bluuebunny/crossref_metadata_2025_split huggingface dataset:
Two Nitrate/Nitrite Transporters Are Encoded within the Mobilizable Plasmid for Nitrate Respiration ofThermus thermophilusHB8
Authors: Ramírez Sandra , Moreno Renata , Zafra Olga , Castán Pablo , Vallés Cristina , Berenguer José 
Published: July 2002

ABSTRACTThermus thermophilusHB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes,narK1andnarK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a doublenarK1 narK2mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed.
Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon
Pyrococcus furiosus
and Its Role in Alanine Production
Authors: Ward Donald E. , Kengen Servé W. M. , van der Oost John , de Vos Willem M. 
Published: July 2002

ABSTRACT

Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon
Pyrococcus furiosus
by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (
aat
) in the
P. furiosus
genome database. The gene was expressed in
Escherichia coli
, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from
P. furiosus
. The
k
cat
/

K
m

values for alanine and pyruvate formation were 41 and 33 s
−1
mM
−1
, respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the
aat
gene. In addition, both the
aat
and
gdh
(encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the
aat
and
gdh
genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in
P. furiosus
.
Role of Arginines in Coenzyme A Binding and Catalysis by the Phosphotransacetylase from
Methanosarcina thermophila
Authors: Iyer Prabha P. , Ferry James G. 
Published: July 2002

ABSTRACT

Phosphotransacetylase (EC
2.3.1.8
) catalyzes the reversible transfer of the acetyl group from acetyl phosphate to coenzyme A (CoA): CH
3
COOPO
3
2−
+ CoASH ⇆ CH
3
COSCoA + HPO
4
2−
. The role of arginine residues was investigated for the phosphotransacetylase from
Methanosarcina thermophila
. Kinetic analysis of a suite of variants indicated that Arg 87 and Arg 133 interact with the substrate CoA. Arg 87 variants were reduced in the ability to discriminate between CoA and the CoA analog 3′-dephospho-CoA, indicating that Arg 87 forms a salt bridge with the 3′-phosphate of CoA. Arg 133 is postulated to interact with the 5′-phosphate of CoA. Large decreases in
k
cat
and
k
cat
/

K
m

for all of the Arg 87 and Arg 133 variants indicated that these residues are also important, although not essential, for catalysis. Large decreases in
k
cat
and
k
cat
/

K
m

were also observed for the variants in which lysine replaced Arg 87 and Arg 133, suggesting that the bidentate interaction of these residues with CoA or their greater bulk is important for optimal activity. Desulfo-CoA is a strong competitive inhibitor of the enzyme, suggesting that the sulfhydryl group of CoA is important for the optimization of CoA-binding energy but not for tight substrate binding. Chemical modification of the wild-type enzyme by 2,3-butanedione and substrate protection by CoA indicated that at least one reactive arginine is in the active site and is important for activity. The inhibition pattern of the R87Q variant indicated that Arg 87 is modified, which contributes to the inactivation; however, at least one additional active-site arginine is modified leading to enzyme inactivation, albeit at a lower rate.
Isolation and Characterization of a
Shewanella putrefaciens
MR-1 Electron Transport Regulator
etrA
Mutant: Reassessment of the Role of EtrA
Authors: Maier Tamara M. , Myers Charles R. 
Published: July 2002

ABSTRACT

Shewanella putrefaciens
MR-1 has emerged as a good model to study anaerobic respiration and electron transport-linked metal reduction. Its remarkable respiratory plasticity suggests the potential for a complex regulatory system to coordinate electron acceptor use in the absence of O
2
. It had previously been suggested that EtrA (electron transport regulator A), an analog of Fnr (fumarate nitrate regulator) from
Escherichia coli
, may regulate gene expression for anaerobic electron transport. An
etrA
knockout strain (ETRA-153) was isolated from MR-1 using a gene replacement strategy. Reverse transcription-PCR analysis of total RNA demonstrated the loss of the
etrA
mRNA in ETRA-153. ETRA-153 cells retained the ability to grow on all electron acceptors tested, including fumarate, trimethylamine
N
-oxide (TMAO), thiosulfate, dimethyl sulfoxide, ferric citrate, nitrate, and O
2
, as well as the ability to reduce ferric citrate, manganese(IV), nitrate, and nitrite. EtrA is therefore not necessary for growth on, or the reduction of, these electron acceptors. However, ETRA-153 had reduced initial growth rates on fumarate and nitrate but not on TMAO. The activities for fumarate and nitrate reductase were lower in ETRA-153, as were the levels of fumarate reductase protein and transcript. ETRA-153 was also deficient in one type of ubiquinone. These results are in contrast to those previously reported for the putative
etrA
mutant METR-1. Molecular analysis of METR-1 indicated that its
etrA
gene is not interrupted; its reported phenotype was likely due to the use of inappropriate anaerobic growth conditions.
Regulated Secretion of YopN by the Type III Machinery of
Yersinia enterocolitica
Authors: Cheng Luisa W. , Kay Olga , Schneewind Olaf 
Published: July 2002

ABSTRACT

During infection,
Yersinia enterocolitica
exports Yop proteins via a type III secretion pathway. Secretion is activated when the environmental concentration of calcium ions is below 100 μM (low-calcium response). Yersiniae lacking
yopN (lcrE), yscB, sycN
, or
tyeA
do not inactivate the type III pathway even when the concentration of calcium is above 100 μM (calcium-blind phenotype). Purified YscB and SycN proteins form cytoplasmic complexes that bind a region including amino acids 16 to 100 of YopN, whereas TyeA binds YopN residues 101 to 294. Translational fusion of
yopN
gene sequences to the 5′ end of the
npt
reporter generates hybrid proteins that are transported by the type III pathway. The signal necessary and sufficient for the type III secretion of hybrid proteins is located within the first 15 codons of
yopN
. Expression of plasmid-borne
yopN
, but not of
yopN
1–294
-
npt
, complements the calcium-blind phenotype of
yopN
mutants. Surprisingly,
yopN
mutants respond to environmental changes in calcium concentration and secrete YopN
1–294
-Npt in the absence but not in the presence of calcium.
tyeA
is required for the low-calcium regulation of YopN
1–294
-Npt secretion, whereas
sycN
and
yscB
mutants fail to secrete YopN
1–294
-Npt in the presence of calcium. Experiments with
yopN-npt
fusions identified two other signals that regulate the secretion of YopN.
yopN
codons 16 to 100 prevent the entry of YopN into the type III pathway, a negative regulatory effect that is overcome by expression of
yscB
and
sycN
. The portion of YopN encoded by codons 101 to 294 prevents transport of the polypeptide across the bacterial double membrane envelope in the presence of functional
tyeA
. These data support a model whereby YopN transport may serve as a regulatory mechanism for the activity of the type III pathway. YscB/SycN binding facilitates the initiation of YopN into the type III pathway, whereas TyeA binding prevents transport of the polypeptide across the bacterial envelope. Changes in the environmental calcium concentration relieve the TyeA-mediated regulation, triggering YopN transport and activating the type III pathway.
Expression of
cspH
, Encoding the Cold Shock Protein in
Salmonella enterica
Serovar Typhimurium UK-1
Authors: Kim Bae Hoon , Bang Iel Soo , Lee Soo Youn , Hong Seong Karp , Bang Seong Ho , Lee In Soo , Park Yong Keun 
Published: July 2002

ABSTRACT

Both
Salmonella enterica
serovar Typhimurium and
Escherichia coli
contain the
cspH
gene encoding CspH, one of the cold shock proteins (CSPs). In this study, we investigated the expression of
cspH
in
S. enterica
serovar Typhimurium and found that it was induced in response to a temperature downshift during exponential phase. The
cspH
promoter was activated at 37°C, and its mRNA was more stable than the other
csp
mRNAs at 37°C. Moreover,
lacZ
expression of the translational
cspH-lacZ
fusion was induced at that temperature. Interestingly, the
cspH
mRNA had a much shorter 5′-untranslated region than those in the other cold-shock-inducible genes, and the promoter sequence, which was only 55 bp, was sufficient for
cspH
expression. The 14-base downstream box located 12 bases downstream of the initiation codon of
cspH
mRNA was essential for its cold shock activation.
Transcriptional Activation of
Bordetella
Alcaligin Siderophore Genes Requires the AlcR Regulator with Alcaligin as Inducer
Authors: Brickman Timothy J. , Kang Ho Young , Armstrong Sandra K. 
Published: July 2002

ABSTRACT

Genetic and biochemical studies have established that Fur and iron mediate repression of
Bordetella
alcaligin siderophore system (
alc
) genes under iron-replete nutritional growth conditions. In this study, transcriptional analyses using
Bordetella
chromosomal
alc-lacZ
operon fusions determined that maximal
alc
gene transcriptional activity under iron starvation stress conditions is dependent on the presence of alcaligin siderophore. Mutational analysis and genetic complementation confirmed that alcaligin-responsive transcriptional activation of
Bordetella
alcaligin system genes is dependent on AlcR, a Fur-regulated AraC-like positive transcriptional regulator encoded within the alcaligin gene cluster. AlcR-mediated transcriptional activation is remarkably sensitive to inducer, occurring at extremely low alcaligin concentrations. This positive autogenous control circuit involving alcaligin siderophore as the inducer for AlcR-mediated transcriptional activation of alcaligin siderophore biosynthesis and transport genes coordinates environmental and intracellular signals for maximal expression of these genes under conditions in which the presence of alcaligin in the environment is perceived.
ihfA
Gene of the Bacterium
Myxococcus xanthus
and Its Role in Activation of Carotenoid Genes by Blue Light
Authors: Moreno Alberto J. , Fontes Marta , Murillo Francisco J. 
Published: July 2002

ABSTRACT

Myxococcus xanthus
responds to blue light by producing carotenoids. Several regulatory genes are known that participate in the light action mechanism, which leads to the transcriptional activation of the carotenoid genes. We had already reported the isolation of a carotenoid-less, Tn
5
-induced strain (MR508), whose mutant site was unlinked to the indicated regulatory genes. Here, we show that ΩMR508::Tn
5
affects all known light-inducible promoters in different ways. It blocks the activation of two of them by light but makes the activity of a third one light independent. The ΩMR508 locus has been cloned and sequenced. The mutation had occurred at the promoter of a gene we propose is the
M. xanthus
ortholog of
ihfA
. This encodes the α subunit of the histone-like integration host factor protein. An in-frame deletion within
ihfA
causes the same effects as the ΩMR508::Tn
5
insertion. Like other IhfA proteins, the deduced amino acid sequence of
M. xanthus
IhfA shows much similarity to HU, another histone-like protein. Sequence comparison data, however, and the finding that the
M. xanthus
gene is preceded by gene
pheT
, as happens in other gram-negative bacteria, strongly argue for the proposed orthology relationship. The
M. xanthus ihfA
gene shows some unusual features, both from structural and physiological points of view. In particular, the protein is predicted to have a unique, long acidic extension at the carboxyl terminus, and it appears to be necessary for normal cell growth and even vital for a certain wild-type strain of
M. xanthus
.
Role of Glutamine Synthetase in Nitrogen Metabolite Repression in
Aspergillus nidulans
Authors: Margelis Soula , D'Souza Cletus , Small Anna J. , Hynes Michael J. , Adams Thomas H. , Davis Meryl A. 
Published: July 2002

ABSTRACT

Glutamine synthetase (GS), EC
6.3.1.2
, is a central enzyme in the assimilation of nitrogen and the biosynthesis of glutamine. We have isolated the
Aspergillus nidulans glnA
gene encoding GS and have shown that
glnA
encodes a highly expressed but not highly regulated mRNA. Inactivation of
glnA
results in an absolute glutamine requirement, indicating that GS is responsible for the synthesis of this essential amino acid. Even when supplemented with high levels of glutamine, strains lacking a functional
glnA
gene have an inhibited morphology, and a wide range of compounds have been shown to interfere with repair of the glutamine auxotrophy. Heterologous expression of the prokaryotic
Anabaena glnA
gene from the
A. nidulans alcA
promoter allowed full complementation of the
A. nidulans glnA
Δ mutation. However, the
A. nidulans fluG
gene, which encodes a protein with similarity to prokaryotic GS, did not replace
A. nidulans glnA
function when similarly expressed. Our studies with the
glnA
Δ mutant confirm that glutamine, and not GS, is the key effector of nitrogen metabolite repression. Additionally, ammonium and its immediate product glutamate may also act directly to signal nitrogen sufficiency.
Heat-Shock-Induced Proteins from
Myxococcus xanthus
Authors: Otani Mieko , Tabata Junko , Ueki Toshiyuki , Sano Keiji , Inouye Sumiko 
Published: July 2002

ABSTRACT

Optimal conditions for two-dimensional gel electrophoresis of total cellular proteins from
Myxococcus xanthus
were established. Using these conditions, we analyzed protein patterns of heat-shocked
M. xanthus
cells. Eighteen major spots and 15 minor spots were found to be induced by heat shock. From N-terminal sequences of 15 major spots, DnaK, GroEL, GroES, alkyl hydroperoxide reductase, aldehyde dehydrogenase, succinyl coenzyme A (CoA) synthetase, 30S ribosomal protein S6, and ATP synthase α subunit were identified. Three of the 18 major spots had an identical N-terminal sequence, indicating that they may be different forms of the same protein. Although a DnaK homologue, SglK, has been identified in
M. xanthus
(R. M. Weimer, C. Creghton, A. Stassinopoulos, P. Youderian, and P. L. Hartzell, J. Bacteriol. 180:5357–5368, 1998; Z. Yang, Y. Geng, and W. Shi, J. Bacteriol. 180:218–224, 1998), SglK was not induced by heat shock. In addition, there were seven substitutions within the N-terminal 30-residue sequence of the newly identified DnaK. This is the first report to demonstrate that succinyl CoA synthetase, 30S ribosomal protein S6, and ATP synthase α subunit are heat shock inducible.
Connection between Poly-β-Hydroxybutyrate Biosynthesis and Growth on C
1
and C
2
Compounds in the Methylotroph
Methylobacterium extorquens
AM1
Authors: Korotkova Natalia , Lidstrom Mary E. 
Published: July 2002

ABSTRACT

Several DNA regions containing genes involved in poly-β-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph
Methylobacterium extorquens
AM1. Genes involved in PHB biosynthesis include those encoding β-ketothiolase (
phaA
), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (
phaB
), and PHB synthase (
phaC
).
phaA
and
phaB
are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as
orf3. phaC
was unlinked to
phaA
and
phaB
. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (
depA
and
depB
). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding β-hydroxybutyrate dehydrogenase (
hbd
) was identified. Insertion mutations were introduced into
depA, depB, phaA, phaB, phaC
, and
hbd
and also in a gene predicted to encode crotonase (
croA
), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in
depA, depB, hbd
, and
croA
all produced normal levels of PHB, and the only growth phenotype observed was the inability of the
hbd
mutant to grow on β-hydroxybutyrate. However, the
phaA, phaB
, and
phaC
mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C
1
and C
2
compounds and, for
phaB
, these defects were rescued by glyoxylate supplementation. These results suggest that β-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and
Streptomyces
spp.
Identification and Characterization of the
dif
Site from
Bacillus subtilis
Authors: Sciochetti Stephen A. , Piggot Patrick J. , Blakely Garry W. 
Published: July 2002

ABSTRACT

Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in
Escherichia coli
is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as
dif
(Ec
dif
). In
Bacillus subtilis
the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bs
dif
), lying 6° counterclockwise from the
B. subtilis
terminus of replication (172°), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bs
dif
in vivo recombination did not require the
B. subtilis
FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the
B. subtilis
SPβ-bacteriophage, and in particular its
yopP
gene product, appears to strongly modulate the extent of the partitioning defects seen in
codV
strains and, to a lesser extent, those seen in
ripX
and
dif
strains.
Analysis of Lipooligosaccharide Biosynthesis in the
Neisseriaceae
Authors: Arking Dan , Tong Yanhong , Stein Daniel C. 
Published: July 2002

ABSTRACT

Neisserial lipooligosaccharide (LOS) contains three oligosaccharide chains, termed the α, β, and γ chains. We used Southern hybridization experiments on DNA isolated from various
Neisseria
spp. to determine if strains considered to be nonpathogenic possessed DNA sequences homologous with genes involved in the biosynthesis of these oligosaccharide chains. The presence or absence of specific genes was compared to the LOS profiles expressed by each strain, as characterized by their mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and their reactivities with various LOS-specific monoclonal antibodies. A great deal of heterogeneity was seen with respect to the presence of genes encoding glycosyltransferases in
Neisseria
. All pathogenic species were found to possess DNA sequences homologous with the
lgt
gene cluster, a group of genes needed for the synthesis of the α chain. Some of these genes were also found to be present in strains considered to be nonpathogenic, such as
Neisseria lactamica, N. subflava
, and
N. sicca
. Some nonpathogenic
Neisseria
spp. were able to express high-molecular-mass LOS structures, even though they lacked the DNA sequences homologous with
rfaF
, a gene whose product must act before gonococcal and meningococcal LOS can be elongated. Using a PCR amplification strategy, in combination with DNA sequencing, we demonstrated that
N. subflava
44 possessed
lgtA, lgtB
, and
lgtE
genes. The predicted amino acid sequence encoded by each of these genes suggested that they encoded functional proteins; however, structural analysis of LOS isolated from this strain indicated that the bulk of its LOS was not modified by these gene products. This suggests the existence of an additional regulatory mechanism that is responsible for the limited expression of these genes in this strain.