bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification, Genomic Organization, and Analysis of the Group III Capsular Polysaccharide Genes
kpsD
,
kpsM
,
kpsT
, and
kpsE
from an Extraintestinal Isolate of
Escherichia coli
(CP9, O4/K54/H5)
Authors: Russo Thomas A. , Wenderoth Suzanne , Carlino Ulrike B. , Merrick Joseph M. , Lesse Alan J. 
Published: December 2019

ABSTRACT

Group III capsular polysaccharides (e.g., K54) of extraintestinal isolates of
Escherichia coli
, similar to group II capsules (e.g., K1), are important virulence traits that confer resistance to selected host defense components in vitro and potentiate systemic infection in vivo. The genomic organization of group II capsule gene clusters has been established as a serotype-specific region 2 flanked by regions 1 and 3, which contain transport genes that are highly homologous between serotypes. In contrast, the organization of group III capsule gene clusters is not well understood. However, they are defined in part by an absence of genes with significant nucleotide homology to group II capsule transport genes in regions 1 and 3. Evaluation of isogenic, Tn
phoA
-generated, group III capsule-minus derivatives of a clinical blood isolate (CP9, O4/K54/H5) has led to the identification of homologs of the group II capsule transport genes
kpsDMTE
. These genes and their surrounding regions were sequenced and analyzed. The genomic organization of these genes is distinctly different from that of their group II counterparts. Although
kps
K54
DMTE
are significantly divergent from their group II homologs at both the DNA and protein levels
phoA
fusions and computer-assisted analyses suggest that their structures and functions are similar. The putative proteins Kps
K54
M and Kps
K54
T appear to be the integral membrane component and the peripheral ATP-binding component of the ABC-2 transporter family, respectively. The putative Kps
K54
E possesses features similar to those of the membrane fusion protein family that facilitates the passage of large molecules across the periplasm. At one boundary of the capsule gene cluster, a truncated
kpsM
(
kpsM
truncated
) and its 5′ noncoding regulatory sequence were identified. In contrast to the complete
kps
K54
M
, this region was highly homologous to the group II
kpsM
. Fifty-three base pairs 3′ from the end of
kpsM
truncated
was a sequence 75% homologous to the 39-bp inverted repeat in the IS
110
insertion element from
Streptomyces coelicolor
. Southern analysis established that two copies of this element are present in CP9. These findings are consistent with the hypothesis that CP9 previously possessed group II capsule genes and acquired group III capsule genes via IS
110
-mediated horizontal transfer.
Construction and Characterization of a
Helicobacter pylori clpB
Mutant and Role of the Gene in the Stress Response
Authors: Allan Elaine , Mullany Peter , Tabaqchali Soad 
Published: December 2019

ABSTRACT

Antiserum raised against whole
Helicobacter pylori
cells identified a novel 94-kDa antigen. The nucleotide sequence of the gene encoding the 94-kDa antigen was determined, and analysis of the deduced amino acid sequence revealed structural features typical of the ClpB ATPase family of stress response proteins. An isogenic
H. pylori clpB
mutant showed increased sensitivity to high-temperature stress, indicating that the
clpB
gene product functions as a stress response protein in
H. pylori.
Characterization of Multiple Regions Involved in Replication and Mobilization of Plasmid pNZ4000 Coding for Exopolysaccharide Production in
Lactococcus lactis
Authors: van Kranenburg Richard , de Vos Willem M. 
Published: December 2019

ABSTRACT

We characterized the regions involved in replication and mobilization of the 40-kb plasmid pNZ4000, encoding exopolysaccharide (EPS) production in
Lactococcus lactis
NIZO B40. The plasmid contains four highly conserved replication regions with homologous
rep
genes (
repB1
,
repB2
,
repB3
, and
repB4
) that belong to the lactococcal theta replicon family. Subcloning of each replicon individually showed that all are functional and compatible in
L. lactis
. Plasmid pNZ4000 and genetically labeled derivatives could be transferred to different
L. lactis
strains by conjugation, and pNZ4000 was shown to be a mobilization plasmid. Two regions involved in mobilization were identified near two of the replicons; both included an
oriT
sequence rich in inverted repeats. Conjugative mobilization of the nonmobilizable plasmid pNZ124 was promoted by either one of these
oriT
sequences, demonstrating their functionality. One
oriT
sequence was followed by a
mobA
gene, coding for a
trans
-acting protein, which increased the frequency of conjugative transfer 100-fold. The predicted MobA protein and the
oriT
sequences show protein and nucleotide similarity, respectively, with the relaxase and with the inverted repeat and
nic
site of the
oriT
from the
Escherichia coli
plasmid R64. The presence on pNZ4000 of four functional replicons, two
oriT
sequences, and several insertion sequence-like elements strongly suggests that this EPS plasmid is a naturally occurring cointegrate.
Conserved Structural Regions Involved in the Catalytic Mechanism of
Escherichia coli
K-12 WaaO (RfaI)
Authors: Shibayama Keigo , Ohsuka Shinji , Tanaka Toshihiko , Arakawa Yoshichika , Ohta Michio 
Published: December 2019

ABSTRACT

Escherichia coli
K-12 WaaO (formerly known as RfaI) is a nonprocessive α-1,3 glucosyltransferase, involved in the synthesis of the R core of lipopolysaccharide. By comparing the amino acid sequence of WaaO with those of 11 homologous α-glycosyltransferases, four strictly conserved regions, I, II, III, and IV, were identified. Since functionally related transferases are predicted to have a similar architecture in the catalytic sites, it is assumed that these four regions are directly involved in the formation of α-glycosidic linkage from α-linked nucleotide diphospho-sugar donor. Hydrophobic cluster analysis revealed a conserved domain at the N termini of these α-glycosyltransferases. This domain was similar to that previously reported for β-glycosyltransferases. Thus, this domain is likely to be involved in the formation of β-glycosidic linkage between the donor sugar and the enzyme at the first step of the reaction. Site-directed mutagenesis analysis of
E. coli
K-12 WaaO revealed four critical amino acid residues.
Regulation of the
Bacillus subtilis
GlcT Antiterminator Protein by Components of the Phosphotransferase System
Authors: Bachem Steffi , Stülke Jörg 
Published: December 2019

ABSTRACT

Bacillus subtilis
utilizes glucose as the preferred source of carbon and energy. The sugar is transported into the cell by a specific permease of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) encoded by the
ptsGHI
operon. Expression of this operon is induced by glucose and requires the action of a positive transcription factor, the GlcT antiterminator protein. Glucose availability is sensed by glucose-specific enzyme II (EII
Glc
), the product of
ptsG
. In the absence of inducer, the glucose permease negatively controls the activity of the antiterminator. The GlcT antiterminator has a modular structure. The isolated N-terminal part contains the RNA-binding protein and acts as a constitutively acting antiterminator. GlcT contains two PTS regulation domains (PRDs) at the C terminus. One (PRD-I) is the target of negative control exerted by EII
Glc
. A conserved His residue (His-104 in GlcT) is involved in inactivation of GlcT in the absence of glucose. It was previously proposed that PRD-containing transcriptional antiterminators are phosphorylated and concomitantly inactivated in the absence of the substrate by their corresponding PTS permeases. The results obtained with
B. subtilis
glucose permease with site-specific mutations suggest, however, that the permease might modulate the phosphorylation reaction without being the phosphate donor.
Analogs of the Autoinducer 3-Oxooctanoyl-Homoserine Lactone Strongly Inhibit Activity of the TraR Protein of
Agrobacterium tumefaciens
Authors: Zhu Jun , Beaber John W. , Moré Margret I. , Fuqua Clay , Eberhard Anatol , Winans Stephen C. 
Published: December 2019

ABSTRACT

The TraR and TraI proteins of
Agrobacterium tumefaciens
mediate cell-density-dependent expression of the Ti plasmid
tra
regulon. TraI synthesizes the autoinducer pheromone
N
-(3-oxooctanoyl)-
l
-homoserine lactone (3-oxo-C
8
-HSL), while TraR is an 3-oxo-C
8
-HSL-responsive transcriptional activator. We have compared the abilities of 3-oxo-C
8
-HSL and 32 related compounds to activate expression of a TraR-regulated promoter. In a strain that expresses wild-type levels of TraR, only 3-oxo-C
8
-HSL was strongly stimulatory, four compounds were detectably active only at high concentrations, and the remaining 28 compounds were inactive. Furthermore, many of these compounds were potent antagonists. In contrast, almost all of these compounds were stimulatory in a congenic strain that overexpresses TraR and no compound was a potent antagonist. We propose a model in which autoinducers enhance the affinity of TraR either for other TraR monomers or for DNA binding sites and that overexpression of TraR potentiates this interaction by mass action. Wild-type
A. tumefaciens
released a rather broad spectrum of autoinducers, including several that antagonize induction of a wild-type strain. However, under all conditions tested, 3-oxo-C
8
-HSL was more abundant than any other analog, indicating that other released autoinducers do not interfere with
tra
gene induction. We conclude that (i) in wild-type strains, only 3-oxo-C
8
-HSL significantly stimulates
tra
gene expression, while many autoinducer analogs are potent antagonists; (ii) TraR overexpression increases agonistic activity of autoinducer analogs, allowing sensitive biodetection of many autoinducers; and (iii) autoinducer stimulatory activity is potentiated by TraR overproduction, suggesting that autoinducers may shift an equilibrium between TraR monomers and dimers or oligomers. When autoinducer specificities of other quorum-sensing proteins are tested, care should be taken not to overexpress those proteins.
Methanococcus jannaschii
Flap Endonuclease: Expression, Purification, and Substrate Requirements
Authors: Rao H. G. V. , Rosenfeld Amy , Wetmur James G. 
Published: December 2019

ABSTRACT

The flap endonuclease (FEN) of the hyperthermophilic archaeon
Methanococcus jannaschii
was expressed in
Escherichia coli
and purified to homogeneity. FEN retained activity after preincubation at 95°C for 15 min. A pseudo-Y-shaped substrate was formed by hybridization of two partially complementary oligonucleotides. FEN cleaved the strand with the free 5′ end adjacent to the single-strand–duplex junction. Deletion of the free 3′ end prevented cleavage. Hybridization of a complementary oligonucleotide to the free 3′ end moved the cleavage site by 1 to 2 nucleotides. Hybridization of excess complementary oligonucleotide to the free 5′ end failed to block cleavage, although this substrate was refractory to cleavage by the 5′-3′ exonuclease activity of
Taq
DNA polymerase. For verification, the free 5′ end was replaced by an internally labeled hairpin structure. This structure was a substrate for FEN but became a substrate for
Taq
DNA polymerase only after exonucleolytic cleavage had destabilized the hairpin. A circular duplex substrate with a 5′ single-stranded branch was formed by primer extension of a partially complementary oligonucleotide on virion φX174. This denaturation-resistant substrate was used to examine the effects of temperature and solution properties, such as pH, salt, and divalent ion concentration on the turnover number of the enzyme.
Fumarate Regulation of Gene Expression in
Escherichia coli
by the DcuSR (
dcuSR
Genes) Two-Component Regulatory System
Authors: Zientz Evelyn , Bongaerts Johannes , Unden Gottfried 
Published: December 2019

ABSTRACT

In
Escherichia coli
the genes encoding the anaerobic fumarate respiratory system are transcriptionally regulated by C
4
-dicarboxylates. The regulation is effected by a two-component regulatory system, DcuSR, consisting of a sensory histidine kinase (DcuS) and a response regulator (DcuR). DcuS and DcuR are encoded by the
dcuSR
genes (previously
yjdHG
) at 93.7 min on the calculated
E. coli
map. Inactivation of the
dcuR
and
dcuS
genes caused the loss of C
4
-dicarboxylate-stimulated synthesis of fumarate reductase (
frdABCD
genes) and of the anaerobic fumarate-succinate antiporter DcuB (
dcuB
gene). DcuS is predicted to contain a large periplasmic domain as the supposed site for C
4
-dicarboxylate sensing. Regulation by DcuR and DcuS responded to the presence of the C
4
-dicarboxylates fumarate, succinate, malate, aspartate, tartrate, and maleate. Since maleate is not taken up by the bacteria under these conditions, the carboxylates presumably act from without. Genes of the aerobic C
4
-dicarboxylate pathway encoding succinate dehydrogenase (
sdhCDAB
) and the aerobic succinate carrier (
dctA
) are only marginally or negatively regulated by the DcuSR system. The CitAB two-component regulatory system, which is highly similar to DcuSR, had no effect on C
4
-dicarboxylate regulation of any of the genes.
Different Phenotypic Classes of
Sinorhizobium meliloti
Mutants Defective in Synthesis of K Antigen
Authors: Campbell Gordon R. O. , Reuhs Bradley L. , Walker Graham C. 
Published: December 2019

ABSTRACT

For
Sinorhizobium meliloti
(also known as
Rhizobium meliloti
) AK631 to establish effective symbiosis with alfalfa, it must be able to synthesize a symbiotically active form of its K antigen, a capsular polysaccharide containing a Kdo (3-deoxy-
d
-manno-octulosonic acid) derivative. Previously isolated mutants defective in the synthesis of K antigen are resistant to bacteriophage φ16-3. By screening ca. 100,000 Tn
5
-mutagenized
R. meliloti
bacteria for resistance to bacteriophage φ16-3, we isolated 119 mutants, 31 of which could not be complemented by genes previously identified as being required for K-antigen synthesis. Of these 31 new mutants, 13 were symbiotically defective and lacked the K antigen. Through genetic and phenotypic analyses, we have grouped these mutants into four distinct classes. Although all of these mutants lack the K antigen, many also have altered lipopolysaccharides (LPS), suggesting that the biochemical pathways for the synthesis of K antigen and LPS have common enzymatic steps. In addition, we have found that these and other classes of K-antigen-defective mutants of
S. meliloti
AK631 exhibit unique patterns of sensitivities to phage strains to which the parental strain was resistant. Our studies have identified new classes of genes required for both the synthesis of K antigen and the symbiotic proficiency of
S. meliloti
AK631. Some of these classes of genes also play a role in LPS synthesis.
Analysis of the Novel Benzylsuccinate Synthase Reaction for Anaerobic Toluene Activation Based on Structural Studies of the Product
Authors: Beller Harry R. , Spormann Alfred M. 
Published: December 2019

ABSTRACT

Recent studies of anaerobic toluene catabolism have demonstrated a novel reaction for anaerobic hydrocarbon activation: the addition of the methyl carbon of toluene to fumarate to form benzylsuccinate. In vitro studies of the anaerobic benzylsuccinate synthase reaction indicate that the H atom abstracted from the toluene methyl group during addition to fumarate is retained in the succinyl moiety of benzylsuccinate. Based on structural studies of benzylsuccinate formed during anaerobic, in vitro assays with denitrifying, toluene-mineralizing strain T, we now report the following characteristics of the benzylsuccinate synthase reaction: (i) it is highly stereospecific, resulting in >95% formation of the (+)-benzylsuccinic acid enantiomer [(
R
)-2-benzyl-3-carboxypropionic acid], and (ii) active benzylsuccinate synthase does not contain an abstracted methyl H atom from toluene at the beginning or at the end of a catalytic cycle.
Oxygen-Insensitive Nitroreductases: Analysis of the Roles of
nfsA
and
nfsB
in Development of Resistance to 5-Nitrofuran Derivatives in
Escherichia coli
Authors: Whiteway J. , Koziarz P. , Veall J. , Sandhu N. , Kumar P. , Hoecher B. , Lambert I. B. 
Published: December 2019

ABSTRACT

Nitroheterocyclic and nitroaromatic compounds constitute an enormous range of chemicals whose potent biological activity has significant human health and environmental implications. The biological activity of nitro-substituted compounds is derived from reductive metabolism of the nitro moiety, a process catalyzed by a variety of nitroreductase activities. Resistance of bacteria to nitro-substituted compounds is believed to result primarily from mutations in genes encoding oxygen-insensitive nitroreductases. We have characterized the
nfsA
and
nfsB
genes of a large number of nitrofuran-resistant mutants of
Escherichia coli
and have correlated mutation with cell extract nitroreductase activity. Our studies demonstrate that first-step resistance to furazolidone or nitrofurazone results from an
nfsA
mutation, while the increased resistance associated with second-step mutants is a consequence of an
nfsB
mutation. Inferences made from mutation about the structure-function relationships of NfsA and NfsB are discussed, especially with regard to the identification of flavin mononucleotide binding sites. We show that expression of plasmid-carried
nfsA
and
nfsB
genes in resistant mutants restores sensitivity to nitrofurans. Among the 20 first-step and 53 second-step mutants isolated in this study, 65 and 49%, respectively, contained insertion sequence elements in
nfsA
and
nfsB
. IS
1
integrated in both genes, while IS
30
and IS
186
were found only in
nfsA
and IS
2
and IS
5
were observed only in
nfsB
. Insertion hot spots for IS
30
and IS
186
are indicated in
nfsA
, and a hot spot for IS
5
insertion is evident in
nfsB
. We discuss potential regional and sequence-specific determinants for insertion sequence element integration in
nfsA
and
nfsB
.
Characterization of Circular Plasmid Dimers in
Borrelia burgdorferi
Authors: Tilly Kit , Lubke Lori , Rosa Patricia 
Published: December 2019

ABSTRACT

We have inactivated the
ospC
,
oppAIV
, and
guaB
genes on the 26-kb circular plasmid of
Borrelia burgdorferi
(cp26) by allelic exchange. On several occasions following such transformations, the cp26 of transformants had an aberrant mobility through agarose gels. Characterization of these cp26 molecules showed that the plasmid had dimerized. These dimers were quite stable during either selective or nonselective passage. Subsequent transformations with dimer DNA supported the hypothesis that in
B. burgdorferi
, transforming cp26 DNA most likely does not displace the resident homologous plasmid but rather must recombine in order to donate sequences that it carries. These serendipitous findings provide a mechanism for obtaining heterozygous complemented control strains when mutant phenotypes are characterized.
A Cytochrome
cbb
3
(Cytochrome c) Terminal Oxidase in
Azospirillum brasilense
Sp7 Supports Microaerobic Growth
Authors: Marchal Kathleen , Sun Jun , Keijers Veerle , Haaker Huub , Vanderleyden Jos 
Published: December 2019

ABSTRACT

Spectral analysis indicated the presence of a cytochrome
cbb
3
oxidase under microaerobic conditions in
Azospirillum brasilense
Sp7 cells. The corresponding genes (
cytNOQP
) were isolated by using PCR. These genes are organized in an operon, preceded by a putative anaerobox. The phenotype of an
A. brasilense cytN
mutant was analyzed. Under aerobic conditions, the specific growth rate during exponential phase (μ
e
) of the
A. brasilense cytN
mutant was comparable to the wild-type specific growth rate (μ
e
of approximately 0.2 h
−1
). In microaerobic NH
4
+
-supplemented conditions, the low respiration of the
A. brasilense cytN
mutant affected its specific growth rate (μ
e
of approximately 0.02 h
−1
) compared to the wild-type specific growth rate (μ
e
of approximately 0.2 h
−1
). Under nitrogen-fixing conditions, both the growth rates and respiration of the wild type were significantly diminished in comparison to those under NH
4
+
-supplemented conditions. Differences in growth rates and respiration between the wild type and the
A. brasilense cytN
mutant were less pronounced under these nitrogen-fixing conditions (μ
e
of approximately 0.03 h
−1
for the wild type and 0.02 h
−1
for the
A. brasilense cytN
mutant). The nitrogen-fixing capacity of the
A. brasilense cytN
mutant was still approximately 80% of that determined for the wild-type strain. This leads to the conclusion that the
A. brasilense
cytochrome
cbb
3
oxidase is required under microaerobic conditions, when a high respiration rate is needed, but that under nitrogen-fixing conditions the respiration rate does not seem to be a growth-limiting factor.
α-Galactoside Uptake in
Rhizobium meliloti
: Isolation and Characterization of
agpA
, a Gene Encoding a Periplasmic Binding Protein Required for Melibiose and Raffinose Utilization
Authors: Gage Daniel J. , Long Sharon R. 
Published: December 2019

ABSTRACT

Rhizobium meliloti
can occupy at least two distinct ecological niches; it is found in the soil as a free-living saprophyte, and it also lives as a nitrogen-fixing intracellular symbiont in root nodules of alfalfa and related legumes. One approach to understanding how
R. meliloti
alters its physiology in order to become an integral part of a developing nodule is to identify and characterize genes that are differentially expressed by bacteria living inside nodules. We used a screen to identify genes under the control of the
R. meliloti
regulatory protein NodD3, SyrM, or SyrA. These regulatory proteins are expressed by bacteria growing inside the root nodule. One gene isolated in this screen was mapped to pSymB and displayed complex regulation. The gene was downregulated by the
syrA
gene product and also by glucose and succinate. This gene, referred to as
agpA
, encodes a periplasmic binding protein that is most similar to proteins from the periplasmic oligopeptide binding protein family. It is likely that AgpA binds α-galactosides, because α-galactosides induce the expression of
agpA
, and
agpA
mutants cannot utilize or transport these sugars. Activity of an
agpA
::Tn
phoA
fusion was downregulated by SyrA. Because
syrA
is known to be expressed at high levels in intracellular symbiotic
R. meliloti
and at low levels in the free-living bacteria, we propose that AgpA may belong to the class of gene products whose expression decreases when
R. meliloti
becomes an intracellular symbiont.
Genes Involved in Cell Wall Localization and Side Chain Formation of Rhamnose-Glucose Polysaccharide inStreptococcus mutans
Authors: Yamashita Yoshihisa , Tsukioka Yuichi , Tomihisa Kiyotaka , Nakano Yoshio , Koga Toshihiko 
Published: December 2019

ABSTRACTWe identified inStreptococcus mutanssix new genes (rgpAthroughrgpF), whose disruption results in a loss of serotype-specific antigenicity, specified by the glucose side chains of rhamnose-glucose polysaccharide from the cell wall. Rhamnose and glucose content of the cell wall decreased drastically in all these disruption mutants, except that in thergpEmutant only the glucose content decreased. RgpC and RgpD are homologous to ATP-binding cassette transporter components and may be involved in polysaccharide export, whereas RgpE may be a transferase of side chain glucose.