of integral β-structured membrane proteins. In order to dissect the structural and functional roles of the surface-exposed loops, they were shortened separately and in all possible combinations. All 16 loop deletion mutants assembled into the outer membrane with high efficiency and adopted the wild-type membrane topology. This systematic approach proves the absence of topogenic signals (e.g., in the form of loop sizes or charge distributions) in these loops. The shortening of surface-exposed loops did not reduce the thermal stability of the protein. However, none of the mutant proteins, with the exception of the variant with the fourth loop shortened, served as a receptor for the OmpA-specific bacteriophage K3. Furthermore, all loops were necessary for the OmpA protein to function in the stabilization of mating aggregates during F conjugation. An OmpA deletion variant with all four loops shortened, consisting of only 135 amino acid residues, constitutes the smallest β-structured integral membrane protein known to date. These results represent a further step toward the development of artificial outer membrane proteins.
Proliferation of Intrahyphal Hyphae Caused by Disruption ofcsmA, Which Encodes a Class V Chitin Synthase with a Myosin Motor-Like Domain inAspergillus nidulans
Authors: Horiuchi Hiroyuki , Fujiwara Makoto , Yamashita Shuichi , Ohta Akinori , Takagi Masamichi 
Published: December 2019

ABSTRACTWe have found that theAspergillus nidulans csmAgene encodes a novel protein which consists of an N-terminal myosin motor-like domain and a C-terminal chitin synthase domain (M. Fujiwara, H. Horiuchi, A. Ohta, and M. Takagi, Biochem. Biophys. Res. Commun. 236:75–78, 1997). To clarify the roles ofcsmAin fungal morphogenesis, we constructedcsmAnull mutants. The growth rate of the mutant colonies was almost the same as that of the wild-type strain, but hyphal growth was severely inhibited when a chitin-binding reagent, Calcofluor white or Congo red, was added to the medium. Moreover, morphological abnormalities in tip growth and septum formation were identified microscopically. Proliferation of intracellular new hyphae, called intrahyphal hyphae, which behaved as intrinsic hyphae, was the most striking phenotypic feature among them. These phenotypes were not suppressed when the only chitin synthase domain ofcsmAwas expressed under the control of thealcApromoter, whereas they were suppressed when the intact form ofcsmAwas expressed. Therefore, it was concluded that the product ofcsmA(CsmA) has important roles in polarized cell wall synthesis and maintenance of cell wall integrity and that the myosin motor-like domain is indispensable for these functions.
A Mutant
Escherichia coli
Primase Defective in Elongation of Primer RNA Chains
Authors: Sun Wuliang , Schoneich Jerzy , Godson G. Nigel 
Published: December 2019

ABSTRACT

Earlier we showed by affinity cross-linking of initiating substrates to
Escherichia coli
primase that one or more of the residues Lys211, Lys229, and Lys241 were involved in the catalytic center of the enzyme (A. A. Mustaev and G. N. Godson, J. Biol. Chem. 270:15711–15718, 1995). We now demonstrate by mutagenesis that only Lys241 but not Lys211 and Lys229 is part of the catalytic center. Primase with a mutation of Arg to Lys at position 241 (defined as K241R-primase) is almost unable to synthesize primer RNA (pRNA) on the single-stranded DNA-binding protein (SSB)/R199G4oric template. However, it is able to synthesize a pppApG dimer plus trace amounts of 8- to 11-nucleotide (nt) pRNA transcribed from the 5′ CTG 3′ pRNA initiation site on phage G4 oric DNA. The amount of dimer synthesized by K241R-primase is similar to that synthesized by the wild-type primase, demonstrating that the K241R mutant can initiate pRNA synthesis normally but is deficient in chain elongation. In the general priming system, the K241R-primase also can synthesize only the dimer and very small amounts of 11-nt pRNA. The results of gel retardation experiments suggested that this deficiency in pRNA chain elongation of the K241R mutant primase is unlikely to be caused by impairment of the DNA binding activity. The K241R mutant primase, however, can still prime DNA synthesis in vivo and in vitro.
Porphyrin-Mediated Binding to Hemoglobin by the HA2 Domain of Cysteine Proteinases (Gingipains) and Hemagglutinins from the Periodontal PathogenPorphyromonas gingivalis
Authors: DeCarlo Arthur A. , Paramaesvaran Mayuri , Yun Peter L. W. , Collyer Charles , Hunter Neil 
Published: December 2019

ABSTRACTHeme binding and uptake are considered fundamental to the growth and virulence of the gram-negative periodontal pathogenPorphyromonas gingivalis. We therefore examined the potential role of the dominantP. gingivaliscysteine proteinases (gingipains) in the acquisition of heme from the environment. A recombinant hemoglobin-binding domain that is conserved between two predominant gingipains (domain HA2) demonstrated tight binding to hemin (Kd= 16 nM), and binding was inhibited by iron-free protoporphyrin IX (Ki= 2.5 μM). Hemoglobin binding to the gingipains and the recombinant HA2 (rHA2) domain (Kd= 2.1 nM) was also inhibited by protoporphyrin IX (Ki= 10 μM), demonstrating an essential interaction between the HA2 domain and the heme moiety in hemoglobin binding. Binding of rHA2 with either hemin, protoporphyrin IX, or hematoporphyrin was abolished by establishing covalent linkage of the protoporphyrin propionic acid side chains to fixed amines, demonstrating specific and directed binding of rHA2 to these protoporphyrins. A monoclonal antibody which recognizes a peptide epitope within the HA2 domain was employed to demonstrate that HA2-associated hemoglobin-binding activity was expressed and released byP. gingivaliscells in a batch culture, in parallel with proteinase activity. Cysteine proteinases fromP. gingivalisappear to be multidomain proteins with functions for hemagglutination, erythrocyte lysis, proteolysis, and heme binding, as demonstrated here. Detailed understanding of the biochemical pathways for heme acquisition inP. gingivalismay allow precise targeting of this critical metabolic aspect for periodontal disease prevention.
An Intracellular Iron Chelator Pleiotropically Suppresses Enzymatic and Growth Defects of Superoxide Dismutase-Deficient
Escherichia coli
Authors: Maringanti Sujatha , Imlay James A. 
Published: December 2019

ABSTRACT

Mutants of
Escherichia coli
that lack cytoplasmic superoxide dismutase (SOD) exhibit auxotrophies for sulfur-containing, branched-chain, and aromatic amino acids and cannot catabolize nonfermentable carbon sources. A secondary-site mutation substantially relieved all of these growth defects. The requirement for fermentable carbon and the branched-chain auxotrophy occur because superoxide (O
2
−
) leaches iron from the [4Fe-4S] clusters of a family of dehydratases, thereby inactivating them; the suppression of these phenotypes was mediated by the restoration of activity to these dehydratases, evidently without changing the intracellular concentration of O
2
−
. Cloning, complementation, and sequence analysis identified the suppressor mutation to be in
dapD
, which encodes tetrahydrodipicolinate succinylase, an enzyme involved in diaminopimelate and lysine biosynthesis. A block in
dapB
, which encodes dihydrodipicolinate reductase in the same pathway, conferred similar protection. Genetic analysis indicated that the protection stems from the intracellular accumulation of tetrahydro- or dihydrodipicolinate. Heterologous expression in the SOD mutants of the dipicolinate synthase of
Bacillus subtilis
generated dipicolinate and similarly protected them. Dipicolinates are excellent iron chelators, and their accumulation in the cell triggered derepression of the Fur regulon and a large increase in the intracellular pool of free iron, presumably as a dipicolinate chelate. A
fur
mutation only partially relieved the auxotrophies, indicating that Fur derepression assists but is not sufficient for suppression. It seems plausible that the abundant internal iron permits efficient reactivation of superoxide-damaged iron-sulfur clusters. This result provides circumstantial evidence that the sulfur and aromatic auxotrophies of SOD mutants are also directly or indirectly linked to iron metabolism.
Construction and Initial Characterization of
Escherichia coli
Strains with Few or No Intact Chromosomal rRNA Operons
Authors: Asai Tsuneaki , Condon Ciarán , Voulgaris Justina , Zaporojets Dmitry , Shen Binghua , Al-Omar Michaal , Squires Craig , Squires Catherine L. 
Published: December 2019

ABSTRACT

The
Escherichia coli
genome carries seven rRNA (
rrn
) operons, each containing three rRNA genes. The presence of multiple operons has been an obstacle to many studies of rRNA because the effect of mutations in one operon is diluted by the six remaining wild-type copies. To create a tool useful for manipulating rRNA, we sequentially inactivated from one to all seven of these operons with deletions spanning the 16S and 23S rRNA genes. In the final strain, carrying no intact rRNA operon on the chromosome, rRNA molecules were expressed from a multicopy plasmid containing a single rRNA operon (prrn). Characterization of these
rrn
deletion strains revealed that deletion of two operons was required to observe a reduction in the growth rate and rRNA/protein ratio. When the number of deletions was extended from three to six, the decrease in the growth rate was slightly more than the decrease in the rRNA/protein ratio, suggesting that ribosome efficiency was reduced. This reduction was most pronounced in the Δ7 prrn strain, in which the growth rate, unlike the rRNA/protein ratio, was not completely restored to wild-type levels by a cloned rRNA operon. The decreases in growth rate and rRNA/protein ratio were surprisingly moderate in the
rrn
deletion strains; the presence of even a single operon on the chromosome was able to produce as much as 56% of wild-type levels of rRNA. We discuss possible applications of these strains in rRNA studies.
Identification of Two New Proteins in Spermidine Nucleoids Isolated from
Escherichia coli
Authors: Murphy Lizabeth D. , Rosner Judah L. , Zimmerman Steven B. , Esposito Dominic 
Published: December 2019

ABSTRACT

The
Escherichia coli
nucleoid contains DNA in a condensed but functional form. Analysis of proteins released from isolated spermidine nucleoids after treatment with DNase I reveals significant amounts of two proteins not previously detected in wild-type
E. coli
. Partial amino-terminal sequencing has identified them as the products of
rdgC
and
yejK
. These proteins are strongly conserved in gram-negative bacteria, suggesting that they have important cellular roles.
Fused and Overlapping
rpoB
and
rpoC
Genes in Helicobacters, Campylobacters, and Related Bacteria
Authors: Zakharova Natalya , Paster Bruce J. , Wesley Irene , Dewhirst Floyd E. , Berg Douglas E. , Severinov Konstantin V. 
Published: December 2019

ABSTRACT

The genes coding for the β (
rpoB
) and β′ (
rpoC
) subunits of RNA polymerase are fused in the gastric pathogen
Helicobacter pylori
but separate in other taxonomic groups. To better understand how the unique fused structure evolved, we determined DNA sequences at and around the
rpoB-rpoC
junction in 10 gastric and nongastric species of
Helicobacter
and in members of the related genera
Wolinella
,
Arcobacter
,
Sulfurospirillum
, and
Campylobacter
. We found the fusion to be specific to
Helicobacter
and
Wolinella
genera;
rpoB
and
rpoC
overlap in the other genera. The fusion may have arisen by a frameshift mutation at the site of
rpoB
and
rpoC
overlap. Loss of good Shine-Dalgarno sequences might then have fixed the fusion in the
Helicobacteraceae
, even if fusion itself did not confer a selective advantage.
Effects of Ethyl and Benzyl Analogues of Spermine on
Escherichia coli
Peptidyltransferase Activity, Polyamine Transport, and Cellular Growth
Authors: Karahalios Panagiotis , Amarantos Ioannis , Mamos Petros , Papaioannou Dionysios , Kalpaxis Dimitrios L. 
Published: December 2019

ABSTRACT

Various ethyl and benzyl spermine analogues, including the anticancer agent
N
1
,
N
12
-bis(ethyl)spermine, were studied for their ability to affect the growth of cultured
Escherichia coli
cells, to inhibit [
3
H]putrescine and [
3
H]spermine uptake into cells, and to modulate the peptidyltransferase activity (EC 2. 3. 2. 12). Relative to other cell lines, growth of
E. coli
was uniquely insensitive to these analogues. Nevertheless, these analogues conferred similar modulation of in vitro protein synthesis and inhibition of [
3
H]putrescine and [
3
H]spermine uptake, as is seen in other cell types. Thus, both ethyl and benzyl analogues of spermine not only promote the formation and stabilization of the initiator ribosomal ternary complex, but they also have a sparing effect on the Mg
2+
requirements. Also, in a complete cell-free protein-synthesizing system, these analogues at low concentrations stimulated peptide bond formation, whereas at higher concentrations, they inhibited the reaction. The ranking order for stimulation of peptide-bond formation by the analogues was
N
4
,
N
9
-dibenzylspermine >
N
4
,
N
9
-bis(ethyl)spermine ≅
N
1
-ethylspermine >
N
1
,
N
12
-bis(ethyl)spermine, whereas the order of analogue potency regarding the inhibitory effect was inverted, with inhibition constant values of 10, 3.1, 1.5, and 0.98 μM, respectively. Although the above analogues failed to interact with the putrescine-specific uptake system, they exhibited high affinity for the polyamine uptake system encoded by the
potABCD
operon. Despite this fact, none of the analogues could be internalized by the polyamine transport system, and therefore they could not influence the intracellular polyamine pools and growth of
E. coli
cells.
Structure-Function Analysis of XcpP, a Component Involved in General Secretory Pathway-Dependent Protein Secretion inPseudomonas aeruginosa
Authors: Bleves Sophie , Gérard-Vincent Manon , Lazdunski Andrée , Filloux Alain 
Published: December 2019

ABSTRACTThe general secretory pathway ofPseudomonas aeruginosais required for the transport of signal peptide-containing exoproteins across the cell envelope. After completion of the Sec-dependent translocation of exoproteins across the inner membrane and cleavage of the signal peptide, the Xcp machinery mediates translocation across the outer membrane. This machinery consists of 12 components, of which XcpQ (GspD) is the sole outer membrane protein. XcpQ forms a multimeric ring-shaped structure, with a central opening through which exoproteins could pass to reach the medium. Surprisingly, all of the other Xcp proteins are located in or are associated with the cytoplasmic membrane. This study is focused on the characteristics of one such cytoplasmic membrane protein, XcpP. AnxcpPmutant demonstrated that the product of this gene is indeed an essential element of theP. aeruginosasecretion machinery. Construction and analysis of truncated forms of XcpP made it possible to define essential domains for the function of the protein. Some of these domains, such as the N-terminal transmembrane domain and a coiled-coil structure identified at the C terminus of XcpP, may be involved in protein-protein interaction during the assembly of the secretory apparatus.