bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reinvestigation of a New Type of Aerobic Benzoate Metabolism in the Proteobacterium
Azoarcus evansii
Authors: Mohamed Magdy El-Said , Zaar Annette , Ebenau-Jehle Christa , Fuchs Georg 
Published: July 2002

ABSTRACT

The aerobic metabolism of benzoate in the proteobacterium
Azoarcus evansii
was reinvestigated. The known pathways leading to catechol or protocatechuate do not operate in this bacterium. The presumed degradation via 3-hydroxybenzoyl-coenzyme A (CoA) and gentisate could not be confirmed. The first committed step is the activation of benzoate to benzoyl-CoA by a specifically induced benzoate-CoA ligase (AMP forming). This enzyme was purified and shown to differ from an isoenzyme catalyzing the same reaction under anaerobic conditions. The second step postulated involves the hydroxylation of benzoyl-CoA to a so far unknown product by a novel benzoyl-CoA oxygenase, presumably a multicomponent enzyme system. An iron-sulfur flavoprotein, which may be a component of this system, was purified and characterized. The homodimeric enzyme had a native molecular mass of 98 kDa as determined by gel filtration and contained 0.72 mol flavin adenine dinucleotide (FAD), 10.4 to 18.4 mol of Fe, and 13.3 to 17.9 mol of acid-labile sulfur per mol of native protein, depending on the method of protein determination. This benzoate-induced enzyme catalyzed a benzoyl-CoA-, FAD-, and O
2
-dependent NADPH oxidation surprisingly without hydroxylation of the aromatic ring; however, H
2
O
2
was formed. The gene (
boxA
, for benzoate oxidation) coding for this protein was cloned and sequenced. It coded for a protein of 46 kDa with two amino acid consensus sequences for two [4Fe-4S] centers at the N terminus. The deduced amino acid sequence showed homology with subunits of ferredoxin-NADP reductase, nitric oxide synthase, NADPH-cytochrome P450 reductase, and phenol hydroxylase. Upstream of the
boxA
gene, another gene,
boxB
, encoding a protein of 55 kDa was found. The
boxB
gene exhibited homology to open reading frames in various other bacteria which code for components of a putative aerobic phenylacetyl-CoA oxidizing system. The
boxB
gene product was one of at least five proteins induced when
A. evansii
was grown on benzoate.
Snapshot of the Genome of the Pseudo-T-Even Bacteriophage RB49
Authors: Desplats Carine , Dez Christophe , Tétart Françoise , Eleaume Heïdy , Krisch H. M. 
Published: July 2002

ABSTRACT

RB49 is a virulent bacteriophage that infects
Escherichia coli
. Its virion morphology is indistinguishable from the well-known T-even phage T4, but DNA hybridization indicated that it was phylogenetically distant from T4 and thus it was classified as a pseudo-T-even phage. To further characterize RB49, we randomly sequenced small fragments corresponding to about 20% of the ≈170-kb genome. Most of these nucleotide sequences lacked sufficient homology to T4 to be detected in an NCBI BlastN analysis. However, when translated, about 70% of them encoded proteins with homology to T4 proteins. Among these sequences were the numerous components of the virion and the phage DNA replication apparatus. Mapping the RB49 genes revealed that many of them had the same relative order found in the T4 genome. The complete nucleotide sequence was determined for the two regions of RB49 genome that contain most of the genes involved in DNA replication. This sequencing revealed that RB49 has homologues of all the essential T4 replication genes, but, as expected, their sequences diverged considerably from their T4 homologues. Many of the nonessential T4 genes are absent from RB49 and have been replaced by unknown sequences. The intergenic sequences of RB49 are less conserved than the coding sequences, and in at least some cases, RB49 has evolved alternative regulatory strategies. For example, an analysis of transcription in RB49 revealed a simpler pattern of regulation than in T4, with only two, rather than three, classes of temporally controlled promoters. These results indicate that RB49 and T4 have diverged substantially from their last common ancestor. The different T4-type phages appear to contain a set of common genes that can be exploited differently, by means of plasticity in the regulatory sequences and the precise choice of a large group of facultative genes.
Expression of Mycobacteriophage Ms6 Lysis Genes Is Driven by Two σ70-Like Promoters and Is Dependent on a Transcription Termination Signal Present in the Leader RNA
Authors: Garcia Miguel , Pimentel Madalena , Moniz-Pereira José 
Published: July 2002

ABSTRACTA mycobacteriophage Ms6 strong promoter region (Plys) was isolated by using transcriptional fusions with thelacZreporter gene. Two tandem σ70-like promoter sequences (P1 and P2) were found in this region. DNA sequencing of the promoter downstream region revealed a 214-bp leader sequence followed by five adjacent coding regions of 231 bp (ORF1), 1,152 bp (ORF2), 996 bp (ORF3), 231 bp (ORF4), and 372 (ORF5). ORF1 has the potential to encode a 77-amino-acid protein which revealed similarity to mycobacteriophage TM4 gp90, a predicted protein with unknown function. ORF2 encodes a 384-amino-acid protein which is related to several bacteriophage amidases. This protein induced cell lysis upon addition of chloroform, confirming its mureinolytic activity. ORF3 encodes a 332-amino-acid protein which is related to TM4 gp30, a protein with sequence similarity to amidases. ORF4 encodes a 77-amino-acid holin-like protein with significant similarity to the holin ofLactococcus lactisr1t bacteriophage. ORF5 encodes a 124-amino-acid protein which is related to mycobacteriophage L5 gp30, a protein with unknown function. These data indicate that the promoter regionPlysdrives the transcription of the Ms6 lysis genes. An intrinsic transcription termination signal was identified in the leader sequence. Experiments usinglacZfusions showed that β-galactosidase synthesis is inhibited when this transcription termination signal is present in the leader sequence. In conclusion, mycobacteriophage Ms6 cell lysis genes are expressed by their own promoter region, independently of virion structure and assembly protein genes. Moreover, an antitermination mechanism might be involved in their transcription regulation.
CcaR Is an Autoregulatory Protein That Binds to the
ccaR
and
cefD-cmcI
Promoters of the Cephamycin C-Clavulanic Acid Cluster in
Streptomyces clavuligerus
Authors: Santamarta Irene , Rodríguez-García Antonio , Pérez-Redondo Rosario , Martín Juan F. , Liras Paloma 
Published: July 2002

ABSTRACT

The putative regulatory CcaR protein, which is encoded in the β-lactam supercluster of
Streptomyces clavuligerus
, has been partially purified by ammonium sulfate precipitation and heparin affinity chromatography. In addition, it was expressed in
Escherichia coli
, purified as a His-tagged recombinant protein (rCcaR), and used to raise anti-rCcaR antibodies. The partially purified CcaR protein from
S. clavuligerus
was able to bind DNA fragments containing the promoter regions of the
ccaR
gene itself and the bidirectional
cefD-cmcI
promoter region. In contrast, CcaR did not bind to DNA fragments with the promoter regions of other genes of the cephamycin-clavulanic acid supercluster including
lat
,
blp
,
claR
,
car-cyp
, and the unlinked
argR
gene. The DNA shifts obtained with CcaR were prevented by anti-rCcaR immunoglobulin G (IgG) antibodies but not by anti-rabbit IgG antibodies.
ccaR
and the bidirectional
cefD-cmcI
promoter region were fused to the
xylE
reporter gene and expressed in
Streptomyces lividans
and
S. clavuligerus
. These constructs produced low catechol dioxygenase activity in the absence of CcaR; activity was increased 1.7- to 4.6-fold in cultures expressing CcaR. Amplification of the
ccaR
promoter region lacking its coding sequence in a high-copy-number plasmid in
S. clavuligerus
ATCC 27064 resulted in a reduced production of cephamycin C and clavulanic acid, by 12 to 20% and 40 to 60%, respectively, due to titration of the CcaR regulator. These findings confirm that CcaR is a positively acting autoregulatory protein able to bind to its own promoter as well as to the
cefD-cmcI
bidirectional promoter region.
Chloromethane-Induced Genes Define a Third C
1
Utilization Pathway in
Methylobacterium chloromethanicum
CM4
Authors: Studer Alex , McAnulla Craig , Büchele Rainer , Leisinger Thomas , Vuilleumier Stéphane 
Published: July 2002

ABSTRACT

Methylobacterium chloromethanicum
CM4 is an aerobic α-proteobacterium capable of growth with chloromethane as the sole carbon and energy source. Two proteins, CmuA and CmuB, were previously purified and shown to catalyze the dehalogenation of chloromethane and the vitamin B
12
-mediated transfer of the methyl group of chloromethane to tetrahydrofolate. Three genes located near
cmuA
and
cmuB
, designated
metF
,
folD
and
purU
and encoding homologs of methylene tetrahydrofolate (methylene-H
4
folate) reductase, methylene-H
4
folate dehydrogenase-methenyl-H
4
folate cyclohydrolase and formyl-H
4
folate hydrolase, respectively, suggested the existence of a chloromethane-specific oxidation pathway from methyl-tetrahydrofolate to formate in strain CM4. Hybridization and PCR analysis indicated that these genes were absent in
Methylobacterium extorquens
AM1, which is unable to grow with chloromethane. Studies with transcriptional
xylE
fusions demonstrated the chloromethane-dependent expression of these genes. Transcriptional start sites were mapped by primer extension and allowed to define three transcriptional units, each likely comprising several genes, that were specifically expressed during growth of strain CM4 with chloromethane. The DNA sequences of the deduced promoters display a high degree of sequence conservation but differ from the
Methylobacterium
promoters described thus far. As shown previously for
purU
, inactivation of the
metF
gene resulted in a CM4 mutant unable to grow with chloromethane. Methylene-H
4
folate reductase activity was detected in a cell extract of strain CM4 only in the presence of chloromethane but not in the
metF
mutant. Taken together, these data provide evidence that
M. chloromethanicum
CM4 requires a specific set of tetrahydrofolate-dependent enzymes for growth with chloromethane.
Transcriptional Activation of the
Rhodobacter sphaeroides
Cytochrome
c
2
Gene P2 Promoter by the Response Regulator PrrA
Authors: Comolli James C. , Carl Audrey J. , Hall Christine , Donohue Timothy 
Published: July 2002

ABSTRACT

Anoxygenic photosynthetic growth of
Rhodobacter sphaeroides
, a member of the α subclass of the class
Proteobacteria
, requires the response regulator PrrA. PrrA and the sensor kinase PrrB are part of a two-component signaling pathway that influences a wide range of processes under oxygen-limited conditions. In this work we characterized the pathway of transcription activation by PrrB and PrrA by purifying these proteins, analyzing them in vitro, and characterizing a mutant PrrA protein in vivo and in vitro. When purified, a soluble transmitter domain of PrrB (cPrrB) could autophosphorylate, rapidly transfer phosphate to PrrA, and stimulate dephosphorylation of phospho-PrrA. Unphosphorylated PrrA activated transcription from a target cytochrome
c
2
gene (
cycA
) promoter, P2, which contained sequences from −73 to +22 relative to the transcription initiation site. However, phosphorylation of PrrA increased its activity since activation of
cycA
P2 was enhanced up to 15-fold by treatment with the low-molecular-weight phosphodonor acetyl phosphate. A mutant PrrA protein containing a single amino acid substitution in the presumed phosphoacceptor site (PrrA-D63A) was not phosphorylated in vitro but also was not able to stimulate
cycA
P2 transcription. PrrA-D63A also had no apparent in vivo activity, demonstrating that aspartate 63 is necessary both for the function of PrrA and for its phosphorylation-dependent activation. The cellular level of wild-type PrrA was negatively autoregulated so that less PrrA was present in the absence of oxygen, conditions in which the activities of many PrrA target genes increase. PrrA-D63A failed to repress expression of the
prrA
gene under anaerobic conditions, suggesting that this single amino acid change also eliminated PrrA function in vivo.
Early Detection of Acute Rhinovirus Infections by a Rapid Reverse Transcription-PCR Assay
Authors: Steininger Christoph , Aberle Stephan W. , Popow-Kraupp Theresia 
Published: July 2002

ABSTRACT
The development of a rhinovirus (RV)-RNA-specific reverse transcription (RT)-PCR assay is complicated by the close homology between the RV and enterovirus (EV) genomes in the highly conserved 5′-noncoding region, which is chosen for primer design in most RT-PCR assays. We have developed a sensitive, rapid, and RV-specific nested RT-PCR assay and have used it to test nasopharyngeal aspirates from 556 patients presenting with acute respiratory tract infections. RV RNA was detected by nested RT-PCR not only in all of 52 samples that were RV positive by virus isolation methods but also in 124 of 367 samples that were negative by virus isolation methods and enzyme-linked immunosorbent assay (ELISA). In addition, in 23 of 137 samples that were positive for a different respiratory virus by virus isolation and/or ELISA, RV RNA was detected by RT-PCR. EVs, adenoviruses, respiratory syncytial viruses, coronaviruses, and influenza and parainfluenza viruses, including clinical isolates as well as stock viruses, were not amplified in our RV-specific RT-PCR assay, indicating that this assay was highly specific. The processing time was less than 2 days for the RT-PCR, as opposed to up to 2 weeks for virus isolation. These results indicate that nested RT-PCR is more sensitive than conventional methods for the detection of RV in patients experiencing acute respiratory tract infections and represents the only reliable tool for the early laboratory diagnosis of RV infections. This is especially important in light of new opportunities for therapy currently being developed.
Detection of a New
Mycobacterium
Species in Wild Striped Bass in the Chesapeake Bay
Authors: Heckert Robert A. , Elankumaran S. , Milani Alessandra , Baya Ana 
Published: July 2002

ABSTRACT

Investigation into recent declines in striped bass health in the Chesapeake Bay in Maryland resulted in the isolation of a putative new species of
Mycobacterium
. This isolate was obtained from fish showing skin ulcers and internal granulomas in various organs. The isolate was slow growing at 28°C; was nonchromogenic; showed no activities of nitrate reduction, catalase activity, Tween 80 hydrolysis, tellurite reduction, or arylsulfatase reduction; grew best at low salt concentrations; and was urease and pyrazinamidase positive. By PCR a unique insertional sequence was identified which matched nothing in any database. Analysis of the nearly complete 16S rRNA gene sequence also indicated a unique sequence which had 87.7% sequence homology to
Mycobacterium ulcerans
, 87.6% homology to
Mycobacterium tuberculosis
, and 85.9% homology to
Mycobacterium marinum
. Phylogenetic analysis placed the organism close to the tuberculosis complex. These data support the conclusion that the isolate probably represents a new mycobacterial species.
Three-Year Assessment of Methicillin-Resistant
Staphylococcus aureus
Clones in Latin America from 1996 to 1998
Authors: Aires de Sousa Marta , Miragaia Maria , Santos Sanches Ilda , Ávila Sofia , Adamson Inger , Casagrande Silvana T. , Brandileone Maria Cristina C. , Palacio Rosario , Dell'Acqua Lillia , Hortal Maria , Camou Teresa , Rossi Alicia , Velazquez-Meza Maria Elena , Echaniz-Aviles Gabriela , Solorzano-Santos Fortino , Heitmann Ingrid , de Lencastre Hermı́nia 
Published: July 2002

ABSTRACT

Four hundred ninety-nine methicillin-resistant
Staphylococcus aureus
(MRSA) isolates recovered from 1996 to 1998 from 22 hospitals in five countries of Latin America—Argentina, Brazil, Chile, Uruguay and Mexico—were examined for antimicrobial susceptibility and clonal type in order to define the endemic clones in those hospitals. The hybridization of
Cla
I restriction digests with the
mecA
- and Tn
554
-specific DNA probes combined with pulsed-field gel electrophoresis of chromosomal
Sma
I digests (
Cla
I-
mecA
::
Cla
I-Tn
554
::PFGE clonal types) documented not only the predominance and persistence of the Brazilian clone (XI::B::B) in Brazil (97%) and Argentina (86%) but also its massive dissemination to Uruguay (100%). Moreover, a close relative of the Brazilian clone (XI::κ::B) was highly represented in Chile (53%) together with a novel clone (47%) (II::E′::F) resistant to pencillin, oxacillin, ciprofloxacin, chloramphenicol, clindamycin, erythromycin, and gentamicin. A unique clonal type (I::NH::M) was detected in Mexico among pediatric isolates and was resistant to penicillin, oxacillin, and gentamicin only. This study clearly documented the very large capacity for geographic expansion and the persistence of the Brazilian clone, contributing not only to the increasing uniformity of the MRSA in South America but worldwide as well.
Antibody Response to Shiga Toxins Stx2 and Stx1 in Children with Enteropathic Hemolytic-Uremic Syndrome
Authors: Ludwig Kerstin , Karmali Mohamed A. , Sarkim Volkan , Bobrowski Christoph , Petric Martin , Karch Helge , Müller-Wiefel Dirk E. ,   Arbeitsgemeinschaft für Pädiatrische Nephrologie
Published: July 2002

ABSTRACT

A Western blot (immunoblot) assay (WBA) for the detection of immunoglobulin G antibodies to Shiga toxins Stx2 and Stx1 in sera from 110 patients with enteropathic hemolytic-uremic syndrome (53 culture confirmed to have Shiga toxin-producing
Escherichia coli
[STEC] infection) and 110 age-matched controls was established by using a chemiluminescence detection system. Thirty-nine (74%) of the 53 culture-confirmed cases were infections with STEC serotype O157, and 14 (26%) were associated with infection by other STEC serotypes. The frequency of an anti-Stx2 response following infection by a Stx2-producing strain (34 of 48 cases; 71%) was higher than that of an anti-Stx1 response following Stx1-producing STEC infection (4 of 10). Furthermore, the frequency of an anti-Stx2 response in 110 control sera (10%) was significantly higher than the frequency of an anti-Stx1 response (1.8%) (
P
= 0.0325). For STEC O157 culture-confirmed cases WBA for toxin detection had a diagnostic sensitivity of 71% and a specificity of 90%. Because of its high specificity the assay might be a helpful tool for diagnosing suspected STEC infection when tests of stool samples or serological tests against various lipopolysaccharide antigens are negative. Furthermore, the prevalence of anti-Stx antibodies in healthy controls probably reflects the population immunity to systemic Stx-associated disease. It can thus serve as a basis for comparing immunity levels in different populations and for considering future Stx toxoid immunization strategies.
High-Frequency Recovery of Quinupristin-Dalfopristin-Resistant
Enterococcus faecium
Isolates from the Poultry Production Environment
Authors: Hayes Joshua R. , McIntosh Angela C. , Qaiyumi Sadaf , Johnson Judith A. , English Linda L. , Carr Lewis E. , Wagner David D. , Joseph Sam W. 
Published: July 2002

ABSTRACT

The occurrence of resistance to the streptogramin quinupristin-dalfopristin in
Enterococcus faecium
isolates from chickens on the Eastern Seaboard, was evaluated. Quinupristin-dalfopristin resistance was found in 51 to 78% of
E. faecium
isolates from the food production environment. The high level of resistance in this organism suggests that this reservoir of resistance may compromise the therapeutic potential of quinupristin-dalfopristin.
Antimicrobial Susceptibilities of
Helicobacter pylori
Isolates under Microaerophilic Atmospheres Established by Two Different Methods
Authors: Kobayashi Intetsu , Muraoka Hiroe , Saika Takeshi , Nishida Minoru , Fujioka Toshio , Nasu Masaru 
Published: July 2002

ABSTRACT

The MICs of clarithromycin, amoxicillin, and metronidazole for 150
Helicobacter pylori
isolates were determined using the AnaeroPack system and were compared with those determined using a microaerophilic incubator. The MICs of clarithromycin, amoxicillin, and metronidazole determined under both microaerophilic atmospheres were mostly within one twofold dilution for 146 (97.3%), 150 (100%), and 149 (99.3%) of the isolates, respectively.
16S/23S rRNA Intergenic Spacer Regions for Phylogenetic Analysis, Identification, and Subtyping of
Bartonella
Species
Authors: Houpikian Pierre , Raoult Didier 
Published: July 2002

ABSTRACT

Species of the genus
Bartonella
are currently recognized in growing numbers and are involved in an increasing variety of human diseases, mainly trench fever, Carrion's disease, bacillary angiomatosis, endocarditis, cat scratch disease, neuroretinitis, and asymptomatic bacteremia. Such a wide spectrum of infections makes it necessary to develop species and strain identification tools in order to perform phylogenetic and epidemiological studies. The 16S/23S rRNA intergenic spacer region (ITS) was sequenced for four previously untested species,
B. vinsonii
subsp.
arupensis, B. tribocorum, B. alsatica
, and
B. koehlerae
, as well as for 28 human isolates of
B. quintana
(most of them from French homeless people), six human or cat isolates of
B. henselae
, five cat isolates of
B. clarridgeiae
, and four human isolates of
B. bacilliformis
. Phylogenetic trees inferred from full ITS sequences of the 14 recognized
Bartonella
species using parsimony and distance methods revealed high statistical support, as bootstrap values were higher than those observed with other tested genes. Five well-supported lineages were identified within the genus and the proposed phylogenetic organization was consistent with that resulting from protein-encoding gene sequence comparisons. The ITS-derived phylogeny appears, therefore, to be a useful tool for investigating the evolutionary relationships of
Bartonella
species and to identify
Bartonella
species. Further, partial ITS amplification and sequencing offers a sensitive means of intraspecies differentiation of
B. henselae, B. clarridgeiae
, and
B. bacilliformis
isolates, as each strain had a specific sequence. The usefulness of this approach in epidemiological investigations should be highlighted. Among
B. quintana
strains, however, the genetic heterogenity was low, as only three ITS genotypes were identified. It was nevertheless sufficient to show that the
B. quintana
population infecting homeless people in France was not clonal.
Rapid Method for Detection of Gram-Positive and -Negative Bacteria in Milk from Cows with Moderate or Severe Clinical Mastitis
Authors: Yazdankhah Siamak P. , Sørum Henning , Larsen Hans Jørgen S. , Gogstad Geir 
Published: July 2002

ABSTRACTA rapid method for demonstration of gram-positive and gram-negative bacteria in milk is described. The technique is based on dilution of the sample in a medium, followed by filtration through a porous polysulfone membrane with a pore size retaining and concentrating bacteria from the sample. The bacteria concentrated on the surface of the membrane are stained with a cationic dye (toluidine blue) that can be visualized by the naked eye. After staining, the membrane is treated with ethanol-acetic acid (pH 2.8 to 3.0), which causes decolorization of gram-negative bacteria, whereas gram-positive bacteria retain the stain. The method does not require heat fixation, electrical power, microscopic examination, or specially trained personnel. The time needed to perform the test is approximately 5 min. The technique was applied to artificially infected milk and milk from cows with moderate or severe clinical mastitis for detection and differentiation of bacteria. The sensitivity of the filtration method was 92 and 100% for gram-positive and gram-negative bacteria, respectively, compared with traditional bacteriological culture of milk samples. The detection limit was 5 × 106CFU/ml forStaphylococcus aureusand 1 × 106CFU/ml forEscherichia coliin spiked milk samples. The overall specificity of the method was 86%. This diagnostic method can provide on-site guidance to the veterinarian to optimize use of antibiotics in mastitis therapy.