bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of the Gene Encoding Sulfopyruvate Decarboxylase, an Enzyme Involved in Biosynthesis of Coenzyme M
Authors: Graupner Marion , Xu Huimin , White Robert H. 
Published: July 2002

ABSTRACT

The products of two adjacent genes in the chromosome of
Methanococcus jannaschii
are similar to the amino and carboxyl halves of phosphonopyruvate decarboxylase, the enzyme that catalyzes the second step of fosfomycin biosynthesis in
Streptomyces wedmorensis
. These two
M. jannaschii
genes were recombinantly expressed in
Escherichia coli
, and their gene products were tested for the ability to catalyze the decarboxylation of a series of α-ketoacids. Both subunits are required to form an α
6
β
6
dodecamer that specifically catalyzes the decarboxylation of sulfopyruvic acid to sulfoacetaldehyde. This transformation is the fourth step in the biosynthesis of coenzyme M, a crucial cofactor in methanogenesis and aliphatic alkene metabolism. The
M. jannaschii
sulfopyruvate decarboxylase was found to be inactivated by oxygen and reactivated by reduction with dithionite. The two subunits, designated ComD and ComE, comprise the first enzyme for the biosynthesis of coenzyme M to be described.
Swarming of
Pseudomonas aeruginosa
Is Dependent on Cell-to-Cell Signaling and Requires Flagella and Pili
Authors: Köhler Thilo , Curty Lasta Kocjancic , Barja Francisco , van Delden Christian , Pechère Jean-Claude 
Published: July 2002

ABSTRACT

We describe swarming in
Pseudomonas aeruginosa
as a third mode of surface translocation in addition to the previously described swimming and twitching motilities. Swarming in
P. aeruginosa
is induced on semisolid surfaces (0.5 to 0.7% agar) under conditions of nitrogen limitation and in response to certain amino acids. Glutamate, aspartate, histidine, or proline, when provided as the sole source of nitrogen, induced swarming, while arginine, asparagine, and glutamine, among other amino acids, did not sustain swarming. Cells from the edge of the swarm were about twice as long as cells from the swarm center. In both instances, bacteria possessing two polar flagella were observed by light and electron microscopy. While a
fliC
mutant of
P. aeruginosa
displayed slightly diminished swarming, a
pilR
and a
pilA
mutant, both deficient in type IV pili, were unable to swarm. Furthermore, cells with mutations in the
las
cell-to-cell signaling system showed diminished swarming behavior, while
rhl
mutants were completely unable to swarm. Evidence is presented for rhamnolipids being the actual surfactant involved in swarming motility, which explains the involvement of the cell-to-cell signaling circuitry of
P. aeruginosa
in this type of surface motility.
Identification of an Operon Required for Ferrichrome Iron Utilization in
Vibrio cholerae
Authors: Rogers Marc B. , Sexton Jessica A. , DeCastro G. Joel , Calderwood Stephen B. 
Published: July 2002

ABSTRACT

Mutagenesis of
Vibrio cholerae
with Tn
phoA
, followed by screening for fusions that were activated under low-iron conditions, led to the identification of seven independent fusion strains, each of which was deficient in the ability to utilize ferrichrome as a sole iron source for growth in a plate bioassay and had an insertion in genes encoding products homologous to
Escherichia coli
FhuA or FhuD. Expression of the gene fusions was independent of IrgB but regulated by Fur. We report here a map of the operon and the predicted amino acid sequence of FhuA, based on the nucleotide sequence. Unlike those of the
E. coli fhu
operon, the
V. cholerae
ferrichrome utilization genes are located adjacent and opposite in orientation to a gene encoding an ATP-binding cassette transporter homolog, but this gene, if disrupted, does not affect the utilization of ferrichrome in vitro.
Biofilm Formation by the Fungal Pathogen
Candida albicans
: Development, Architecture, and Drug Resistance
Authors: Chandra Jyotsna , Kuhn Duncan M. , Mukherjee Pranab K. , Hoyer Lois L. , McCormick Thomas , Ghannoum Mahmoud A. 
Published: July 2002

ABSTRACT

Biofilms are a protected niche for microorganisms, where they are safe from antibiotic treatment and can create a source of persistent infection. Using two clinically relevant
Candida albicans
biofilm models formed on bioprosthetic materials, we demonstrated that biofilm formation proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in a polysaccharide matrix. Fluorescence and confocal scanning laser microscopy revealed that
C. albicans
biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements. In both models, antifungal resistance of biofilm-grown cells increased in conjunction with biofilm formation. The expression of agglutinin-like (
ALS
) genes, which encode a family of proteins implicated in adhesion to host surfaces, was differentially regulated between planktonic and biofilm-grown cells. The ability of
C. albicans
to form biofilms contrasts sharply with that of
Saccharomyces cerevisiae
, which adhered to bioprosthetic surfaces but failed to form a mature biofilm. The studies described here form the basis for investigations into the molecular mechanisms of
Candida
biofilm biology and antifungal resistance and provide the means to design novel therapies for biofilm-based infections.
One of Two
hemN
Genes in
Bradyrhizobium japonicum
Is Functional during Anaerobic Growth and in Symbiosis
Authors: Fischer Hans-Martin , Velasco Leonardo , Delgado Maria J. , Bedmar Eulogio J. , Schären Simon , Zingg Daniel , Göttfert Michael , Hennecke Hauke 
Published: July 2002

ABSTRACT

Previously, we screened the symbiotic gene region of the
Bradyrhizobium japonicum
chromosome for new NifA-dependent genes by competitive DNA-RNA hybridization (A. Nienaber, A. Huber, M. Göttfert, H. Hennecke, and H. M. Fischer, J. Bacteriol. 182:1472–1480, 2000). Here we report more details on one of the genes identified, a
hemN
-like gene (now called
hemN
1
) whose product exhibits significant similarity to oxygen-independent coproporphyrinogen III dehydrogenases involved in heme biosynthesis in facultatively anaerobic bacteria. In the course of these studies, we discovered that
B. japonicum
possesses a second
hemN
-like gene (
hemN
2
), which was then cloned by using
hemN
1
as a probe. The
hemN
2
gene maps outside of the symbiotic gene region; it is located 1.5 kb upstream of
nirK
, the gene for a Cu-containing nitrite reductase. The two deduced HemN proteins are similar in size (445 and 450 amino acids for HemN
1
and HemN
2
, respectively) and share 53% identical (68% similar) amino acids. Expression of both
hemN
genes was monitored with the help of chromosomally integrated translational
lacZ
fusions. No significant expression of either gene was detected in aerobically grown cells, whereas both genes were strongly induced (≥20-fold) under microaerobic or anaerobic conditions. Induction was in both cases dependent on the transcriptional activator protein FixK
2
. In addition, maximal anaerobic
hemN
1
expression was partially dependent on NifA, which explains why this gene had been identified by the competitive DNA-RNA hybridization approach. Strains were constructed carrying null mutations either in individual
hemN
genes or simultaneously in both genes. All mutants showed normal growth in rich medium under aerobic conditions. Unlike the
hemN
1
mutant, strains lacking a functional
hemN
2
gene were unable to grow anaerobically under nitrate-respiring conditions and largely failed to fix nitrogen in symbiosis with the soybean host plant. Moreover, these mutants lacked several
c
-type cytochromes which are normally detectable by heme staining of proteins from anaerobically grown wild-type cells. Taken together, our results revealed that
B. japonicum hemN
2
, but not
hemN
1
, encodes a protein that is functional under the conditions tested, and this conclusion was further corroborated by the successful complementation of a
Salmonella enterica
serovar Typhimurium
hemF hemN
mutant with
hemN
2
only.
Ribonuclease M5 Has Few, If Any, mRNA Substrates inBacillus subtilis
Authors: Condon Ciarán , Rourera Jordi , Brechemier-Baey Dominique , Putzer Harald 
Published: July 2002

ABSTRACTInBacillus subtilis, maturation of 5S rRNA is catalyzed by an enzyme called RNase M5. We searched for potential mRNA substrates for RNase M5 by gene array technology, based on the premise that most endonucleolytic cleavages have an effect on the stability of RNA and hence on steady-state levels of expression. Only a handful of genes had significantly altered expression inrnmVmutants compared to wild-type strains that could subsequently be confirmed by Northern blotting. The effect of RNase M5 on the expression of the best candidates, theodhABandsucCDoperons, is indirect, by a mechanism we do not yet understand. We show that an effect of RNase M5 on the expression of the remaining candidate,ctsR, is due to the failure to process the 5S rRNA contained in therrnWlying directly upstream. We thus conclude that RNase M5 has very few or possibly no mRNA substrates inB. subtilis.
Regulation ofSalmonella entericaSerovar TyphimuriummntHTranscription by H2O2, Fe2+, and Mn2+
Authors: Kehres David G. , Janakiraman Anu , Slauch James M. , Maguire Michael E. 
Published: July 2002

ABSTRACTMntH, a bacterial homolog of mammalian natural resistance associated macrophage protein 1 (Nramp1), is a primary transporter for Mn2+influx inSalmonellaentericaserovar Typhimurium andEscherichia coli. S. entericaserovar Typhimurium MntH contributes to H2O2resistance and is important for full virulence. Consistent with its phenotype and function,mntHis regulated at the transcriptional level by both H2O2and substrate cation. We have now identified threetrans-acting regulatory factors and the three correspondingcis-actingmntHpromoter motifs that mediate this regulation. In the presence of hydrogen peroxide,mntHis activated by OxyR, acting through an OxyR-binding motif centered just upstream of the likely −35 RNA polymerase-binding site. In the presence of Fe2+,mntHis repressed primarily by Fur, acting through a Fur-binding motif overlapping the −35 region. In the presence of Mn2+,mntHis repressed primarily by theSalmonellaequivalent ofE. colib0817, a distant homolog of theBacillus subtilismanganese transport repressor, MntR, acting through an inverted-repeat motif located between the likely −10 polymerase binding site and the ribosome binding site.E. colib0817 was recently shown to bind the identical inverted-repeat motif in theE. coli mntHpromoter and hence has been renamed MntR (S. I. Patzer and K. Hantke, J. Bacteriol.183:4806-4813, 2001). Using Δfur, ΔmntR, and ΔfurΔmntRmutant strains as well as mutations in the Fur- and MntR-binding motif elements, we found that Fe2+can also mediate repression through the Mn2+repressor MntR.
mig
-
14
Is a
Salmonella
Gene That Plays a Role in Bacterial Resistance to Antimicrobial Peptides
Authors: Brodsky Igor E. , Ernst Robert K. , Miller Samuel I. , Falkow Stanley 
Published: July 2002

ABSTRACT

It was previously demonstrated that the
mig
-
14
gene of
Salmonella enterica
serovar Typhimurium is necessary for bacterial proliferation in the liver and spleen of mice following intragastric inoculation and that
mig
-
14
expression, which is induced within macrophages, is under the control of the global regulator PhoP. Here we demonstrate that the
mig
-
14
promoter is induced by growth in minimal medium containing low magnesium or acidic pH, consistent with regulation by PhoP. In addition,
mig
-
14
is strongly induced by polymyxin B, protamine, and the mammalian antimicrobial peptide protegrin-1. While
phoP
is necessary for the induction of
mig
-
14
in response to protamine and protegrin,
mig
-
14
is still induced by polymyxin B in a
phoP
background. We also demonstrate that
mig
-
14
is necessary for resistance of
S. enterica
serovar Typhimurium to both polymyxin B and protegrin-1. Gram-negative resistance to a variety of antimicrobial peptides has been correlated with modifications of lipopolysaccharide structure. However, we show that
mig
-
14
is not required for one of these modifications, the addition of 4-aminoarabinose to lipid A. Additionally, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of wild-type and
mig
-
14
lipopolysaccharide also shows no detectable differences between the two strains. Therefore,
mig
-
14
contributes to
Salmonella
resistance to antimicrobial peptides by a mechanism that is not yet fully understood.
Variable Surface Protein Vmm ofMycoplasma mycoidessubsp.mycoidesSmall Colony Type
Authors: Persson Anja , Jacobsson Karin , Frykberg Lars , Johansson Karl-Erik , Poumarat François 
Published: July 2002

ABSTRACTA variable surface protein, Vmm, of the bovine pathogenMycoplasma mycoidessubsp.mycoidessmall colony type (M. mycoidesSC) has been identified and characterized. Vmm was specific for the SC biotype and was expressed by 68 of 69 analyzedM. mycoidesSC strains. The protein was found to undergo reversible phase variation at a frequency of 9 × 10−4to 5 × 10−5per cell per generation. Thevmmgene was present in all of the 69 testedM. mycoidesSC strains and encodes a lipoprotein precursor of 59 amino acids (aa), where the mature protein was predicted to be 36 aa and was anchored to the membrane by only the lipid moiety, as no transmembrane region could be identified. DNA sequencing of thevmmgene region from ON and OFF clones showed that the expression of Vmm was regulated at the transcriptional level by dinucleotide insertions or deletions in a repetitive region of the promoter spacer.Vmm-like genes were also found in four closely related mycoplasmas,Mycoplasma capricolumsubsp.capricolum,M. capricolumsubsp. capripneumoniae,Mycoplasmasp. bovine serogroup 7, andMycoplasma putrefaciens. However, Vmm could not be detected in whole-cell lysates of these species, suggesting that the proteins encoded by thevmm-like genes lack the binding epitope for the monoclonal antibody used in this study or, alternatively, that the Vmm-like proteins were not expressed.
Partitioning of Chromosomal DNA during Establishment of Cellular Asymmetry inBacillus subtilis
Authors: Pogliano Joe , Sharp Marc D. , Pogliano Kit 
Published: July 2002

ABSTRACTThe switch from symmetric to asymmetric cell division is a key feature of development in many organisms, includingBacillus subtilissporulation. Here we demonstrate that, prior to the onset of asymmetric cell division, theB. subtilischromosome is partitioned into two unequally sized domains, with the origin-proximal one-third of the future forespore chromosome condensed near one pole of the cell. Asymmetric chromosome partitioning is independent of polar division, as it occurs in cells depleted of FtsZ but depends on two transcription factors that govern the initiation of sporulation, σHand Spo0A-P. It is also independent of chromosome partitioning proteins Spo0J and Soj, suggesting the existence of a novel mechanism controlling chromosome structure. Thus, our results demonstrate that, during sporulation, two separable events prepareB. subtilisfor asymmetric cell division: the relocation of cell division sites to the cell poles and the asymmetric partitioning of the future forespore chromosome.
Quantification of Human Cytomegalovirus DNA by Real-Time PCR
Authors: Gault Elyanne , Michel Yanne , Dehée Axelle , Belabani Chahrazed , Nicolas Jean-Claude , Garbarg-Chenon Antoine 
Published: July 2002

ABSTRACT

A quantitative real-time PCR assay was developed to measure human cytomegalovirus (HCMV) DNA load in peripheral blood leukocytes (PBLs). The HCMV DNA load in PBLs was normalized by means of the quantification of a cellular gene (albumin). The results of the real-time PCR assay correlated with those of the HCMV pp65-antigenemia assay (
P
< 0.0001).
Simultaneous Detection of
Neisseria meningitidis, Haemophilus influenzae,
and
Streptococcus pneumoniae
in Suspected Cases of Meningitis and Septicemia Using Real-Time PCR
Authors: Corless C. E. , Guiver M. , Borrow R. , Edwards-Jones V. , Fox A. J. , Kaczmarski E. B. 
Published: July 2002

ABSTRACT

A single-tube 5′ nuclease multiplex PCR assay was developed on the ABI 7700 Sequence Detection System (TaqMan) for the detection of
Neisseria meningitidis, Haemophilus influenzae,
and
Streptococcus pneumoniae
from clinical samples of cerebrospinal fluid (CSF), plasma, serum, and whole blood. Capsular transport (
ctrA
)
,
capsulation (
bexA
), and pneumolysin (
ply
) gene targets specific for
N. meningitidis, H. influenzae,
and
S. pneumoniae,
respectively, were selected. Using sequence-specific fluorescent-dye-labeled probes and continuous real-time monitoring, accumulation of amplified product was measured. Sensitivity was assessed using clinical samples (CSF, serum, plasma, and whole blood) from culture-confirmed cases for the three organisms. The respective sensitivities (as percentages) for
N. meningitidis, H. influenzae,
and
S. pneumoniae
were 88.4, 100, and 91.8. The primer sets were 100% specific for the selected culture isolates. The
ctrA
primers amplified meningococcal serogroups A, B, C, 29E, W135, X, Y, and Z; the
ply
primers amplified pneumococcal serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10A, 11A, 12, 14, 15B, 17F, 18C, 19, 20, 22, 23, 24, 31, and 33; and the
bexA
primers amplified
H. influenzae
types b and c. Coamplification of two target genes without a loss of sensitivity was demonstrated. The multiplex assay was then used to test a large number (
n
= 4,113) of culture-negative samples for the three pathogens. Cases of meningococcal,
H. influenzae,
and pneumococcal disease that had not previously been confirmed by culture were identified with this assay. The
ctrA
primer set used in the multiplex PCR was found to be more sensitive (
P
< 0.0001) than the
ctrA
primers that had been used for meningococcal PCR testing at that time.
Antigenic Heterogeneity of the Hepatitis C Virus NS5A Protein
Authors: Dou Xiao-Guang , Talekar Ganesh , Chang Joy , Dai Xing , Li Lixia , Bonafonte Maria-Teresa , Holloway Brian , Fields Howard A. , Khudyakov Yury E. 
Published: July 2002

ABSTRACTThe effect of sequence variability between different types of hepatitis C virus (HCV) on the antigenic properties of the NS5 protein was studied by using recombinant proteins. A strong antigenic region was identified within the HCV NS5A protein at amino acids 2212 to 2313. Forty-five unique sequences encompassing this region were selected from GenBank and were compared to each other. The results of this analysis showed that the primary structure of this strong antigenic region is highly variable. Percent homology between different genotype sequences varied from 40.4 to 72.5%. Thirteen representative sequences from all six HCV genotypes were selected to design synthetic genes coding for this antigenic region. These genes were assembled by PCR from synthetic oligonucleotides and expressed inEscherichia colias hybrid proteins with glutathioneS-transferase. All 13 fusion proteins were purified from bacterial lysates and used to test a panel of anti-HCV positive sera (n= 91) obtained from patients infected with HCV genotypes 1 through 6. All but two proteins immunoreacted with 62 to 93% of HCV anti-NS5-positive serum samples. Although a variable degree of genotype-specific antigenic reactivity was detected, only one protein demonstrated a noticeable preference to immunoreact with antibodies against the homologous HCV genotype. On the other hand, closely related proteins derived from the same subtype or genotype immunoreacted with significantly different efficiency with HCV antibodies. Thus, sequence variability has a profound effect on the antigenic properties of the NS5A immunodominant regions. This observation should be taken into consideration in the development of diagnostic tests for the efficient detection of anti-HCV activity in serum specimens.
A Cell-Line-Specific Defect in the Intracellular Transport and Release of Assembled Retroviral Capsids
Authors: Parker Scott D. , Hunter Eric 
Published: July 2002

ABSTRACT
Retrovirus assembly involves a complex series of events in which a large number of proteins must be targeted to a point on the plasma membrane where immature viruses bud from the cell. Gag polyproteins of most retroviruses assemble an immature capsid on the cytoplasmic side of the plasma membrane during the budding process (C-type assembly), but a few assemble immature capsids deep in the cytoplasm and are then transported to the plasma membrane (B- or D-type assembly), where they are enveloped. With both assembly phenotypes, Gag polyproteins must be transported to the site of viral budding in either a relatively unassembled form (C type) or a completely assembled form (B and D types). The molecular nature of this transport process and the host cell factors that are involved have remained obscure. During the development of a recombinant baculovirus/insect cell system for the expression of both C-type and D-type Gag polyproteins, we discovered an insect cell line (High Five) with two distinct defects that resulted in the reduced release of virus-like particles. The first of these was a pronounced defect in the transport of D-type but not C-type Gag polyproteins to the plasma membrane. High Five cells expressing wild-type Mason-Pfizer monkey virus (M-PMV) Gag precursors accumulate assembled immature capsids in large cytoplasmic aggregates similar to a transport-defective mutant (MA-A18V). In contrast, a larger fraction of the Gag molecules encoded by the M-PMV C-type morphogenesis mutant (MA-R55W) and those of human immunodeficiency virus were transported to the plasma membrane for assembly and budding of virions. When pulse-labeled Gag precursors from High Five cells were fractionated on velocity gradients, they sedimented more rapidly, indicating that they are sequestered in a higher-molecular-mass complex. Compared to Sf9 insect cells, the High Five cells also demonstrate a defect in the release of C-type virus particles. These findings support the hypothesis that host cell factors are important in the process of Gag transport and in the release of enveloped viral particles.
Three Spliced mRNAs of TT Virus Transcribed from a Plasmid Containing the Entire Genome in COS1 Cells
Authors: Kamahora Toshio , Hino Shigeo , Miyata Hironori 
Published: July 2002

ABSTRACT

A permuted whole-genome construct of a TT virus (TTV), named VT416, had 3,852 nucleotides (nt) 98.2% similar to the prototype TA278 genome. To allow the transcription of TTV from the internal promoter, pBK*VT416(1.3G), carrying 1.3 units of VT416, was constructed. The poly(A)
+
RNAs expressed in COS1 cells 48 h posttransfection contained three TTV mRNA species 3.0, 1.2, and 1.0 kb in length, which were recovered in the 13 DNA clones from a λ phage cDNA library. These mRNAs in the antigenomic orientation possessed in common the 3′ terminus downstream of a poly(A) signal (A
3073
ATAAA) and the 5′ terminus downstream of a cap site (C
98
ACTTC). A common splicing to join nt 185 with nt 277 was detected in all mRNAs. The coding region of the largest open reading frame (ORF) was maintained in 3.0-kb mRNA, because this splicing was located upstream of its initiation codon (A
589
TG). The second splicing was detected in 1.2-kb mRNA to join nt 711 with nt 2374 and in 1.0-kb mRNA to bind nt 711 to nt 2567. They linked a proposed ORF2 to another ORF for creating new ORFs over nt 2374 to 2872 in frame 2 and nt 2567 to 3074 in frame 3. The donor and acceptor sites of all three splicings matched the consensus sequence and were conserved in most of the 16 TTVs of distinct genotypes retrieved from the database. The observed transcription profile is unique to TTV among known members in the family
Circoviridae
.