bluuebunny/crossref_metadata_2025 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

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

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

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

Streptomyces 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

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

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

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

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

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

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

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

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

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

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

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

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

GenergpGis 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

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.
Structural Characterization of the Symbiotically Important Low-Molecular-Weight Succinoglycan of
Sinorhizobium meliloti
Authors: Wang Lai-Xi , Wang Ying , Pellock Brett , Walker Graham C.
Published: December 2019

The production of succinoglycan by
Sinorhizobium meliloti
Rm1021 is required for successful nodule invasion by the bacterium of its host plant, alfalfa. Rm1021 produces succinoglycan, an acidic exopolysaccharide composed of an octasaccharide repeating unit modified with acetyl, succinyl, and pyruvyl moieties, in both low- and high-molecular-weight forms. Low-molecular-weight (LMW) succinoglycan, previously thought to consist of monomers, trimers, and tetramers of the repeating unit, has been reported as being capable of promoting the formation of nitrogen-fixing nodules by succinoglycan-deficient derivatives of strain Rm1021. We have determined that the three size classes of LMW succinoglycan species are in fact monomers, dimers, and trimers of the repeating unit and that the trimer is the species active in promoting nodule invasion. A detailed structural analysis of the components of LMW succinoglycan by using various chromatographic techniques, along with nuclear magnetic resonance analyses, has revealed that there is considerable heterogeneity within the LMW succinoglycan oligomers in terms of noncarbohydrate substitutions, and we have determined the structural basis of this heterogeneity.
Cloning and Expression of
Mycobacterium tuberculosis
and
Mycobacterium leprae
Dihydropteroate Synthase in
Escherichia coli
Authors: Nopponpunth Vanida , Sirawaraporn Worachart , Greene Patricia J. , Santi Daniel V.
Published: December 2019

The genes for dihydropteroate synthase of
Mycobacterium tuberculosis
and
Mycobacterium leprae
were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for
M. tuberculosis
dihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for
M. leprae
dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain of
Escherichia coli
. Using three chromatography steps, we purified both
M. tuberculosis
and
M. leprae
dihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for
M. tuberculosis
dihydropteroate synthase and 30 kDa for
M. leprae
dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from both
M. tuberculosis
and
M. leprae
were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent
p
-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.
CzcD Is a Heavy Metal Ion Transporter Involved in Regulation of Heavy Metal Resistance in
Ralstonia
sp. Strain CH34
Authors: Anton Andreas , Große Cornelia , Reißmann Jana , Pribyl Thomas , Nies Dietrich H.
Published: December 2019

The Czc system of
Ralstonia
sp. strain CH34 mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB
2
A cation-proton antiporter. The CzcD protein is involved in the regulation of the Czc system. It is a membrane-bound protein with at least four transmembrane α-helices and is a member of a subfamily of the cation diffusion facilitator (CDF) protein family, which occurs in all three domains of life. The deletion of
czcD
in a
Ralstonia
sp. led to partially constitutive expression of the Czc system due to an increased transcription of the structural
czcCBA
genes, both in the absence and presence of inducers. The
czcD
deletion could be fully complemented in
trans
by CzcD and two other CDF proteins from
Saccharomyces cerevisiae
, ZRC1p and COT1p. All three proteins mediated a small but significant resistance to cobalt, zinc, and cadmium in
Ralstonia
, and this resistance was based on a reduced accumulation of the cations. Thus, CzcD appeared to repress the Czc system by an export of the inducing cations.
Immunochemical Characterization and Taxonomic Evaluation of the O Polysaccharides of the Lipopolysaccharides ofPseudomonas syringaeSerogroup O1 Strains
Authors: Ovod Vladimir V. , Knirel Yuriy A. , Samson Regine , Krohn Kai J.
Published: December 2019

The O polysaccharide (OPS) of the lipopolysaccharide (LPS) ofPseudomonas syringaepv. atrofaciens IMV 7836 and some other strains that are classified in serogroup O1 was shown to be a novel linear α-d-rhamnan with the tetrasaccharide O repeat →3)-α-d-Rhap-(1→3)-α-d-Rhap-(1→2)-α-d-Rhap-(1→2)-α-d-Rhap-(1→ (chemotype 1A). The same α-d-rhamnan serves as the backbone in branched OPSs with lateral (α1→3)-linkedd-Rhap, (β1→4)-linkedd-GlcpNAc, and (α1→4)-linkedd-Fucfresidues (chemotypes 1B, 1C, and 1D, respectively). Strains of chemotype 1C demonstrated variations resulting in a decrease of the degree of substitution of the backbone 1A with the laterald-GlcNAc residue (chemotype 1C-1A), which may be described as branched regular ↔ branched irregular → linear OPS structure alterations (1C↔1C-1A → 1A). Based on serological data, chemotype 1D was suggested to undergo a 1D ↔ 1D-1A alteration, whereas chemotype 1B showed no alteration. A number of OPS backbone-specific monoclonal antibodies (MAbs), Ps(1-2)a, Ps(1-2)a1, Ps1a, Ps1a1, and Ps1a2, as well as MAbs Ps1b, Ps1c, Ps1c1, Ps1d, Ps(1-2)d, and Ps(1-2)d1specific to epitopes related to the lateral sugar substituents of the OPSs, were produced againstP. syringaeserogroup O1 strains. By using MAbs, some specific epitopes were inferred, serogroup O1 strains were serotyped in more detail, and thus, the serological classification scheme ofP. syringaewas improved. Screening with MAbs of about 800 strains representing all 56 knownP. syringaepathovars showed that the strains classified in serogroup O1 were found among 15 pathovars and the strains with the linear OPSs of chemotype 1A were found among 9 of the 15 pathovars. A possible role for the LPS ofP. syringaeand related pseudomonads as a phylogenetic marker is discussed.
A Novel Lipolytic Enzyme Located in the Outer Membrane of
Pseudomonas aeruginosa
Authors: Wilhelm Susanne , Tommassen Jan , Jaeger Karl-Erich
Published: December 2019

A lipase-negative deletion mutant of
Pseudomonas aeruginosa
PAO1 still showed extracellular lipolytic activity toward short-chain
p
-nitrophenylesters. By screening a genomic DNA library of
P. aeruginosa
PAO1, an esterase gene,
estA
, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product of
estA
is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for
35
S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The
estA
gene showed high similarity to an open reading frame of unknown function located in the
trpE-trpG
region of
P. putida
and to a gene encoding an outer membrane esterase of
Salmonella typhimurium
. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal β-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of
estA
in
P. aeruginosa
and
Escherichia coli
and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in
P. aeruginosa
(i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.
Role of CcpA in Regulation of the Central Pathways of Carbon Catabolism in
Bacillus subtilis
Authors: Tobisch Steffen , Zühlke Daniela , Bernhardt Jörg , Stülke Jörg , Hecker Michael
Published: December 2019

The
Bacillus subtilis
two-dimensional (2D) protein index contains almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes, among them the most abundant housekeeping proteins of growing cells. Therefore, a comprehensive study on the regulation of glycolysis and the TCA cycle was initiated. Whereas expression of genes encoding the upper and lower parts of glycolysis (
pgi
,
pfk
,
fbaA
, and
pykA
) is not affected by the glucose supply, there is an activation of the glycolytic
gap
gene and the
pgk
operon by glucose. This activation seems to be dependent on the global regulator CcpA, as shown by 2D polyacrylamide gel electrophoresis analysis as well as by transcriptional analysis. Furthermore, a high glucose concentration stimulates production and excretion of organic acids (overflow metabolism) in the wild type but not in the
ccpA
mutant. Finally, CcpA is involved in strong glucose repression of almost all TCA cycle genes. In addition to TCA cycle and glycolytic enzymes, the levels of many other proteins are affected by the
ccpA
mutation. Our data suggest (i) that
ccpA
mutants are unable to activate glycolysis or carbon overflow metabolism and (ii) that CcpA might be a key regulator molecule, controlling a superregulon of glucose catabolism.
Characterization of the Essential Transport Function of the AIDA-I Autotransporter and Evidence Supporting Structural Predictions
Authors: Maurer Jochen , Jose Joachim , Meyer Thomas F.
Published: December 2019

The current model for autodisplay suggests a mechanism that allows a passenger protein to be translocated across the outer membrane by coordinate action of a C-terminal β-barrel and its preceding linking region. The passenger protein, linker, and β-barrel are together termed the autotransporter, while the linker and β-barrel are here referred to as the translocation unit (TU). We characterized the minimal TU necessary for autodisplay with the adhesin-involved-in-diffuse-adherence (AIDA-I) autotransporter. The assumed β-barrel structure at the C terminus of the AIDA-I autotransporter was studied by constructing a set of seven AIDA-I–cholera toxin B subunit fusion proteins containing various portions of AIDA-I. Surface exposure of the cholera toxin B moiety was assessed by dot blot experiments and trypsin accessibility of the chimeric proteins expressed in
Escherichia coli
JK321 or UT5600. Export of cholera toxin B strictly depended on a complete predicted β-barrel region. The absolute necessity for export of a linking region and its influence on expression as an integral part of the TU was also demonstrated. The different electrophoretic mobilities of native and denatured chimeras indicated that the proposed β-barrel resides within the C-terminal 312 amino acids of AIDA-I. Together these data provide evidence for the predicted β-barrel structure and support our formerly proposed model of membrane topology of the AIDA-I autotransporter.
Transcriptional Control of the Low-Temperature-Inducible
des
Gene, Encoding the Δ5 Desaturase of
Bacillus subtilis
Authors: Aguilar Pablo S. , Lopez Paloma , de Mendoza Diego
Published: December 2019

The
Bacillus subtilis des
gene encodes the cold-inducible Δ5 lipid desaturase involved in the formation of unsaturated fatty acids from saturated phospholipid precursors. Here, we describe the expression pattern of the
des
gene in response to a temperature downshift from 37 to 20°C. We found that the synthesis of
des
mRNA is undetectable at 37°C but dramatically induced upon the temperature downshift. Decay characteristics of the
des
transcript as well as the in vivo decay of
B. subtilis
bulk mRNA were investigated. The results showed that the stability of the
des
transcript as well as of bulk mRNA lasted substantially longer at 20°C than at 37°C. Functional expression of
des
at 37°C was achieved by exchanging its promoter with the non-cold shock
spac
promoter. These data provide the first direct evidence that temperature-mediated control of transcription is the major mechanism regulating the mRNA levels of the
B. subtilis
desaturase. The present results also demonstrate that the only component of the desaturation system regulated by temperature is the desaturase enzyme.
Synergistic Operation of theCAR2(Ornithine Transaminase) Promoter Elements inSaccharomyces cerevisiae
Authors: Park Heui-Dong , Scott Stephanie , Rai Rajendra , Dorrington Rosemary , Cooper Terrance G.
Published: December 2019

Dal82p binds to theUISALLsites of allophanate-induced genes of the allantoin-degradative pathway and functions synergistically with the GATA family Gln3p and Gat1p transcriptional activators that are responsible for nitrogen catabolite repression-sensitive gene expression.CAR2, which encodes the arginine-degradative enzyme ornithine transaminase, is not nitrogen catabolite repression sensitive, but its expression can be modestly induced by the allantoin pathway inducer. The dominant activators ofCAR2transcription have been thought to be the ArgR and Mcm1 factors, which mediate arginine-dependent induction. These observations prompted us to investigate the structure of theCAR2promoter with the objectives of determining whether other transcription factors were required forCAR2expression and, if so, of ascertaining their relative contributions toCAR2’s expression and control. We show that Rap1p binds upstream ofCAR2and plays a central role in its induced expression irrespective of whether the inducer is arginine or the allantoin pathway inducer analogue oxalurate (OXLU). Our data also explain the early report that ornithine transaminase production is induced when cells are grown with urea. OXLU induction derives from the Dal82p binding site, which is immediately downstream of the Rap1p site, and Dal82p functions synergistically with Rap1p. This synergism is unlike all other known instances of Dal82p synergism, namely, that with the GATA family transcription activators Gln3p and Gat1p, which occurs only in the presence of an inducer. The observations reported suggest thatCAR2gene expression results from strong constitutive transcriptional activation mediated by Rap1p and Dal82p being balanced by the down regulation of an equally strong transcriptional repressor, Ume6p. This balance is then tipped in the direction of expression by the presence of the inducer. The formal structure of theCAR2promoter and its operation closely follow the model proposed forCAR1.
Transcriptional Organization of the Erythromycin Biosynthetic Gene Cluster of
Saccharopolyspora erythraea
Authors: Reeves Andrew R. , English R. Samuel , Lampel J. S. , Post David A. , Vanden Boom Thomas J.
Published: December 2019

The transcriptional organization of the erythromycin biosynthetic gene (
ery
) cluster of
Saccharopolyspora erythraea
has been examined by a variety of methods, including S1 nuclease protection assays, Northern blotting, Western blotting, and bioconversion analysis of erythromycin intermediates. The analysis was facilitated by the construction of novel mutants containing a
S. erythraea
transcriptional terminator within the
eryAI
,
eryAIII
,
eryBIII
,
eryBIV
,
eryBV
,
eryBVI
,
eryCIV
, and
eryCVI
genes and additionally by an
eryAI
−10 promoter mutant. All mutant strains demonstrated polar effects on the transcription of downstream
ery
biosynthetic genes. Our results demonstrate that the
ery
gene cluster contains four major polycistronic transcriptional units, the largest one extending approximately 35 kb from
eryAI
to
eryG
. Two overlapping polycistronic transcripts extending from
eryBIV
to
eryBVII
were identified. In addition, seven
ery
cluster promoter transcription start sites, one each beginning at
eryAI
,
eryBI
,
eryBIII
,
eryBVI
, and
eryK
and two beginning at
eryBIV
, were determined.
Chromosomal Integration of Heterologous DNA in
Escherichia coli
with Precise Removal of Markers and Replicons Used during Construction
Authors: Martinez-Morales F. , Borges A. C. , Martinez A. , Shanmugam K. T. , Ingram L. O.
Published: December 2019

A set of vectors which facilitates the sequential integration of new functions into the
Escherichia coli
chromosome by homologous recombination has been developed. These vectors are based on plasmids described by Posfai et al. (J. Bacteriol. 179:4426–4428, 1997) which contain conditional replicons (pSC101 or R6K), a choice of three selectable markers (ampicillin, chloramphenicol, or kanamycin), and a single
FRT
site. The modified vectors contain two
FRT
sites which bracket a modified multiple cloning region for DNA insertion. After integration, a helper plasmid expressing the flippase (FLP) recombinase allows precise in vivo excision of the replicon and the marker used for selection. Sites are also available for temporary insertion of additional functions which can be subsequently deleted with the replicon. Only the DNA inserted into the multiple cloning sites (passenger genes and homologous fragment for targeting) and a single
FRT
site (68 bp) remain in the chromosome after excision. The utility of these vectors was demonstrated by integrating
Zymomonas mobilis
genes encoding the ethanol pathway behind the native chromosomal
adhE
gene in strains of
E. coli
K-12 and
E. coli
B. With these vectors, a single antibiotic selection system can be used repeatedly for the successive improvement of
E. coli
strains with precise deletion of extraneous genes used during construction.
AlgT (ς
22
) Controls Alginate Production and Tolerance to Environmental Stress in
Pseudomonas syringae
Authors: Keith Lisa M. W. , Bender Carol L.
Published: December 2019

Pseudomonas aeruginosa
and the phytopathogen
P. syringae
produce the exopolysaccharide alginate, which is a copolymer of
d
-mannuronic and
l
-guluronic acids. One of the key regulatory genes controlling alginate biosynthesis in
P. aeruginosa
is
algT
, which encodes the alternate sigma factor, ς
22
. In the present study, the
algT
gene product from
P. syringae
pv. syringae showed 90% amino acid identity with its
P. aeruginosa
counterpart, and sequence analysis of the region flanking
algT
in
P. syringae
revealed the presence of
nadB
,
mucA
, and
mucB
in an arrangement virtually identical to that of
P. aeruginosa
. An
algT
mutant of
P. syringae
was defective in alginate production but could be complemented with wild-type
algT
from
P. syringae
or
P. aeruginosa
when expressed in
trans
. The
algT
mutant also displayed increased sensitivity to heat, paraquat, and hydrogen peroxide (H
2
O
2
); the latter two compounds are known to generate reactive oxygen intermediates. Signals for activation of
algT
gene expression in
P. syringae
were investigated with an
algT
::
uidA
transcriptional fusion. Like that in
P. aeruginosa
,
algT
transcription in
P. syringae
was activated by heat shock. However,
algT
expression in
P. syringae
was also stimulated by osmotic stress and by exposure to paraquat, H
2
O
2
, and copper sulfate. The latter two compounds are frequently encountered during colonization of plant tissue and may be unique signals for
algT
activation in
P. syringae
.
Physical Mapping and Functional Assignment of the Geranylgeranyl-Bacteriochlorophyll Reductase Gene,
bchP
, of
Rhodobacter sphaeroides
Authors: Addlesee Hugh A. , Hunter C. Neil
Published: December 2019

The bacteriochlorophyll of the purple photosynthetic bacterium
Rhodobacter sphaeroides
is esterified with phytol. The presence of this alcohol moiety is essential for the correct assembly of the photosynthetic apparatus. Despite this, and the fact that
R. sphaeroides
is widely used for the study of structure-function relationships in photosynthesis, the molecular genetics of the steps in which the alcohol is added and modified have not previously been investigated in this organism. Sequencing near the center of the photosynthesis gene cluster has now revealed the existence of an open reading frame encoding a putative 394-amino-acid polypeptide displaying strong homology with the products of a number of genes from other photosynthetic organisms, each proposed to be responsible for the reduction of the alcohol moiety of (bacterio)chlorophyll to phytol. An
R. sphaeroides
transposon mutant in this gene,
bchP
, possessed a structurally modified photosystem assembled with bacteriochlorophyll esterified with geranylgeraniol, rather than with phytol, implying that the product of this gene was geranylgeranyl-bacteriochlorophyll reductase. This identification was confirmed by the performance of in vitro assays using heterologously expressed protein, providing the first direct demonstration of the activity of a
bchP
gene product.
Molecular Evidence for a New Bacteriophage of
Borrelia burgdorferi
Authors: Eggers Christian H. , Samuels D. Scott
Published: December 2019

We have recovered a DNase-protected, chloroform-resistant molecule of DNA from the cell-free supernatant of a
Borrelia burgdorferi
culture. The DNA is a 32-kb double-stranded linear molecule that is derived from the 32-kb circular plasmids (cp32s) of the
B. burgdorferi
genome. Electron microscopy of samples from which the 32-kb DNA molecule was purified revealed bacteriophage particles. The bacteriophage has a polyhedral head with a diameter of 55 nm and appears to have a simple 100-nm-long tail. The phage is produced constitutively at low levels from growing cultures of some
B. burgdorferi
strains and is inducible to higher levels with 10 μg of 1-methyl-3-nitroso-nitroguanidine (MNNG) ml
−1
. In addition, the prophage can be induced with MNNG from some
Borrelia
isolates that do not naturally produce phage. We have isolated and partially characterized the phage associated with
B. burgdorferi
CA-11.2A. To our knowledge, this is the first molecular characterization of a bacteriophage of
B. burgdorferi
.
Helicobacter pylori rocF
Is Required for Arginase Activity and Acid Protection In Vitro but Is Not Essential for Colonization of Mice or for Urease Activity
Authors: McGee David J. , Radcliff Fiona J. , Mendz George L. , Ferrero Richard L. , Mobley Harry L. T.
Published: December 2019

Arginase of the
Helicobacter pylori
urea cycle hydrolyzes
l
-arginine to
l
-ornithine and urea.
H. pylori
urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved in
H. pylori
nitrogen metabolism. The roles of arginase in the physiology of
H. pylori
were investigated in vitro and in vivo, since arginase in
H. pylori
is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The
H. pylori
gene
hp1399
, which is orthologous to the
Bacillus subtilis rocF
gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three
H. pylori
strains were made by using two different constructs: 236-2 and
rocF::aphA3
. In contrast to wild-type (WT) strains, all
rocF
mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of
H. pylori
, the
rocF::aphA3
mutant was ∼1,000-fold more sensitive to acid exposure. The acid sensitivity of the
rocF::aphA3
mutant was not reversed by the addition of
l
-arginine, in contrast to the WT, and yielded a ∼10,000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that
rocF
is not required for urease activity in vitro. Finally,
H. pylori
mouse-adapted strain SS1 and the 236-2
rocF
isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the
rocF::aphA3
mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of
H. pylori
recovered from these mice (in log
10
CFU) were 6.1, 5.5, and 4.1, respectively. Thus,
H. pylori rocF
is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity.