bluuebunny/crossref_metadata_2025_split huggingface dataset:
Activity of the Molybdopterin-Containing Xanthine Dehydrogenase of
Rhodobacter capsulatus
Can Be Restored by High Molybdenum Concentrations in a
moeA
Mutant Defective in Molybdenum Cofactor Biosynthesis
Authors: Leimkühler Silke , Angermüller Sieglinde , Schwarz Günter , Mendel Ralf R. , Klipp Werner 
Published: December 2019

ABSTRACT

During the screening for
Rhodobacter capsulatus
mutants defective in xanthine degradation, one Tn
5
mutant which was able to grow with xanthine as a sole nitrogen source only in the presence of high molybdate concentrations (1 mM), a phenotype resembling
Escherichia coli mogA
mutants, was identified. Unexpectedly, the corresponding Tn
5
insertion was located within the
moeA
gene. Partial DNA sequence analysis and interposon mutagenesis of regions flanking
R. capsulatus moeA
revealed that no further genes essential for molybdopterin biosynthesis are located in the vicinity of
moeA
and revealed that
moeA
forms a monocistronic transcriptional unit in
R. capsulatus
. Amino acid sequence alignments of
R. capsulatus
MoeA (414 amino acids [aa]) with
E. coli
MogA (195 aa) showed that MoeA contains an internal domain homologous to MogA, suggesting similar functions of these proteins in the biosynthesis of the molybdenum cofactor. Interposon mutants defective in
moeA
did not exhibit dimethyl sulfoxide reductase or nitrate reductase activity, which both require the molybdopterin guanine dinucleotide (MGD) cofactor, even after addition of 1 mM molybdate to the medium. In contrast, the activity of xanthine dehydrogenase, which binds the molybdopterin (MPT) cofactor, was restored to wild-type levels after the addition of 1 mM molybdate to the growth medium. Analysis of fluorescent derivatives of the molybdenum cofactor of purified xanthine dehydrogenase isolated from
moeA
and
modA
mutant strains, respectively, revealed that MPT is inserted into the enzyme only after molybdenum chelation, and both metal chelation and Mo-MPT insertion can occur only under high molybdate concentrations in the absence of MoeA. These data support a model for the biosynthesis of the molybdenum cofactor in which the biosynthesis of MPT and MGD are split at a stage when the molybdenum atom is added to MPT.
The
ripX
Locus of
Bacillus subtilis
Encodes a Site-Specific Recombinase Involved in Proper Chromosome Partitioning
Authors: Sciochetti Stephen A. , Piggot Patrick J. , Sherratt David J. , Blakely Garry 
Published: December 2019

ABSTRACT

The
Bacillus subtilis ripX
gene encodes a protein that has 37 and 44% identity with the XerC and XerD site-specific recombinases of
Escherichia coli
. XerC and XerD are hypothesized to act in concert at the
dif
site to resolve dimeric chromosomes formed by recombination during replication. Cultures of
ripX
mutants contained a subpopulation of unequal-size cells held together in long chains. The chains included anucleate cells and cells with aberrantly dense or diffuse nucleoids, indicating a chromosome partitioning failure. This result is consistent with RipX having a role in the resolution of chromosome dimers in
B. subtilis
. Spores contain a single uninitiated chromosome, and analysis of germinated, outgrowing spores showed that the placement of FtsZ rings and septa is affected in
ripX
strains by the first division after the initiation of germination. The introduction of a
recA
mutation into
ripX
strains resulted in only slight modifications of the
ripX
phenotype, suggesting that chromosome dimers can form in a RecA-independent manner in
B. subtilis
. In addition to RipX, the CodV protein of
B. subtilis
shows extensive similarity to XerC and XerD. The RipX and CodV proteins were shown to bind in vitro to DNA containing the
E. coli dif
site. Together they functioned efficiently in vitro to catalyze site-specific cleavage of an artificial Holliday junction containing a
dif
site. Inactivation of
codV
alone did not cause a discernible change in phenotype, and it is speculated that RipX can substitute for CodV in vivo.
Characterization of a Group of Anaerobically Induced,
fnr
-Dependent Genes of
Salmonella typhimurium
Authors: Wei Yan , Miller Charles G. 
Published: December 2019

ABSTRACT

We have previously reported the isolation of a group of anaerobically regulated,
fnr
-dependent
lac
fusions in
Salmonella typhimurium
and have grouped these
oxd
genes into classes based on map position. In order to identify these genes, we have replaced the original Mud-
lac
fusion in a member of each
oxd
class with the much smaller Mud-
cam
element, cloned the fusion, and determined DNA sequence sufficient to define the
oxd
gene. Several of the fusions correspond to previously known genes from
S. typhimurium
or
Escherichia coli
:
oxd-4 = cbiA
and
oxd-11 = cbiK
,
oxd-5 = hybB
,
oxd-7 = dcuB
,
oxd-8 = moaB
,
oxd-12 = dmsA
, and
oxd-14 = napB
(
aeg-46.5
). Two other fusions correspond to previously unknown loci:
oxd-2
encodes an acetate/propionate kinase, and
oxd-6
encodes a putative ABC transporter present in
S. typhimurium
but not in
E. coli
.
AStreptomyces coelicolorAntibiotic Regulatory Gene,absB, Encodes an RNase III Homolog
Authors: Price Brenda , Adamidis Trifon , Kong Renqui , Champness Wendy 
Published: December 2019

ABSTRACTStreptomyces coelicolorproduces four genetically and structurally distinct antibiotics in a growth-phase-dependent manner.S. coelicolormutants globally deficient in antibiotic production (Abs−phenotype) have previously been isolated, and some of these were found to define theabsBlocus. In this study, we isolatedabsB-complementing DNA and show that it encodes theS. coelicolorhomolog of RNase III (rnc). Several lines of evidence indicate that theabsBmutant global defect in antibiotic synthesis is due to a deficiency in RNase III. In marker exchange experiments, theS. coelicolor rncgene rescuedabsBmutants, restoring antibiotic production. Sequencing the DNA ofabsBmutants confirmed that theabsBmutations lay in therncopen reading frame. Constructed disruptions ofrncin bothS. coelicolor1501 andStreptomyces lividans1326 caused an Abs−phenotype. AnabsBmutation caused accumulation of 30S rRNA precursors, as had previously been reported forE. coli rncmutants. TheabsBgene is widely conserved in streptomycetes. We speculate on why an RNase III deficiency could globally affect the synthesis of antibiotics.
Interstrain Variation of the Polysaccharide B Biosynthesis Locus of
Bacteroides fragilis
: Characterization of the Region from Strain 638R
Authors: Comstock Laurie E. , Coyne Michael J. , Tzianabos Arthur O. , Kasper Dennis L. 
Published: December 2019

ABSTRACT

The sequence and analysis of the capsular polysaccharide biosynthesis locus, PS B2, of
Bacteroides fragilis
638R are described, and the sequence is compared with that of the PS B1 biosynthesis locus of
B. fragilis
NCTC 9343. Two genes of the region,
wcgD
and
wcgC
, are shown by complementation to encode a UDP-
N
-acetylglucosamine 2-epimerase and a UDP-
N
-acetylmannosamine dehydrogenase, respectively.
Characterization of the Phthalate Permease OphD from
Burkholderia cepacia
ATCC 17616
Authors: Chang Hung-Kuang , Zylstra Gerben J. 
Published: December 2019

ABSTRACT

The
ophD
gene, encoding a permease for phthalate transport, was cloned from
Burkholderia cepacia
ATCC 17616. Expression of the gene in
Escherichia coli
results in the ability to transport phthalate rapidly into the cell. Uptake inhibition experiments show that 4-hydroxyphthalate, 4-chlorophthalate, 4-methylphthalate, and cinchomeronate compete for the phthalate permease. An
ophD
knockout mutant of 17616 grows slightly more slowly on phthalate but is still able to take up phthalate at rates equivalent to that of the wild-type strain. This means that 17616 must have a second phthalate-inducible phthalate uptake system.
Rhizobium
(
Sinorhizobium
)
meliloti phn
Genes: Characterization and Identification of Their Protein Products
Authors: Parker George F. , Higgins Timothy P. , Hawkes Timothy , Robson Robert L. 
Published: December 2019

ABSTRACT

In
Escherichia coli
, the
phn
operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA from
Rhizobium
(
Sinorhizobium
)
meliloti
that contains homologues of the
E. coli phnG
, -
H
, -
I
, -
J
, and -
K
genes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the
R. meliloti
C-P lyase. Furthermore, the phenotype of this
phn
mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the
R. meliloti
and
E. coli phn
genes and their predicted products gave new information about C-P lyase. The putative
R. meliloti
PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by
R. meliloti
grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.
Escherichia coli
RcsA, a Positive Activator of Colanic Acid Capsular Polysaccharide Synthesis, Functions To Activate Its Own Expression
Authors: Ebel Wolfgang , Trempy Janine E. 
Published: December 2019

ABSTRACT

Capsule (
cps
) gene expression in
Escherichia coli
is controlled by a complex network of regulators. Transcription of the
cps
operon is controlled by at least two positive regulators, RcsA and RcsB. We show here that RcsA functions to activate its own expression, as seen by the 100-fold-increased expression of a
rcsA
::
lacZ
transcriptional fusion in strains with high levels of RcsA protein, either due to a mutation in
lon
or due to overexpression of RcsA from a multicopy plasmid. Expression of the
rcsA
::
lacZ
fusion is increased by but not dependent on the presence of RcsB. In addition, the effects of H-NS and RcsB on the expression of
rcsA
are independent of each other. A sequence motif, conserved between the
E. coli cps
promoter and the
Erwinia amylovora ams
promoter and previously shown to be the RcsA-RcsB binding site, was identified in the
rcsA
promoter region and shown to be required for high-level expression of
rcsA
.
Isolation and Characterization of the
nikR
Gene Encoding a Nickel-Responsive Regulator in
Escherichia coli
Authors: De Pina Karinne , Desjardin Valerie , Mandrand-Berthelot Marie-Andree , Giordano Gerard , Wu Long-Fei 
Published: December 2019

ABSTRACT

Expression of the nickel-specific transport system encoded by the
Escherichia coli nikABCDE
operon is repressed by a high concentration of nickel. By using random transposon Tn
10
insertion, we isolated mutants in which expression of the
nik
operon became constitutive with respect to nickel. We have identified the corresponding
nikR
gene which encodes a nickel-responsive regulator. Expression of
nikR
was partially controlled by Fnr through transcription from the
nikA
promoter region. In addition, a specific transcription start site for the constitutive expression of
nikR
was found 51 bp upstream of the
nikR
gene.
Characterization of a New Sigma-K-Dependent Peptidoglycan Hydrolase Gene That Plays a Role in
Bacillus subtilis
Mother Cell Lysis
Authors: Nugroho Fajar Aji , Yamamoto Hiroki , Kobayashi Yasuo , Sekiguchi Junichi 
Published: December 2019

ABSTRACT

Bacillus subtilis
produces a 30-kDa peptidoglycan hydrolase, CwlH, during the late sporulation phase. Disruption of
yqeE
led to a complete loss of CwlH formation, indicating the identity of
yqeE
with
cwlH
. Northern blot analysis of
cwlH
revealed a 0.8-kb transcript after 6 to 7.5 h for the wild-type strain but not for the ς
F
, ς
E
, ς
G
, and ς
K
mutants. Expression of the ς
K
-dependent
cwlH
gene depended on
gerE
. Primer extension analysis also suggested that
cwlH
is transcribed by Eς
K
RNA polymerase. CwlH produced in
Escherichia coli
harboring a
cwlH
plasmid is an
N
-acetylmuramoyl-
l
-alanine amidase (EC
3.5.1.28
) and exhibited an optimum pH of 7.0 and high-level binding to the
B. subtilis
cell wall. A
cwlC cwlH
double mutation led to a lack of mother cell lysis even after 7 days of incubation in DSM medium, but the single mutations led to mother cell lysis after 24 h.
Multiple Roles for TnpI Recombinase in Regulation of Tn
5401
Transposition in
Bacillus thuringiensis
Authors: Baum James A. , Gilmer Amy Jelen , Light Mettus Anne-Marie 
Published: December 2019

ABSTRACT

Tn
5401
is a class II transposable element derived from the gram-positive bacterium
Bacillus thuringiensis
. The 4,837-bp transposon encodes a Tn
3
-like transposase (TnpA) and an integrase-like recombinase (TnpI) and is notable for its unusually long 53-bp terminal inverted repeats (TIRs). The
tnpA
and
tnpI
genes are transcribed from a common promoter, designated P
R
, that is subject to negative regulation by TnpI. The TIRs of Tn
5401
each contain a 38-bp sequence that can be aligned with the 38- to 40-bp TIR sequences of Tn
3
-like transposons and an adjacent 12-bp sequence that binds TnpI. This unique juxtaposition of TnpA and TnpI binding sites suggests that TnpI may regulate the binding or catalytic activity of TnpA. The results of the present study indicate that TnpI, in addition to functioning as a site-specific recombinase and as a transcriptional repressor, is required for TnpA binding to the TIRs of Tn
5401
.
Mutation Analysis of the 5′ Untranslated Region of the Cold Shock
cspA
mRNA of
Escherichia coli
Authors: Yamanaka Kunitoshi , Mitta Masanori , Inouye Masayori 
Published: December 2019

ABSTRACT

The mRNA for CspA, a major cold shock protein in
Escherichia coli
, contains an unusually long (159 bases) 5′ untranslated region (5′-UTR), and its stability has been shown to play a major role in cold shock induction of CspA. The 5′-UTR of the
cspA
mRNA has a negative effect on its expression at 37°C but has a positive effect upon cold shock. In this report, a series of
cspA-lacZ
fusions having a 26- to 32-base deletion in the 5′-UTR were constructed to examine the roles of specific regions within the 5′-UTR in
cspA
expression. It was found that none of the deletion mutations had significant effects on the stability of mRNA at both 37 and 15°C. However, two mutations (Δ56-86 and Δ86-117) caused a substantial increase of β-galactosidase activity at 37°C, indicating that the deleted regions contain a negative
cis
element(s) for translation. A mutation (Δ2-27) deleting the highly conserved cold box sequence had little effect on cold shock induction of β-galactosidase. Interestingly, three mutations (Δ28-55, Δ86-117, and Δ118-143) caused poor cold shock induction of β-galactosidase. In particular, the Δ118-143 mutation reduced the translation efficiency of the
cspA
mRNA to less than 10% of that of the wild-type construct. The deleted region contains a 13-base sequence named upstream box (bases 123 to 135), which is highly conserved in
cspA
,
cspB
,
cspG
, and
cspI
, and is located 11 bases upstream of the Shine-Dalgarno (SD) sequence. The upstream box might be another
cis
element involved in translation efficiency of the
cspA
mRNA in addition to the SD sequence and the downstream box sequence. The relationship between the mRNA secondary structure and translation efficiency is discussed.
Ras Signaling Is Required for Serum-Induced Hyphal Differentiation in
Candida albicans
Authors: Feng Qinghua , Summers Eric , Guo Bing , Fink Gerald 
Published: December 2019

ABSTRACT

Serum induces
Candida albicans
to make a rapid morphological change from the yeast cell form to hyphae. Contrary to the previous reports, we found that serum albumin does not play a critical role in this morphological change. Instead, a filtrate (molecular mass, <1 kDa) devoid of serum albumin induces hyphae. To study genes controlling this response, we have isolated the
RAS1
gene from
C. albicans
by complementation. The
Candida
Ras1 protein, like Ras1 and Ras2 of
Saccharomyces cerevisiae
, has a long C-terminal extension. Although
RAS1
appears to be the only
RAS
gene present in the
C. albicans
genome, strains homozygous for a deletion of
RAS1
(
ras1-2/ras1-3
) are viable. The Candida
ras1-2/ras1-3
mutant fails to form germ tubes and hyphae in response to serum or to a serum filtrate but does form pseudohyphae. Moreover, strains expressing the dominant active

RAS1
V13

allele manifest enhanced hyphal growth, whereas those expressing a dominant negative

RAS1
A16

allele show reduced hyphal growth. These data show that low-molecular-weight molecules in serum induce hyphal differentiation in
C. albicans
through a Ras-mediated signal transduction pathway.
Growth Phase-Dependent Variation in Protein Composition of the
Escherichia coli
Nucleoid
Authors: Ali Azam Talukder , Iwata Akira , Nishimura Akiko , Ueda Susumu , Ishihama Akira 
Published: December 2019

ABSTRACT

The genome DNA of
Escherichia coli
is associated with about 10 DNA-binding structural proteins, altogether forming the nucleoid. The nucleoid proteins play some functional roles, besides their structural roles, in the global regulation of such essential DNA functions as replication, recombination, and transcription. Using a quantitative Western blot method, we have performed for the first time a systematic determination of the intracellular concentrations of 12 species of the nucleoid protein in
E. coli
W3110, including CbpA (curved DNA-binding protein A), CbpB (curved DNA-binding protein B, also known as Rob [right origin binding protein]), DnaA (DNA-binding protein A), Dps (DNA-binding protein from starved cells), Fis (factor for inversion stimulation), Hfq (host factor for phage Q
β
), H-NS (histone-like nucleoid structuring protein), HU (heat-unstable nucleoid protein), IciA (inhibitor of chromosome initiation A), IHF (integration host factor), Lrp (leucine-responsive regulatory protein), and StpA (suppressor of
td
mutant phenotype A). Intracellular protein levels reach a maximum at the growing phase for nine proteins, CbpB (Rob), DnaA, Fis, Hfq, H-NS, HU, IciA, Lrp, and StpA, which may play regulatory roles in DNA replication and/or transcription of the growth-related genes. In descending order, the level of accumulation, calculated in monomers, in growing
E. coli
cells is Fis, Hfq, HU, StpA, H-NS, IHF*, CbpB (Rob), Dps*, Lrp, DnaA, IciA, and CbpA* (stars represent the stationary-phase proteins). The order of abundance, in descending order, in the early stationary phase is Dps*, IHF*, HU, Hfq, H-NS, StpA, CbpB (Rob), DnaA, Lrp, IciA, CbpA, and Fis, while that in the late stationary phase is Dps*, IHF*, Hfq, HU, CbpA*, StpA, H-NS, CbpB (Rob), DnaA, Lrp, IciA, and Fis. Thus, the major protein components of the nucleoid change from Fis and HU in the growing phase to Dps in the stationary phase. The curved DNA-binding protein, CbpA, appears only in the late stationary phase. These changes in the composition of nucleoid-associated proteins in the stationary phase are accompanied by compaction of the genome DNA and silencing of the genome functions.
Biochemical and Genetic Analyses of the Role of Yeast Casein Kinase 2 in Salt Tolerance
Authors: de Nadal Eulàlia , Calero Fernando , Ramos José , Ariño Joaquín 
Published: December 2019

ABSTRACT

Saccharomyces cerevisiae
cells lacking the regulatory subunit of casein kinase 2 (CK-2), encoded by the gene
CKB1
, display a phenotype of hypersensitivity to Na
+
and Li
+
cations. The sensitivity of a strain lacking
ckb1
is higher than that of a calcineurin mutant and similar to that of a strain lacking
HAL3
, the regulatory subunit of the Ppz1 protein phosphatase. Genetic analysis indicated that Ckb1 participates in regulatory pathways different from that of Ppz1 or calcineurin. Deletion of
CKB1
increased the salt sensitivity of a strain lacking Ena1 ATPase, the major determinant for sodium efflux, suggesting that the function of the kinase is not mediated by Ena1. Consistently,
ckb1
mutants did not show an altered cation efflux. The function of Ckb1 was independent of the
TRK
system, which is responsible for discrimination of potassium and sodium entry, and in the absence of the kinase regulatory subunit, the influx of sodium was essentially normal. Therefore, the salt sensitivity of a
ckb1
mutant cannot be attributed to defects in the fluxes of sodium. In fact, in these cells, both the intracellular content and the cytoplasm/vacuole ratio for sodium were similar to those features of wild-type cells. The possible causes for the salt sensitivity phenotype of casein kinase mutants are discussed in the light of these findings.
Purification of P
II
and P
II
-UMP and In Vitro Studies of Regulation of Glutamine Synthetase in
Rhodospirillum rubrum
Authors: Johansson Magnus , Nordlund Stefan 
Published: December 2019

ABSTRACT

The P
II
protein from
Rhodospirillum rubrum
was fused with a histidine tag, overexpressed in
Escherichia coli
, and purified by Ni
2+
-chelating chromatography. The uridylylated form of the P
II
protein could be generated in
E. coli
. The effects on the regulation of glutamine synthetase by P
II
, P
II
-UMP, glutamine, and α-ketoglutarate were studied in extracts from
R. rubrum
grown under different conditions. P
II
and glutamine were shown to stimulate the ATP-dependent inactivation (adenylylation) of glutamine synthetase, which could be totally inhibited by α-ketoglutarate. Deadenylylation (activation) of glutamine synthetase required phosphate, but none of the effectors studied had any major effect, which is different from their role in the
E. coli
system. In addition, deadenylylation was found to be much slower than adenylylation under the conditions investigated.
A Novel Gene Required for Rhamnose-Glucose Polysaccharide Synthesis inStreptococcus mutans
Authors: Yamashita Yoshihisa , Shibata Yukie , Nakano Yoshio , Tsuda Hiromasa , Kido Nobuo , Ohta Michio , Koga Toshihiko 
Published: December 2019

ABSTRACTGenergpGis required for biosynthesis of rhamnose-glucose polysaccharide (RGP) inStreptococcus mutans. Its deduced amino acid sequence had similarity to WecA, which initiates syntheses of enterobacterial common antigen and some O antigens inEscherichia coli. GenergpGcomplemented awecAmutation ofE. coli, suggesting thatrgpGmay function similarly in RGP synthesis.
Purification and Characterization of (Per)Chlorate Reductase from the Chlorate-Respiring Strain GR-1
Authors: Kengen Servé W. M. , Rikken Geoffrey B. , Hagen Wilfred R. , van Ginkel Cees G. , Stams Alfons J. M. 
Published: December 2019

ABSTRACT

Strain GR-1 is one of several recently isolated bacterial species that are able to respire by using chlorate or perchlorate as the terminal electron acceptor. The organism performs a complete reduction of chlorate or perchlorate to chloride and oxygen, with the intermediate formation of chlorite. This study describes the purification and characterization of the key enzyme of the reductive pathway, the chlorate and perchlorate reductase. A single enzyme was found to catalyze both the chlorate- and perchlorate-reducing activity. The oxygen-sensitive enzyme was located in the periplasm and had an apparent molecular mass of 420 kDa, with subunits of 95 and 40 kDa in an α
3
β
3
composition. Metal analysis showed the presence of 11 mol of iron, 1 mol of molybdenum, and 1 mol of selenium per mol of heterodimer. In accordance, quantitative electron paramagnetic resonance spectroscopy showed the presence of one [3Fe-4S] cluster and two [4Fe-4S] clusters. Furthermore, two different signals were ascribed to Mo(V). The

K
m

values for perchlorate and chlorate were 27 and <5 μM, respectively. Besides perchlorate and chlorate, nitrate, iodate, and bromate were also reduced at considerable rates. The resemblance of the enzyme to nitrate reductases, formate dehydrogenases, and selenate reductase is discussed.