bluuebunny/crossref_metadata_2025_split huggingface dataset:
Haloalkane-UtilizingRhodococcusStrains Isolated from Geographically Distinct Locations Possess a Highly Conserved Gene Cluster Encoding Haloalkane Catabolism
Authors: Poelarends Gerrit J. , Zandstra Marjan , Bosma Tjibbe , Kulakov Leonid A. , Larkin Michael J. , Marchesi Julian R. , Weightman Andrew J. , Janssen Dick B. 
Published: July 2002

ABSTRACTThe sequences of the 16S rRNA and haloalkane dehalogenase (dhaA) genes of five gram-positive haloalkane-utilizing bacteria isolated from contaminated sites in Europe, Japan, and the United States and of the archetypal haloalkane-degrading bacteriumRhodococcussp. strain NCIMB13064 were compared. The 16S rRNA gene sequences showed less than 1% sequence divergence, and all haloalkane degraders clearly belonged to the genusRhodococcus. All strains shared a completely conserveddhaAgene, suggesting that thedhaAgenes were recently derived from a common ancestor. The genetic organization of thedhaAgene region in each of the haloalkane degraders was examined by hybridization analysis and DNA sequencing. Three different groups could be defined on the basis of the extent of the conserveddhaAsegment. The minimal structure present in all strains consisted of a conserved region of 12.5 kb, which included the haloalkane-degradative gene cluster that was previously found in strain NCIMB13064. Plasmids of different sizes were found in all strains. Southern hybridization analysis with adhaAgene probe suggested that all haloalkane degraders carry thedhaAgene region both on the chromosome and on a plasmid (70 to 100 kb). This suggests that an ancestral plasmid was transferred between theseRhodococcusstrains and subsequently has undergone insertions or deletions. In addition, transposition events and/or plasmid integration may be responsible for positioning thedhaAgene region on the chromosome. The data suggest that the haloalkane dehalogenase gene regions of these gram-positive haloalkane-utilizing bacteria are composed of a single catabolic gene cluster that was recently distributed worldwide.
In Vivo Analysis of the Mechanisms for Oxidation of Cytosolic NADH by
Saccharomyces cerevisiae
Mitochondria
Authors: Overkamp Karin M. , Bakker Barbara M. , Kötter Peter , van Tuijl Arjen , de Vries Simon , van Dijken Johannes P. , Pronk Jack T. 
Published: July 2002

ABSTRACT

During respiratory glucose dissimilation, eukaryotes produce cytosolic NADH via glycolysis. This NADH has to be reoxidized outside the mitochondria, because the mitochondrial inner membrane is impermeable to NADH. In
Saccharomyces cerevisiae
, this may involve external NADH dehydrogenases (Nde1p or Nde2p) and/or a glycerol-3-phosphate shuttle consisting of soluble (Gpd1p or Gpd2p) and membrane-bound (Gut2p) glycerol-3-phosphate dehydrogenases. This study addresses the physiological relevance of these mechanisms and the possible involvement of alternative routes for mitochondrial oxidation of cytosolic NADH. Aerobic, glucose-limited chemostat cultures of a
gut2Δ
mutant exhibited fully respiratory growth at low specific growth rates. Alcoholic fermentation set in at the same specific growth rate as in wild-type cultures (0.3 h
−1
). Apparently, the glycerol-3-phosphate shuttle is not essential for respiratory glucose dissimilation. An
nde1Δ nde2Δ
mutant already produced glycerol at specific growth rates of 0.10 h
−1
and above, indicating a requirement for external NADH dehydrogenase to sustain fully respiratory growth. An
nde1Δ nde2Δ gut2Δ
mutant produced even larger amounts of glycerol at specific growth rates ranging from 0.05 to 0.15 h
−1
. Apparently, even at a low glycolytic flux, alternative mechanisms could not fully replace the external NADH dehydrogenases and glycerol-3-phosphate shuttle. However, at low dilution rates, the
nde1Δ nde2Δ gut2Δ
mutant did not produce ethanol. Since glycerol production could not account for all glycolytic NADH, another NADH-oxidizing system has to be present. Two alternative mechanisms for reoxidizing cytosolic NADH are discussed: (i) cytosolic production of ethanol followed by its intramitochondrial oxidation and (ii) a redox shuttle linking cytosolic NADH oxidation to the internal NADH dehydrogenase.
Secretion of the
Caulobacter crescentus
S-Layer Protein: Further Localization of the C-Terminal Secretion Signal and Its Use for Secretion of Recombinant Proteins
Authors: Bingle Wade H. , Nomellini John F. , Smit John 
Published: July 2002

ABSTRACT

The secretion signal of the
Caulobacter crescentus
S-layer protein (RsaA) was localized to the C-terminal 82 amino acids of the molecule. Protein yield studies showed that 336 or 242 C-terminal residues of RsaA mediated secretion of >50 mg of a cellulase passenger protein per liter to the culture fluids.
The Dual-Specificity Protein Phosphatase Yvh1p Regulates Sporulation, Growth, and Glycogen Accumulation Independently of Catalytic Activity in
Saccharomyces cerevisiae
via the Cyclic AMP-Dependent Protein Kinase Cascade
Authors: Beeser Alexander E. , Cooper Terrance G. 
Published: July 2002

ABSTRACT

Yvh1p, a dual-specific protein phosphatase induced specifically by nitrogen starvation, regulates cell growth as well as initiation and completion of sporulation. We demonstrate that
yvh1
disruption mutants are also unable to accumulate glycogen in stationary phase. A catalytically inactive variant of
yvh1
(C117S) and a DNA fragment encoding only the Yvh1p C-terminal 159 amino acids (which completely lacks the phosphatase domain) complement all three phenotypes as well as the wild-type allele; no complementation occurs with a fragment encoding only the C-terminal 74 amino acids. These observations argue that phosphatase activity is not required for the Yvh1p functions we measured. Mutations which decrease endogenous cyclic AMP (cAMP) levels partially suppress the sporulation and glycogen accumulation defects. In addition, reporter gene expression supported by a
DRR2
promoter fragment, containing two stress response elements known to respond to cAMP-protein kinase A, decreases in a
yvh1
disruption mutant. Therefore, our results identify three cellular processes that both require Yvh1p and respond to alterations in cAMP, and they lead us to suggest that Yvh1p may be a participant in and/or a contributor to regulation of the cAMP-dependent protein kinase cascade. The fact that decreasing the levels of cAMP alleviates the need for Yvh1p function supports this suggestion.
Reduction of Cob(III)alamin to Cob(II)alamin in
Salmonella enterica
Serovar Typhimurium LT2
Authors: Fonseca Maris V. , Escalante-Semerena Jorge C. 
Published: July 2002

ABSTRACT

Reduction of the cobalt ion of cobalamin from the Co(III) to the Co(I) oxidation state is essential for the synthesis of adenosylcobalamin, the coenzymic form of this cofactor. A cob(II)alamin reductase activity in
Salmonella enterica
serovar Typhimurium LT2 was isolated to homogeneity. N-terminal analysis of the homogeneous protein identified NAD(P)H:flavin oxidoreductase (Fre) (EC
1.6.8.1
) as the enzyme responsible for this activity. The
fre
gene was cloned, and the overexpressed protein, with a histidine tag at its N terminus, was purified to homogeneity by nickel affinity chromatography. His-tagged Fre reduced flavins (flavin mononucleotide [FMN] and flavin adenine dinucleotide [FAD]) and cob(III)alamin to cob(II)alamin very efficiently. Photochemically reduced FMN substituted for Fre in the reduction of cob(III)alamin to cob(II)alamin, indicating that the observed cobalamin reduction activity was not Fre dependent but FMNH
2
dependent. Enzyme-independent reduction of cob(III)alamin to cob(II)alamin by FMNH
2
occurred at a rate too fast to be measured. The thermodynamically unfavorable reduction of cob(II)alamin to cob(I)alamin was detectable by alkylation of the cob(I)alamin nucleophile with iodoacetate. Detection of the product, caboxymethylcob(III)alamin, depended on the presence of FMNH
2
in the reaction mixture. FMNH
2
failed to substitute for potassium borohydride in in vitro assays for corrinoid adenosylation catalyzed by the ATP:co(I)rrinoid adenosyltransferase (CobA) enzyme, even under conditions where Fre and NADH were present in the reaction mixture to ensure that FMN was always reduced. These results were interpreted to mean that Fre was not responsible for the generation of cob(I)alamin in vivo. Consistent with this idea, a
fre
mutant displayed wild-type cobalamin biosynthetic phenotypes. It is proposed that
S. enterica
serovar Typhimurium LT2 may not have a cob(III)alamin reductase enzyme and that, in vivo, nonadenosylated cobalamin and other corrinoids are maintained as co(II)rrinoids by reduced flavin nucleotides generated by Fre and other flavin oxidoreductases.
In Vivo and In Vitro Effects of (p)ppGpp on the ς
54
Promoter
Pu
of the TOL Plasmid of
Pseudomonas putida
Authors: Carmona Manuel , Rodríguez Maria J. , Martínez-Costa Óscar , de Lorenzo Víctor 
Published: July 2002

ABSTRACT

The connection between the physiological control of the ς
54
-dependent
Pu
promoter of the TOL plasmid pWW0 of
Pseudomonas putida
and the stringent response mediated by the alarmone (p)ppGpp has been examined in vivo an in vitro. To this end, the key regulatory elements of the system were faithfully reproduced in an
Escherichia coli
strain and assayed as
lacZ
fusions in various genetic backgrounds lacking (p)ppGpp or overexpressing
relA
. Neither the responsiveness of
Pu
to 3-methyl benzylalcohol mediated by its cognate activator XylR nor the down-regulation of the promoter by rapid growth were affected in
relA/spoT
strains to an extent which could account for the known physiological control that governs this promoter. Overexpression of the
relA
gene [predicted to increase intracellullar (p)ppGpp levels] did, however, cause a significant gain in
Pu
activity. Since such a gain might be the result of indirect effects, we resorted to an in vitro transcription system to assay directly the effect of ppGpp on the transcriptional machinery. Although we did observe a significant increase in
Pu
performance through a range of ς
54
-RNAP concentrations, such an increase never exceeded twofold. The difference between these results and the behavior of the related
Po
promoter of the phenol degradation plasmid pVI150 could be traced to the different promoter sequences, which may dictate the type of metabolic signals recruited for the physiological control of ς
54
-systems.
Characterization of the Operon Encoding the Alternative ς
B
Factor from
Bacillus anthracis
and Its Role in Virulence
Authors: Fouet Agnès , Namy Olivier , Lambert Guillaume 
Published: July 2002

ABSTRACT

The operon encoding the general stress transcription factor ς
B
and two proteins of its regulatory network, RsbV and RsbW, was cloned from the gram-positive bacterium
Bacillus anthracis
by PCR amplification of chromosomal DNA with degenerate primers, by inverse PCR, and by direct cloning. The gene cluster was very similar to the
Bacillus subtilis sigB
operon both in the primary sequences of the gene products and in the order of its three genes. However, the deduced products of sequences upstream and downstream from this operon showed no similarity to other proteins encoded by the
B. subtilis sigB
operon. Therefore, the
B. anthracis sigB
operon contains three genes rather than eight as in
B. subtilis
. The
B. anthracis
operon is preceded by a ς
B
-like promoter sequence, the expression of which depends on an intact ς
B
transcription factor in
B. subtilis
. It is followed by another open reading frame that is also preceded by a promoter sequence similarly dependent on
B. subtilis
ς
B
. We found that in
B. anthracis
, both these promoters were induced during the stationary phase and induction required an intact
sigB
gene. The
sigB
operon was induced by heat shock. Mutants from which
sigB
was deleted were constructed in a toxinogenic and a plasmidless strain. These mutants differed from the parental strains in terms of morphology. The toxinogenic
sigB
mutant strain was also less virulent than the parental strain in the mouse model.
B. anthracis
ς
B
may therefore be a minor virulence factor.
Essentiality, Expression, and Characterization of the Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase of
Staphylococcus aureus
Authors: Wilding E. Imogen , Kim Dong-Yul , Bryant Alexander P. , Gwynn Michael N. , Lunsford R. Dwayne , McDevitt Damien , Myers Joseph E. , Rosenberg Martin , Sylvester Daniel , Stauffacher Cynthia V. , Rodwell Victor W. 
Published: July 2002

ABSTRACT

Sequence comparisons have implied the presence of genes encoding enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis in the gram-positive pathogen
Staphylococcus aureus
. In this study we showed through genetic disruption experiments that
mvaA
, which encodes a putative class II 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is essential for in vitro growth of
S. aureus
. Supplementation of media with mevalonate permitted isolation of an auxotrophic
mvaA
null mutant that was attenuated for virulence in a murine hematogenous pyelonephritis infection model. The
mvaA
gene was cloned from
S. aureus
DNA and expressed with an N-terminal His tag in
Escherichia coli
. The encoded protein was affinity purified to apparent homogeneity and was shown to be a class II HMG-CoA reductase, the first class II eubacterial biosynthetic enzyme isolated. Unlike most other HMG-CoA reductases, the
S. aureus
enzyme exhibits dual coenzyme specificity for NADP(H) and NAD(H), but NADP(H) was the preferred coenzyme. Kinetic parameters were determined for all substrates for all four catalyzed reactions using either NADP(H) or NAD(H). In all instances optimal activity using NAD(H) occurred at a pH one to two units more acidic than that using NADP(H). pH profiles suggested that His378 and Lys263, the apparent cognates of the active-site histidine and lysine of
Pseudomonas mevalonii
HMG-CoA reductase, function in catalysis and that the general catalytic mechanism is valid for the
S. aureus
enzyme. Fluvastatin inhibited competitively with HMG-CoA, with a

K
i

of 320 μM, over 10
4
higher than that for a class I HMG-CoA reductase. Bacterial class II HMG-CoA reductases thus are potential targets for antibacterial agents directed against multidrug-resistant gram-positive cocci.
Mutational Analysis of Genes Encoding the Early Flagellar Components of
Helicobacter pylori
: Evidence for Transcriptional Regulation of Flagellin A Biosynthesis
Authors: Allan Elaine , Dorrell Nick , Foynes Susan , Anyim Martin , Wren Brendan W. 
Published: July 2002

ABSTRACT

We investigated the roles of
fliF
,
fliS
,
flhB
,
fliQ
,
fliG
, and
fliI
of
Helicobacter pylori
, predicted by homology to encode structural components of the flagellar basal body and export apparatus. Mutation of these genes resulted in nonmotile, nonflagellate strains. Western blot analysis showed that all the mutants had considerably reduced levels of both flagellin subunits and of FlgE, the flagellar hook protein. RNA slot blot hybridization showed reduced levels of
flaA
mRNA, indicating that transcription of the major flagellin gene is inhibited in the absence of the early components of the flagellar-assembly pathway. This is the first demonstration of a checkpoint in
H. pylori
flagellar assembly.
Pathogenic Neisseriae Can Use Hemoglobin, Transferrin, and Lactoferrin Independently of the
tonB
Locus
Authors: Desai Pragnya Jasvantrai , Garges Eric , Genco Caroline Attardo 
Published: July 2002

ABSTRACT

Redundant TonB systems which function in iron transport from TonB-dependent ligands have recently been identified in several gram-negative bacteria. We demonstrate here that in addition to the previously described
tonB
locus, an alternative system exists for the utilization of iron from hemoglobin, transferrin, or lactoferrin in
Neisseria meningitidis
and
Neisseria gonorrhoeae
. Following incubation on media containing hemoglobin,
N. meningitidis
IR3436 (
tonB exbB exbD
deletion mutant) and
N. gonorrhoeae
PD3401 (
tonB
insertional mutant) give rise to colonies which can grow with hemoglobin. Transfer of Hb
+
variants (PD3437 or PD3402) to media containing hemoglobin, transferrin, and/or lactoferrin as sole iron sources resulted in growth comparable to that observed for the wild-type strains. Transformation of
N. meningitidis
IR3436 or
N. gonorrhoeae
PD3401 with chromosomal DNA from the Hb
+
variants yielded transformants capable of growth with hemoglobin. When we inactivated the TonB-dependent outer membrane hemoglobin receptors (HmbR or HpuB) in the
Neisseria
Hb
+
variants, these strains could not grow with hemoglobin; however, growth was observed with transferrin and/or lactoferrin. These results demonstrate that accumulation of iron from hemoglobin, transferrin, and lactoferrin in the pathogenic neisseriae can occur via a system that is independent of the previously described
tonB
locus.
Contribution of NADH Oxidase to Aerobic Metabolism of
Streptococcus pyogenes
Authors: Gibson Carmela M. , Mallett T. Conn , Claiborne Al , Caparon Michael G. 
Published: July 2002

ABSTRACT

An understanding of how the heme-deficient gram-positive bacterium
Streptococcus pyogenes
establishes infections in O
2
-rich environments requires careful analysis of the gene products important in aerobic metabolism. NADH oxidase (NOXase) is a unique flavoprotein of
S. pyogenes
and other lactic acid bacteria which directly catalyzes the four-electron reduction of O
2
to H
2
O. To elucidate a putative role for this enzyme in aerobic metabolism, NOXase-deficient mutants were constructed by insertional inactivation of the gene that encodes NOXase. Characterization of the resulting mutants revealed that growth in rich medium under low-O
2
conditions was indistinguishable from that of the wild type. However, the mutants were unable to grow under high-O
2
conditions and demonstrated enhanced sensitivity to the superoxide-generating agent paraquat. Mutants cultured in liquid medium under conditions of carbohydrate limitation and high O
2
tension were characterized by an extended lag phase, a reduction in growth, and a greater accumulation of H
2
O
2
in the growth medium compared to the wild-type strain. All of these mutant phenotypes could be overcome by the addition of glucose. Either the addition of catalase to the culture medium of the mutants or the introduction of a heterologous NADH peroxidase into the mutants eliminated the accumulation of H
2
O
2
and rescued the growth defect of the mutants under high-O
2
conditions in carbohydrate-limited liquid medium. Taken together, these data show that NOXase is important for aerobic metabolism and essential in environments high in O
2
with carbohydrate limitation.
Identification of RpoS (ς
S
)-Regulated Genes in
Salmonella enterica
Serovar Typhimurium
Authors: Ibanez-Ruiz Magdalena , Robbe-Saule Véronique , Hermant Daniel , Labrude Séverine , Norel Françoise 
Published: July 2002

ABSTRACT

The
rpoS
gene encodes the alternative sigma factor ς
S
(RpoS) and is required for survival of bacteria under starvation and stress conditions. It is also essential for
Salmonella
virulence in mice. Most work on the RpoS regulon has been in the closely related enterobacterial species
Escherichia coli
. To characterize the RpoS regulon in
Salmonella
, we isolated 38 unique RpoS-activated
lacZ
gene fusions from a bank of
Salmonella enterica
serovar Typhimurium mutants harboring random Tn5B21 mutations. Dependence on RpoS varied from 3-fold to over 95-fold, and all gene fusions isolated were regulated by growth phase. The identities of 21 RpoS-dependent fusions were determined by DNA sequence analysis. Seven of the fusions mapped to DNA regions in
Salmonella
serovar Typhimurium that do not match any known
E. coli
sequence, suggesting that the composition of the RpoS regulon differs markedly in the two species. The other 14 fusions mapped to 13 DNA regions very similar to
E. coli
sequences. None of the insertion mutations in DNA regions common to both species appeared to affect
Salmonella
virulence in BALB/c mice. Of these, only three (
otsA
,
katE
, and
poxB
) are located in known members of the RpoS regulon. Ten insertions mapped in nine open reading frames of unknown function (
yciF
,
yehY
,
yhjY
,
yncC
,
yjgB
,
yahO
,
ygaU
,
ycgB
, and
yeaG
) appear to be novel members of the RpoS regulon. One insertion, that in mutant C52::H87, was in the noncoding region upstream from
ogt
, encoding a
O
6
-methylguanine DNA methyltransferase involved in repairing alkylation damage in DNA. The
ogt
coding sequence is very similar to the
E. coli
homolog, but the
ogt
5′ flanking regions were found to be markedly different in the two species, suggesting genetic rearrangements. Using primer extension assays, a specific
ogt
mRNA start site was detected in RNAs of the
Salmonella
serovar Typhimurium wild-type strains C52 and SL1344 but not in RNAs of the mutant strains C52K (
rpoS
), SL1344K (
rpoS
), and C52::H87. In mutant C52::H87, Tn
5
B21 is inserted at the
ogt
mRNA start site, with
lacZ
presumably transcribed from the identified RpoS-regulated promoter. These results indicate that
ogt
gene expression in
Salmonella
is regulated by RpoS in stationary phase of growth in rich medium, a finding that suggests a novel role for RpoS in DNA repair functions.
The Last RNA-Binding Repeat of the
Escherichia coli
Ribosomal Protein S1 Is Specifically Involved in Autogenous Control
Authors: Boni Irina V. , Artamonova Valentina S. , Dreyfus Marc 
Published: July 2002

ABSTRACT

The
ssyF29
mutation, originally selected as an extragenic suppressor of a protein export defect, has been mapped within the
rpsA
gene encoding ribosomal protein S1. Here, we examine the nature of this mutation and its effect on translation. Sequencing of the
rpsA
gene from the
ssyF
mutant has revealed that, due to an IS
10
R insertion, its product lacks the last 92 residues of the wild-type S1 protein corresponding to one of the four homologous repeats of the RNA-binding domain. To investigate how this truncation affects translation, we have created two series of
Escherichia coli
strains (
rpsA
+
and
ssyF
) bearing various translation initiation regions (TIRs) fused to the chromosomal
lacZ
gene. Using a β-galactosidase assay, we show that none of these TIRs differ in activity between
ssyF
and
rpsA
+
cells, except for the
rpsA
TIR: the latter is stimulated threefold in
ssyF
cells, provided it retains at least ca. 90 nucleotides upstream of the start codon. Similarly, the activity of this TIR can be severely repressed in
trans
by excess S1, again provided it retains the same minimal upstream sequence. Thus, the
ssyF
stimulation requires the presence of the
rpsA
translational autogenous operator. As an interpretation, we propose that the
ssyF
mutation relieves the residual repression caused by normal supply of S1 (i.e., that it impairs autogenous control). Thus, the C-terminal repeat of the S1 RNA-binding domain appears to be required for autoregulation, but not for overall mRNA recognition.