bluuebunny/crossref_metadata_2025_split huggingface dataset:
Molecular Characterization of Type-Specific Capsular Polysaccharide Biosynthesis Genes of
Streptococcus agalactiae
Type Ia
Authors: Yamamoto Shin , Miyake Katsuhide , Koike Yoichi , Watanabe Masaki , Machida Yuichi , Ohta Michio , Iijima Shinji 
Published: December 2019

ABSTRACT

The type-specific capsular polysaccharide (CP) of a group B streptococcus,
Streptococcus agalactiae
type Ia, is a high-molecular-weight polymer consisting of the pentasaccharide repeating unit 4)-[α-
d
-Neu
p
NAc-(2→3)-β-
d
-Gal
p
-(1→4)-β-
d
-Glc
p
NAc-(1→3)]-β-
d
-Gal
p
-(1→4)-β-
d
-Glc
p
-(1. Here, cloning, sequencing, and transcription of the type Ia-specific capsular polysaccharide synthesis (
cps
) genes and functional analysis of these gene products are described. A 26-kb DNA fragment containing 18 complete open reading frames (ORFs) was cloned. These ORFs were designated
cpsIaA
to
cpsIaL
,
neu
(neuraminic acid synthesis gene)
A
to
D
,
orf1
and
ung
(uracil DNA glycosylase). The
cps
gene products of
S. agalactiae
type Ia were homologous to proteins involved in CP synthesis of
S. agalactiae
type III and
S. pneumoniae
serotype 14. Unlike the
cps
gene cluster of
S. pneumoniae
serotype 14, transcription of this operon may start from
cpsIaA
,
cpsIaE
, and
orf1
because putative promoter sequences were found in front of these genes. Northern hybridization, reverse transcription-PCR, and primer extension analyses supported this hypothesis. DNA sequence analysis showed that there were two transcriptional terminators in the 3′ end of this operon (downstream of
orf1
and
ung
). The functions of CpsIaE, CpsIaG, CpsIaI, and CpsIaJ were examined by glycosyltransferase assay by using the gene products expressed in
Escherichia coli
JM109 harboring plasmids containing various
S. agalactiae
type Ia
cps
gene fragments. Enzyme assays suggested that the gene products of
cpsIaE
,
cpsIaG
,
cpsIaI
, and
cpsIaJ
are putative glucosyltransferase, β-1,4-galactosyltransferase, β-1,3-
N
-acetylglucosaminyltransferase, and β-1,4-galactosyltransferase, respectively.
Distinct Types of rRNA Operons Exist in the Genome of the Actinomycete
Thermomonospora chromogena
and Evidence for Horizontal Transfer of an Entire rRNA Operon
Authors: Yap Wai Ho , Zhang Zhenshui , Wang Yue 
Published: December 2019

ABSTRACT

We describe here the presence of two distinct types of rRNA operons in the genome of a thermophilic actinomycete
Thermomonospora chromogena
. The genome of
T. chromogena
contains six rRNA operons (
rrn
), of which four complete and two incomplete ones were cloned and sequenced. Comparative analysis revealed that the operon
rrnB
exhibits high levels of sequence variations to the other five nearly identical ones throughout the entire length of the operon. The coding sequences for the 16S and 23S rRNA genes differ by approximately 6 and 10%, respectively, between the two types of operons. Normal functionality of
rrnB
is concluded on the basis of the nonrandom distribution of nucleotide substitutions, the presence of compensating nucleotide covariations, the preservation of secondary and tertiary rRNA structures, and the detection of correctly processed rRNAs in the cell. Comparative sequence analysis also revealed a close evolutionary relationship between
rrnB
operon of
T. chromogena
and
rrnA
operon of another thermophilic actinomycete
Thermobispora bispora
. We propose that
T. chromogena
acquired
rrnB
operon from
T. bispora
or a related organism via horizontal gene transfer.
The Cpx Envelope Stress Response Is Controlled by Amplification and Feedback Inhibition
Authors: Raivio Tracy L. , Popkin Daniel L. , Silhavy Thomas J. 
Published: December 2019

ABSTRACT

In
Escherichia coli
, the Cpx two-component regulatory system activates expression of protein folding and degrading factors in response to misfolded proteins in the bacterial envelope (inner membrane, periplasm, and outer membrane). It is comprised of the histidine kinase CpxA and the response regulator CpxR. This response plays a role in protection from stresses, such as elevated pH, as well as in the biogenesis of virulence factors. Here, we show that the Cpx periplasmic stress response is subject to amplification and repression through positive and negative autofeedback mechanisms. Western blot and operon fusion analyses demonstrated that the
cpxRA
operon is autoactivated. Conditions that lead to elevated levels of phosphorylated CpxR cause a concomitant increase in transcription of
cpxRA
. Conversely, overproduction of CpxP, a small, Cpx-regulated protein of previously unknown function, represses the regulon and can block activation of the pathway. This repression is dependent on an intact CpxA sensing domain. The ability to autoactivate and then subsequently repress allows for a temporary amplification of the Cpx response that may be important in rescuing cells from transitory stresses and cueing the appropriately timed elaboration of virulence factors.
Cloning and Sequence Analysis of a New Cellulase Gene Encoding CelK, a Major Cellulosome Component of
Clostridium thermocellum
: Evidence for Gene Duplication and Recombination
Authors: Kataeva Irina , Li Xin-Liang , Chen Huizhong , Choi Sang-Ki , Ljungdahl Lars G. 
Published: December 2019

ABSTRACT

The cellulolytic and hemicellulolytic complex of
Clostridium thermocellum
, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced. They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide. We report here a new cellulase gene,
celK
, coding for CelK, a 98-kDa major component of the cellulosome. The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da. A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da. The nucleotide sequence is highly similar to that of
cbhA
(V. V. Zverlov et al., J. Bacteriol. 180:3091–3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium. Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively. Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain. A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD. This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA. Upstream and downstream flanking sequences of the two genes show no homology. The data indicate that
celK
and
cbhA
in the genome of
C. thermocellum
have evolved through gene duplication and recombination of domain coding sequences.
celK
without a dockerin domain was expressed in
Escherichia coli
and purified. The enzyme had pH and temperature optima at 6.0 and 65°C, respectively. It hydrolyzed
p
-nitrophenyl-β-
d
-cellobioside with a

K
m

and a
V
max
of 1.67 μM and 15.1 U/mg, respectively. Cellobiose was a strong inhibitor of CelK activity, with a

K
i

of 0.29 mM. The enzyme was thermostable, after 200 h of incubation at 60°C, 97% of the original activity remained. Properties of the enzyme indicated that it is a cellobiohydrolase.
Roles of RuvC and RecG in Phage λ Red-Mediated Recombination
Authors: Poteete Anthony R. , Fenton Anita C. , Murphy Kenan C. 
Published: December 2019

ABSTRACT

The recombination properties of
Escherichia coli
strains expressing the
red
genes of bacteriophage λ and lacking
recBCD
function either by mutation or by expression of λ
gam
were examined. The substrates for recombination were nonreplicating λ chromosomes, introduced by infection; Red-mediated recombination was initiated by a double-strand break created by the action of a restriction endonuclease in the infected cell. In one type of experiment, two phages marked with restriction site polymorphisms were crossed. Efficient formation of recombinant DNA molecules was observed in

ruvC
+
recG
+

,
ruvC recG
+
,

ruvC
+
recG

, and
ruvC recG
hosts. In a second type of experiment, a 1-kb nonhomology was inserted between the double-strand break and the donor chromosome’s restriction site marker. In this case, recombinant formation was found to be partially dependent upon
ruvC
function, especially in a
recG
mutant background. In a third type of experiment, the recombining partners were the host cell chromosome and a 4-kb linear DNA fragment containing the
cat
gene, with flanking
lac
sequences, released from the infecting phage chromosome by restriction enzyme cleavage in the cell; the formation of chloramphenicol-resistant bacterial progeny was measured. Dependence on RuvC varied considerably among the three types of cross. However, in all cases, the frequency of Red-mediated recombination was higher in
recG
than in
recG
+
. These observations favor models in which RecG tends to push invading 3′-ended strands back out of recombination intermediates.
New Sporulation Loci in
Streptomyces coelicolor
A3(2)
Authors: Ryding N. Jamie , Bibb Maureen J. , Molle Virginie , Findlay Kim C. , Chater Keith F. , Buttner Mark J. 
Published: December 2019

ABSTRACT

Sporulation mutants of
Streptomyces coelicolor
appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (
whi
) mutants had been used to define eight sporulation loci,
whiA
,
whiB
,
whiD
,
whiE
,
whiG
,
whiH
,
whiI
, and
whiJ
(K. F. Chater, J. Gen. Microbiol. 72:9–28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new
whi
loci, we mutagenized
S. coelicolor
M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115
whi
mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned
whi
genes, self-transmissible SCP2*-derived plasmids carrying
whiA
,
whiB
,
whiG
,
whiH
, or
whiJ
(but not
whiD
,
whiE
, or
whiI
) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31
whi
mutants, an SCP2* library of wild-type
S. coelicolor
chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated
whiK
,
whiL
,
whiM
,
whiN
, and
whiO
. Each of the five loci was located on the ordered cosmid library:
whiL
,
whiM
,
whiN
, and
whiO
occupied positions distinct from previously cloned
whi
genes;
whiK
was located on the same cosmid overlap as
whiD
, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.
Mutations Affecting Motifs of Unknown Function in the Central Domain of Nitrogen Regulatory Protein C
Authors: Li Jieli , Passaglia Luciane , Rombel Irene , Yan Dalai , Kustu Sydney 
Published: December 2019

ABSTRACT

The positive control function of the bacterial enhancer-binding protein NtrC resides in its central domain, which is highly conserved among activators of ς
54
holoenzyme. Previous studies of a small set of mutant forms specifically defective in transcriptional activation, called NtrC repressor [NtrC(Rep)] proteins, had enabled us to locate various functional determinants in the central domain. In this more comprehensive survey, the DNA encoding a major portion of the central domain was randomly mutagenized and mutated
ntrC
genes were introduced into the cell via multicopy expression plasmids. DNA sequencing of 95 isolates identified by a preliminary phenotypic screen revealed that the lesions in them caused 55 distinct single amino acid substitutions at 44 different positions. Assays of
glnA
transcription in vivo and in vitro yielded two conclusions. First, of the 41 mutant proteins that could be purified, 17 (1 known, 16 new) showed no detectable activity in either assay, thus qualifying them as true NtrC(Rep) proteins. These contained residue changes in six of the seven highly conserved regions in the central domain, including two never studied before. Second, some mutant proteins were inactive in vivo but were either marginally or fully active in vitro. Their surprising lack of activity in vivo may be accounted for by high levels of expression, which apparently decreased activation by these mutant proteins but not by wild-type NtrC (NtrC
WT
). Of particular interest were a subset of these proteins that exhibited greater transcriptional activation than NtrC
WT
at low concentrations. Their elevated activation capacities remain to be explained.
First Evidence for the Presence of a Hydrogenase in the Sulfur-Reducing Bacterium
Desulfuromonas acetoxidans
Authors: Brugna Marianne , Nitschke Wolfgang , Toci René , Bruschi Mireille , Giudici-Orticoni Marie-Thérèse 
Published: December 2019

ABSTRACT

Hydrogenases, which are ubiquitous in sulfate-reducing bacteria, were previously thought to be absent from
Desulfuromonas acetoxidans
. For the first time, a hydrogenase from the strict anaerobic sulfur-respiring bacterium
D. acetoxidans
, grown on ethanol-malate, was detected and enriched. To assay the role of the hydrogenase in the energetic metabolism of
D. acetoxidans
, we examined the reactivity of the enzyme with polyheme cytochromes from the same bacterium.
Population Genetic and Evolutionary Approaches to Analysis of
Neisseria meningitidis
Isolates Belonging to the ET-5 Complex
Authors: Bygraves J. A. , Urwin R. , Fox A. J. , Gray S. J. , Russell J. E. , Feavers I. M. , Maiden M. C. J. 
Published: December 2019

ABSTRACT

Periodically, new disease-associated variants of the human pathogen
Neisseria meningitidis
arise. These meningococci diversify during spread, and related isolates recovered from different parts of the world have different genetic and antigenic characteristics. An example is the ET-5 complex, members of which were isolated globally from the mid-1970s onwards. Isolates from a hyperendemic outbreak of meningococcal disease in Worcester, England, during the late 1980s were characterized by multilocus sequence typing and sequence determination of antigen genes. These data established that the Worcester outbreak was caused by ET-5 complex meningococci which were not closely related to the ET-5 complex bacteria responsible for a hyperendemic outbreak in the nearby town of Stroud during the years preceding the Worcester outbreak. A comparison with other ET-5 complex meningococci established that there were at least three distinct globally distributed subpopulations within the ET-5 complex, characterized by particular housekeeping and antigen gene alleles. The Worcester isolates belonged to one of these subpopulations, the Stroud isolates belonged to another, and at least one representative of the third subpopulation identified in this work was isolated elsewhere in the United Kingdom. The sequence data demonstrated that ET-5 variants have arisen by multiple complex pathways involving the recombination of antigen and housekeeping genes and de novo mutation of antigen genes. The data further suggest that either the ET-5 complex has been in existence for many years, evolving and spreading relatively slowly until its disease-causing potential was recognized, or it has evolved and spread rapidly since its first identification in the 1970s, with each of the subpopulations attaining a distribution spanning several continents.
Cloning and Molecular Characterization of the Genes for Carbon Monoxide Dehydrogenase and Localization of Molybdopterin, Flavin Adenine Dinucleotide, and Iron-Sulfur Centers in the Enzyme of
Hydrogenophaga pseudoflava
Authors: Kang Beom S. , Kim Young M. 
Published: December 2019

ABSTRACT

Carbon monoxide dehydrogenases (CO-DH) are the enzymes responsible for the oxidation of CO to carbon dioxide in carboxydobacteria and consist of three nonidentical subunits containing molybdopterin flavin adenine dinucleotide (FAD), and two different iron-sulfur clusters (O. Meyer, K. Frunzke, D. Gadkari, S. Jacobitz, I. Hugendieck, and M. Kraut, FEMS Microbiol. Rev. 87:253–260, 1990). The three structural genes of CO-DH in
Hydrogenophaga pseudoflava
were cloned and characterized. The genes were clustered on the chromosome in the transcriptional order
cutM-cutS-cutL
. The cloned
cutM
,
cutS
, and
cutL
genes had open reading frames of 864, 492, and 2,412 nucleotides, coding for proteins with calculated molecular weights of 30,694, 17,752, and 87,224, respectively. The overall identities in the nucleotide sequence of the genes and the amino acid sequence of the subunits with those of other carboxydobacteria were 64.5 to 74.3% and 62.8 to 72.3%, respectively. Primer extension analysis revealed that the transcriptional start site of the genes was the nucleotide G located 47 bp upstream of the
cutM
start codon. The deduced amino acid sequences of the three subunits of CO-DH implied the presence of molybdenum cofactor, FAD, and iron-sulfur centers in CutL, CutM, and CutS, respectively. Fluorometric analysis coupled with denaturing polyacrylamide gel electrophoresis of fractions from hydroxyapatite column chromatography in the presence of 8 M urea of active CO-DH and from gel filtration of spontaneously inactivated enzyme revealed that the large and medium subunits of CO-DH in
H. pseudoflava
bind molybdopterin and FAD cofactors, respectively. Iron-sulfur centers of the enzyme were identified to be present in the small subunit on the basis of the iron content in each subunit eluted from the denaturing polyacrylamide gels.
Genetic Diversity and Mosaicism at the
por
Locus of
Neisseria gonorrhoeae
Authors: Fudyk Trevor C. , Maclean Ian W. , Simonsen J. Neil , Njagi Ephantus N. , Kimani Joshua , Brunham Robert C. , Plummer Francis A. 
Published: December 2019

ABSTRACT

The
por
genes of the predominant serovars of
Neisseria gonorrhoeae
circulating in a high-frequency transmitter core group located in Nairobi, Kenya, were examined for nucleotide sequence polymorphism. The level of
por
gene diversity did not differ significantly between core group-derived gonococcal strains and gonococcal strains originating elsewhere. However,
por
mosaicism appeared to be more frequent among core group-derived strains, suggesting that recombination of different
por
sequences may be a important strategy by which
N. gonorrhoeae
generates
por
gene diversity within core group populations. Despite extensive sequence variability,
por
expressed by gonococcal isolates of different geographic origin exhibited conserved patterns of nucleotide change, suggesting that diversity among
por
alleles may also be finite.
P1 ParB Domain Structure Includes Two Independent Multimerization Domains
Authors: Surtees Jennifer A. , Funnell Barbara E. 
Published: December 2019

ABSTRACT

ParB is one of two P1-encoded proteins that are required for active partition of the P1 prophage in
Escherichia coli
. To probe the native domain structure of ParB, we performed limited proteolytic digestions of full-length ParB, as well as of several N-terminal and C-terminal deletion fragments of ParB. The C-terminal 140 amino acids of ParB form a very trypsin-resistant domain. In contrast, the N terminus is more susceptible to proteolysis, suggesting that it forms a less stably folded domain or domains. Because native ParB is a dimer in solution, we analyzed the ability of ParB fragments to dimerize, using both the yeast two-hybrid system and in vitro chemical cross-linking of purified proteins. These studies revealed that the C-terminal 59 amino acids of ParB, a region within the protease-resistant domain, are sufficient for dimerization. Cross-linking and yeast two-hybrid experiments also revealed the presence of a second self-association domain within the N-terminal half of ParB. The cross-linking data also suggest that the C terminus is inhibitory to multimerization through the N-terminal domain in vitro. We propose that the two multimerization domains play distinct roles in partition complex formation.
Identification and Characterization of a Determinant (
eep
) on the
Enterococcus faecalis
Chromosome That Is Involved in Production of the Peptide Sex Pheromone cAD1
Authors: An Florence Y. , Sulavik Mark C. , Clewell Don B. 
Published: December 2019

ABSTRACT

Plasmid-free strains of
Enterococcus faecalis
secrete a peptide sex pheromone, cAD1, which specifically induces a mating response by donors carrying the hemolysin plasmid pAD1 or related elements. A determinant on the
E. faecalis
OG1X chromosome has been found to encode a 46.5-kDa protein that plays an important role in the production of the extracellular cAD1. Wild-type
E. faecalis
OG1X cells harboring a plasmid chimera carrying the determinant exhibited an eightfold enhanced production of cAD1, and plasmid-free cells carrying a mutated chromosomal determinant secreted undetectable or very low amounts of the pheromone. The production of other pheromones such as cPD1, cOB1, and cCF10 was also influenced, although there was no effect on the pheromone cAM373. The determinant, designated
eep
(for enhanced expression of pheromone), did not include the sequence of the pheromone. Its deduced product (Eep) contains apparent membrane-spanning sequences; conceivably it is involved in processing a pheromone precursor structure or in some way regulates expression or secretion.
Transcriptional Control of
Bacillus subtilis hemN
and
hemZ
Authors: Homuth Georg , Rompf Alexandra , Schumann Wolfgang , Jahn Dieter 
Published: December 2019

ABSTRACT

Previous characterization of
Bacillus subtilis hemN
, encoding a protein involved in oxygen-independent coproporphyrinogen III decarboxylation, indicated the presence of a second
hemN
-like gene (B. Hippler, G. Homuth, T. Hoffmann, C. Hungerer, W. Schumann, and D. Jahn, J. Bacteriol. 179:7181–7185, 1997). The corresponding
hemZ
gene was found to be split into the two potential open reading frames
yhaV
and
yhaW
by a sequencing error of the genome sequencing project. The
hemZ
gene, encoding a 501-amino-acid protein with a calculated molecular mass of 57,533 Da, complemented a
Salmonella typhimurium hemF hemN
double mutant under aerobic and anaerobic growth conditions. A
B. subtilis hemZ
mutant accumulated coproporphyrinogen III under anaerobic growth conditions. A
hemN hemZ
double mutant exhibited normal aerobic and anaerobic growth, indicating the presence of a third alternative oxygen-independent enzymatic system for coproporphyrinogen III oxidation. The
hemY
gene, encoding oxygen-dependent protoporphyrinogen IX oxidase with coproporphyrinogen III oxidase side activity, did not significantly contribute to this newly identified system. Growth behavior of
hemY
mutants revealed the presence of an oxygen-independent protoporphyrinogen IX oxidase in
B. subtilis
. A monocistronic
hemZ
mRNA, starting 31 bp upstream of the translational start codon, was detected. Reporter gene fusions of
hemZ
and
hemN
demonstrated a fivefold anaerobic induction of both genes under nitrate ammonifying growth conditions. No anaerobic induction was observed for fermentatively growing
B. subtilis
. The
B. subtilis
redox regulatory systems encoded by
resDE
,
fnr
, and
ywiD
were indispensable for the observed transcriptional induction. A redox regulation cascade proceeding from an unknown sensor via
resDE
, through
fnr
and
ywiD
to
hemN/hemZ
, is suggested for the observed coregulation of heme biosynthesis and the anaerobic respiratory energy metabolism. Finally, only
hemZ
was found to be fivefold induced by the presence of H
2
O
2
, indicating further coregulation of heme biosynthesis with the formation of the tetrapyrrole enzyme catalase.