bluuebunny/crossref_metadata_2025_split huggingface dataset:
Evolution and Functional Characterization of the
RH50
Gene from the Ammonia-Oxidizing Bacterium
Nitrosomonas europaea
Authors: Cherif-Zahar Baya , Durand Anne , Schmidt Ingo , Hamdaoui Nabila , Matic Ivan , Merrick Mike , Matassi Giorgio 
Published: October 2007

ABSTRACT

The family of ammonia and ammonium channel proteins comprises the Amt proteins, which are present in all three domains of life with the notable exception of vertebrates, and the homologous Rh proteins (Rh50 and Rh30) that have been described thus far only in eukaryotes. The existence of an
RH50
gene in bacteria was first revealed by the genome sequencing of the ammonia-oxidizing bacterium
Nitrosomonas europaea
. Here we have used a phylogenetic approach to study the evolution of the
N. europaea RH50
gene, and we show that this gene, probably as a component of an integron cassette, has been transferred to the
N. europaea
genome by horizontal gene transfer. In addition, by functionally characterizing the Rh50

Ne

protein and the corresponding knockout mutant, we determined that
Ne
Rh50 can mediate ammonium uptake. The

RH50
Ne

gene may thus have replaced functionally the
AMT
gene, which is missing in the genome of
N. europaea
and may be regarded as a case of nonorthologous gene displacement.
Rhodobacter capsulatus
OlsA Is a Bifunctional Enyzme Active in both Ornithine Lipid and Phosphatidic Acid Biosynthesis
Authors: Aygun-Sunar Semra , Bilaloglu Rahmi , Goldfine Howard , Daldal Fevzi 
Published: October 2007

ABSTRACT

The
Rhodobacter capsulatus
genome contains three genes (
olsA
[
plsC138
],
plsC316
, and
plsC3498
) that are annotated as lysophosphatidic acid (1-acyl-
sn
-glycerol-3-phosphate) acyltransferase (AGPAT). Of these genes,
olsA
was previously shown to be an
O
-acyltransferase in the second step of ornithine lipid biosynthesis, which is important for optimal steady-state levels of
c
-type cytochromes (S. Aygun-Sunar, S. Mandaci, H.-G. Koch, I. V. J. Murray, H. Goldfine, and F. Daldal. Mol. Microbiol. 61:418-435, 2006). The roles of the remaining
plsC316
and
plsC3498
genes remained unknown. In this work, these genes were cloned, and chromosomal insertion-deletion mutations inactivating them were obtained to define their function. Characterization of these mutants indicated that, unlike the
Escherichia coli plsC
, neither
plsC316
nor
plsC3498
was essential in
R. capsulatus
. In contrast, no
plsC316 olsA
double mutant could be isolated, indicating that an intact copy of either
olsA
or
plsC316
was required for
R. capsulatus
growth under the conditions tested. Compared to OlsA null mutants, PlsC316 null mutants contained ornithine lipid and had no
c
-type cytochrome-related phenotype. However, they exhibited slight growth impairment and highly altered total fatty acid and phospholipid profiles. Heterologous expression in an
E. coli plsC
(Ts) mutant of either
R. capsulatus plsC316
or
olsA
gene products supported growth at a nonpermissive temperature, exhibited AGPAT activity in vitro, and restored phosphatidic acid biosynthesis. The more vigorous AGPAT activity displayed by PlsC316 suggested that
plsC316
encodes the main AGPAT required for glycerophospholipid synthesis in
R. capsulatus
, while
olsA
acts as an alternative AGPAT that is specific for ornithine lipid synthesis. This study therefore revealed for the first time that some OlsA enzymes, like the enzyme of
R. capsulatus
, are bifunctional and involved in both membrane ornithine lipid and glycerophospholipid biosynthesis.
H2O2Is Required for Optimal Establishment of theMedicago sativa/Sinorhizobium melilotiSymbiosis
Authors: Jamet Alexandre , Mandon Karine , Puppo Alain , Hérouart Didier 
Published: October 2007

ABSTRACTThe symbiotic interaction betweenMedicago sativaandSinorhizobium melilotiRmkatB++overexpressing the housekeeping catalasekatBis delayed, and this delay is combined with an enlargement of infection threads. This result provides evidence that H2O2is required for optimal progression of infection threads through the root hairs and plant cell layers.
Unusual Starch Degradation Pathway via Cyclodextrins in the Hyperthermophilic Sulfate-Reducing Archaeon
Archaeoglobus fulgidus
Strain 7324
Authors: Labes Antje , Schönheit Peter 
Published: October 2007

ABSTRACT

The hyperthermophilic archaeon
Archaeoglobus fulgidus
strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, α-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that
A. fulgidus
utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly β-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from
Thermococcus litoralis
. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.
Structure, Function, and Regulation of the Aldouronate Utilization Gene Cluster from
Paenibacillus
sp. Strain JDR-2
Authors: Chow Virginia , Nong Guang , Preston James F. 
Published: October 2007

ABSTRACT

Direct bacterial conversion of the hemicellulose fraction of hardwoods and crop residues to biobased products depends upon extracellular depolymerization of methylglucuronoxylan (MeGAX
n
), followed by assimilation and intracellular conversion of aldouronates and xylooligosaccharides to fermentable xylose.
Paenibacillus
sp. strain JDR-2, an aggressively xylanolytic bacterium, secretes a multimodular cell-associated GH10 endoxylanase (XynA1) that catalyzes depolymerization of MeGAX
n
and rapidly assimilates the principal products, β-1,4-xylobiose, β-1,4-xylotriose, and MeGAX
3
, the aldotetrauronate 4-
O
-methylglucuronosyl-α-1,2-xylotriose. Genomic libraries derived from this bacterium have now allowed cloning and sequencing of a unique aldouronate utilization gene cluster comprised of genes encoding signal transduction regulatory proteins, ABC transporter proteins, and the enzymes AguA (GH67 α-glucuronidase), XynA2 (GH10 endoxylanase), and XynB (GH43 β-xylosidase/α-arabinofuranosidase). Expression of these genes, as well as
xynA1
encoding the secreted GH10 endoxylanase, is induced by growth on MeGAX
n
and repressed by glucose. Sequences in the
yesN, lplA
, and
xynA2
genes within the cluster and in the distal
xynA1
gene show significant similarity to catabolite responsive element (
cre
) defined in
Bacillus subtilis
for recognition of the catabolite control protein (CcpA) and consequential repression of catabolic regulons. The aldouronate utilization gene cluster in
Paenibacillus
sp. strain JDR-2 operates as a regulon, coregulated with the expression of
xynA1
, conferring the ability for efficient assimilation and catabolism of the aldouronate product generated by a multimodular cell surface-anchored GH10 endoxylanase. This cluster offers a desirable metabolic potential for bacterial conversion of hemicellulose fractions of hardwood and crop residues to biobased products.
TrwC-Mediated Site-Specific Recombination Is Controlled by Host Factors Altering Local DNA Topology
Authors: César Carolina Elvira , Llosa Matxalen 
Published: October 2007

ABSTRACT

R388 conjugative relaxase TrwC acts as a site-specific recombinase, promoting recombination between two cognate
oriT
s on double-stranded DNA substrates. The relaxosome component TrwA is also required for efficient recombination. In this work we present data on the in vivo control of this reaction by host proteins that affect local DNA topology. In the absence of TrwA, binding of integration host factor (IHF) to the
oriT
keeps the recombination levels low, probably by keeping the relaxosome complex, formed at recombination locus 1, in a “closed” conformation. In an IHF-deficient (IHF
−
) background, the formation of a transcript elongation complex at this locus still hampers recombination. A mutation abating the promoter sequence at locus 1, or repression of transcription by exposure to rifampin, lifts the inhibition imposed on recombination in an IHF
−
background. We also observe an increase in conjugation efficiency under these conditions. Relieving the inhibition imposed by these host factors allows efficient levels of recombination between short
oriT
loci in the absence of TrwA. The presence of TrwA counteracts these inhibitory effects. TrwA would then activate both recombination and conjugation by switching the conformation of the relaxosome to an “open” form that exposes single-stranded DNA at the
nic
site, promoting the initial TrwC nicking reaction.
Orotate Phosphoribosyltransferase fromCorynebacterium ammoniagenesLacking a Conserved Lysine
Authors: Wang Xing , Ma Cuiqing , Wang Xiuwen , Xu Ping 
Published: October 2007

ABSTRACTThepyrEgene, encoding orotate phosphoribosyltransferase (OPRTase), was cloned by nested PCR and colony blotting fromCorynebacterium ammoniagenesATCC 6872, which is widely used in nucleotide production. Sequence analysis shows that there is a lack of an important conserved lysine (Lys 73 inSalmonella entericaserovar Typhimurium OPRTase) in theC. ammoniagenesOPRTase. This lysine has been considered to contribute to the initiation of catalysis. The enzyme was overexpressed and purified from a recombinantEscherichia colistrain. The molecular mass of the purified OPRTase was determined to be 45.4 ± 1.5 kDa by gel filtration. Since the molecular mass for the subunit of the enzyme was 21.3 ± 0.6 kDa, the native enzyme exists as a dimer. Divalent magnesium was necessary for the activity of the enzyme and can be substituted for by Mn2+and Co2+. The optimal pH for the forward (phosphoribosyl transfer) reaction is 10.5 to 11.5, which is higher than that of other reported OPRTases, and the optimal pH for the reverse (pyrophosphorolysis) reaction is 5.5 to 6.5. TheKmvalues for the four substrates were determined to be 33 μM for orotate, 64 μM for 5-phosphoribosyl-1-pyrophosphate (PRPP), 45 μM for orotidine-5-phosphate (OMP), and 36 μM for pyrophosphate. TheKmvalue for OMP is much larger than those of other organisms. These differences may be due to the absence of Lys 73, which is present in the active sites of other OPRTases and is known to interact with OMP and PRPP.
Effects of Oxygen on Virulence Traits ofStreptococcus mutans
Authors: Ahn Sang-Joon , Wen Zezhang T. , Burne Robert A. 
Published: October 2007

ABSTRACTOxygen profoundly affects the composition of oral biofilms. Recently, we showed that exposure ofStreptococcus mutansto oxygen strongly inhibits biofilm formation and alters cell surface biogenesis. To begin to dissect the underlying mechanisms by which oxygen affects known virulence traits ofS. mutans, transcription profiling was used to show that roughly 5% of the genes of this organism are differentially expressed in response to aeration. Among the most profoundly upregulated genes were autolysis-related genes and those that encode bacteriocins, the ClpB protease chaperone subunit, pyruvate dehydrogenase, the tricarboxylic acid cycle enzymes, NADH oxidase enzymes, and certain carbohydrate transporters and catabolic pathways. Consistent with our observation that the ability ofS. mutansto form biofilms was severely impaired by oxygen exposure, transcription of thegtfBgene, which encodes one of the primary enzymes involved in the production of water-insoluble, adhesive glucan exopolysaccharides, was down-regulated in cells growing aerobically. Further investigation revealed that transcription ofgtfB, but notgtfC, was responsive to oxygen and that aeration causes major changes in the amount and degree of cell association of the Gtf enzymes. Moreover, inactivation of the VicK sensor kinase affected the expression and localization the GtfB and GtfC enzymes. This study provides novel insights into the complex transcriptional and posttranscriptional regulatory networks used byS. mutansto modulate virulence gene expression and exopolysaccharide production in response to changes in oxygen availability.
TheyydFGHIJOperon ofBacillus subtilisEncodes a Peptide That Induces the LiaRS Two-Component System
Authors: Butcher Bronwyn G. , Lin Yi-Pin , Helmann John D. 
Published: October 2007

ABSTRACTTheBacillus subtilisLiaRS two-component system (TCS) responds to perturbations of the cell envelope induced bylipid II-interactingantibiotics, such as vancomycin, ramoplanin, nisin, and bacitracin. Here, we characterize Tn7-generated mutations that induce theliaRSTCS. In addition to insertions inliaF, a known negative regulator of the LiaRS TCS, we identified two disruptions in the last two genes of theyydFGHIJoperon. This operon is predicted to encode a 49-amino-acid peptide (YydF), a modification enzyme (YydG), a membrane-embedded protease (YydH), and an ATP-binding cassette (ABC) transporter (YydIJ). Genome sequence comparisons suggest that theyydFGHIJoperon may have been acquired by horizontal transfer. Inactivation of the YydIJ transporter resulted in increased expression from the LiaR-dependent PliaIpromoter only in the presence of theyydFGHgenes. Cells harboring the completeyydFGHIJoperon induced LiaR activity in cocultured cells lacking either this transporter or the complete operon. These results suggest that this operon is involved in the synthesis and export of a modified peptide (YydF*) that elicits cell envelope stress sensed by the LiaRS TCS.
Influence of the Poly-3-Hydroxybutyrate (PHB) Granule-Associated Proteins (PhaP1 and PhaP2) on PHB Accumulation and Symbiotic Nitrogen Fixation in
Sinorhizobium meliloti
Rm1021
Authors: Wang Chunxia , Sheng Xiaoyan , Equi Raymie C. , Trainer Maria A. , Charles Trevor C. , Sobral Bruno W. S. 
Published: October 2007

ABSTRACT

Sinorhizobium meliloti
cells store excess carbon as intracellular poly-3-hydroxybutyrate (PHB) granules that assist survival under fluctuating nutritional conditions. PHB granule-associated proteins (phasins) are proposed to regulate PHB synthesis and granule formation. Although the enzymology and genetics of PHB metabolism in
S. meliloti
have been well characterized, phasins have not yet been described for this organism. Comparison of the protein profiles of the wild type and a PHB synthesis mutant revealed two major proteins absent from the mutant. These were identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) as being encoded by the SMc00777 (
phaP1
) and SMc02111 (
phaP2
) genes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins associated with PHB granules followed by MALDI-TOF confirmed that PhaP1 and PhaP2 were the two major phasins. Double mutants were defective in PHB production, while single mutants still produced PHB, and unlike PHB synthesis mutants that have reduced exopolysaccharide, the double mutants had higher exopolysaccharide levels.
Medicago truncatula
plants inoculated with the double mutant exhibited reduced shoot dry weight (SDW), although there was no corresponding reduction in nitrogen fixation activity. Whether the phasins are involved in a metabolic regulatory response or whether the reduced SDW is due to a reduction in assimilation of fixed nitrogen rather than a reduction in nitrogen fixation activity remains to be established.
Substitution of a Highly Conserved Histidine in the
Escherichia coli
Heat Shock Transcription Factor, σ
32
, Affects Promoter Utilization In Vitro and Leads to Overexpression of the Biofilm-Associated Flu Protein In Vivo
Authors: Kourennaia Olga V. , deHaseth Pieter L. 
Published: October 2007

ABSTRACT

The heat shock sigma factor (σ
32
in
Escherichia coli
) directs the bacterial RNA polymerase to promoters of a specific sequence to form a stable complex, competent to initiate transcription of genes whose products mitigate the effects of exposure of the cell to high temperatures. The histidine at position 107 of σ
32
is at the homologous position of a tryptophan residue at position 433 of the main sigma factor of
E. coli
, σ
70
. This tryptophan is essential for the strand separation step leading to the formation of the initiation-competent RNA polymerase-promoter complex. The heat shock sigma factors of all gammaproteobacteria sequenced have a histidine at this position, while in the alpha- and deltaproteobacteria, it is a tryptophan. In vitro the alanine-for-histidine substitution at position 107 (H107A) destabilizes complexes between the GroE promoter and RNA polymerase containing σ
32
, implying that H107 plays a role in formation or maintenance of the strand-separated complex. In vivo, the H107A substitution in σ
32
impedes recovery from heat shock (exposure to 42°C), and it also leads to overexpression at lower temperatures (30°C) of the Flu protein, which is associated with biofilm formation.
Identification and Characterization of the
dps
Promoter of
Mycobacterium smegmatis
: Promoter Recognition by Stress-Specific Extracytoplasmic Function Sigma Factors σ
H
and σ
F
Authors: Chowdhury Rakhi Pait , Gupta Surbhi , Chatterji Dipankar 
Published: October 2007

ABSTRACT

The survival of a bacterium with a depleted oxygen or nutrient supply is important for its long-term persistence inside the host under stressful conditions. We studied a gene,
dps
, from
Mycobacterium smegmatis
, encoding a protein, Dps (for DNA binding protein from starved cells), which is overexpressed under oxidative and nutritional stresses and provides bimodal protection to the bacterial DNA. Characterization of the
dps
promoter in vivo is therefore important. We cloned a 1-kb putative promoter region of the
dps
gene of
M. smegmatis
in an
Escherichia coli
-
Mycobacterium
shuttle vector, pSD5B, immediately upstream of the
lacZ
gene. Promoter activities were assayed in vivo both in solid medium and in liquid cultures by quantitative β-galactosidase activity measurements. To characterize the minimal promoter region, a 200-bp fragment from the whole 1-kb sequence was further cloned in the same vector, and in a similar way, β-galactosidase activity was quantitated. Primer extension analysis was performed to determine the +1 transcription start site of the gene. Point mutations were inserted in the putative promoter sequences in the −10 and −20 regions, and the promoter sequence was confirmed. The promoter was not recognized by purified
M. smegmatis
core RNA polymerase reconstituted with purified
Mycobacterium tuberculosis
σ
A
or σ
B
during multiple- and single-round in vitro transcription assays. Promoter-specific in vivo pull-down assays with an immobilized 1-kb DNA fragment containing the
dps
promoter established that extracellular function sigma factors were associated with this starvation-inducible promoter. Single-round transcription at the
dps
promoter further supported the idea that only core RNA polymerase reconstituted with σ
F
or σ
H
can generate proper transcripts.
Identification of a Fourth Formate Dehydrogenase in
Methylobacterium extorquens
AM1 and Confirmation of the Essential Role of Formate Oxidation in Methylotrophy
Authors: Chistoserdova Ludmila , Crowther Gregory J. , Vorholt Julia A. , Skovran Elizabeth , Portais Jean-Charles , Lidstrom Mary E. 
Published: October 2007

ABSTRACT

A mutant of
Methylobacterium extorquens
AM1 with lesions in genes for three formate dehydrogenase (FDH) enzymes was previously described by us (L. Chistoserdova, M. Laukel, J.-C. Portais, J. A. Vorholt, and M. E. Lidstrom, J. Bacteriol. 186:22-28, 2004). This mutant had lost its ability to grow on formate but still maintained the ability to grow on methanol. In this work, we further investigated the phenotype of this mutant. Nuclear magnetic resonance experiments with [
13
C]formate, as well as
14
C-labeling experiments, demonstrated production of labeled CO
2
in the mutant, pointing to the presence of an additional enzyme or a pathway for formate oxidation. The tungsten-sensitive phenotype of the mutant suggested the involvement of a molybdenum-dependent enzyme. Whole-genome array experiments were conducted to test for genes overexpressed in the triple-FDH mutant compared to the wild type, and a gene (
fdh4A
) was identified whose translated product carried similarity to an uncharacterized putative molybdopterin-binding oxidoreductase-like protein sharing relatively low similarity with known formate dehydrogenase alpha subunits. Mutation of this gene in the triple-FDH mutant background resulted in a methanol-negative phenotype. When the gene was deleted in the wild-type background, the mutant revealed diminished growth on methanol with accumulation of high levels of formate in the medium, pointing to an important role of FDH4 in methanol metabolism. The identity of FDH4 as a novel FDH was also confirmed by labeling experiments that revealed strongly reduced CO
2
formation in growing cultures. Mutation of a small open reading frame (
fdh4B
) downstream of
fdh4A
resulted in mutant phenotypes similar to the phenotypes of
fdh4A
mutants, suggesting that
fdh4B
is also involved in formate oxidation.
Mapping Critical Interactive Sites within the Periplasmic Domain of the
Vibrio cholerae
Type II Secretion Protein EpsM
Authors: Johnson Tanya L. , Scott Maria E. , Sandkvist Maria 
Published: October 2007

ABSTRACT

The type II secretion (T2S) system is present in many gram-negative species, both pathogenic and nonpathogenic, where it supports the delivery of a variety of toxins, proteases, and lipases into the extracellular environment. In
Vibrio cholerae
, the T2S apparatus is composed of 12 Eps proteins that assemble into a multiprotein complex that spans the entire cell envelope. Two of these proteins, EpsM and EpsL, are key components of the secretion machinery present in the inner membrane. In addition to likely forming homodimers, EpsL and EpsM have been shown to form a stable complex in the inner membrane and to protect each other from proteolytic degradation. To identify and map the specific regions of EpsM involved in protein-protein interactions with both another molecule of EpsM and EpsL, we tested the interactions of deletion constructs of EpsM with full-length EpsM and EpsL by functional characterization and copurification as well as coimmunoprecipitation. Analysis of the truncated EpsM mutants revealed that the region of EpsM from amino acids 100 to 135 is necessary for EpsM to form homo-oligomers, while residues 84 to 99 appear to be critical for a stable interaction with EpsL.
Expression of the σF-DirectedcsfBLocus Prevents Premature Appearance of σGActivity during Sporulation ofBacillus subtilis
Authors: Chary Vasant K. , Xenopoulos Panagiotis , Piggot Patrick J. 
Published: October 2007

ABSTRACTDuring sporulation, σGbecomes active in the prespore upon the completion of engulfment. We show that the inactivation of the σF-directedcsfBlocus resulted in premature activation of σG. CsfB exerted control distinct from but overlapping with that exerted by LonA to prevent inappropriate σGactivation. The artificial induction ofcsfBseverely compromised spore formation.