bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cloning and Characterization of the Phosphatidylserine Synthase Gene of
Agrobacterium
sp. Strain ATCC 31749 and Effect of Its Inactivation on Production of High-Molecular-Mass (1→3)-β-
d
-Glucan (Curdlan)
Authors: Karnezis Tara , Fisher Helen C. , Neumann Gregory M. , Stone Bruce A. , Stanisich Vilma A. 
Published: July 2002

ABSTRACT

Genes involved in the production of the extracellular (1→3)-β-glucan, curdlan, by
Agrobacterium
sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology
9:
31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon Tn
phoA
was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene,

pss
AG

, encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this
Agrobacterium
strain is not solely dependent on PE in a pathway controlled by the Pss
AG
protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the

pss
AG

::Tn
phoA
mutation was complemented by the intact

pss
AG

gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.
Growth ofCampylobacter jejuniSupported by Respiration of Fumarate, Nitrate, Nitrite, Trimethylamine-N-Oxide, or Dimethyl Sulfoxide Requires Oxygen
Authors: Sellars Michael J. , Hall Stephen J. , Kelly David J. 
Published: July 2002

ABSTRACTThe human gastrointestinal pathogenCampylobacter jejuniis a microaerophilic bacterium with a respiratory metabolism. The genome sequence ofC. jejunistrain 11168 reveals the presence of genes that encode terminal reductases that are predicted to allow the use of a wide range of alternative electron acceptors to oxygen, including fumarate, nitrate, nitrite, andN- orS-oxides. All of these reductase activities were present in cells of strain 11168, and the molybdoenzyme encoded byCj0264cwas shown by mutagenesis to be responsible for both trimethylamine-N-oxide (TMAO) and dimethyl sulfoxide (DMSO) reduction. Nevertheless, growth ofC. jejuniunder strictly anaerobic conditions (with hydrogen or formate as electron donor) in the presence of any of the electron acceptors tested was insignificant. However, when fumarate, nitrate, nitrite, TMAO, or DMSO was added to microaerobic cultures in which the rate of oxygen transfer was severely restricted, clear increases in both the growth rate and final cell density compared to what was seen with the control were obtained, indicative of electron acceptor-dependent energy conservation. TheC. jejunigenome encodes a single class I-type ribonucleotide reductase (RNR) which requires oxygen to generate a tyrosyl radical for catalysis. Electron microscopy of cells that had been incubated under strictly anaerobic conditions with an electron acceptor showed filamentation due to an inhibition of cell division similar to that induced by the RNR inhibitor hydroxyurea. An oxygen requirement for DNA synthesis can thus explain the lack of anaerobic growth ofC. jejuni. The results indicate that strict anaerobiosis is a stress condition forC. jejunibut that alternative respiratory pathways can contribute significantly to energy conservation under oxygen-limited conditions, as might be found in vivo.
Origins of the 2,4-Dinitrotoluene Pathway
Authors: Johnson Glenn R. , Jain Rakesh K. , Spain Jim C. 
Published: July 2002

ABSTRACT

The degradation of synthetic compounds requires bacteria to recruit and adapt enzymes from pathways for naturally occurring compounds. Previous work defined the steps in 2,4-dinitrotoluene (2,4-DNT) metabolism through the ring fission reaction. The results presented here characterize subsequent steps in the pathway that yield the central metabolic intermediates pyruvate and propionyl coenzyme A (CoA). The genes encoding the degradative pathway were identified within a 27-kb region of DNA cloned from
Burkholderia cepacia
R34, a strain that grows using 2,4-DNT as a sole carbon, energy, and nitrogen source. Genes for the lower pathway in 2,4-DNT degradation were found downstream from
dntD
, the gene encoding the extradiol ring fission enzyme of the pathway. The region includes genes encoding a CoA-dependent methylmalonate semialdehyde dehydrogenase (
dntE
), a putative NADH-dependent dehydrogenase (ORF13), and a bifunctional isomerase/hydrolase (
dntG
). Results from analysis of the gene sequence, reverse transcriptase PCR, and enzyme assays indicated that
dntD dntE
ORF13
dntG
composes an operon that encodes the lower pathway. Additional genes that were uncovered encode the 2,4-DNT dioxygenase (
dntAaAbAcAd
), methylnitrocatechol monooxygenase (
dntB
), a putative LysR-type transcriptional (ORF12) regulator, an intradiol ring cleavage enzyme (ORF3), a maleylacetate reductase (ORF10), a complete ABC transport complex (ORF5 to ORF8), a putative methyl-accepting chemoreceptor protein (ORF11), and remnants from two transposable elements. Some of the additional gene products might play as-yet-undefined roles in 2,4-DNT degradation; others appear to remain from recruitment of the neighboring genes. The presence of the transposon remnants and vestigial genes suggests that the pathway for 2,4-DNT degradation evolved relatively recently because the extraneous elements have not been eliminated from the region.
pH-Dependent Expression of Periplasmic Proteins and Amino Acid Catabolism inEscherichia coli
Authors: Stancik Lauren M. , Stancik Dawn M. , Schmidt Brian , Barnhart D. Michael , Yoncheva Yuliya N. , Slonczewski Joan L. 
Published: July 2002

ABSTRACTEscherichia coligrows over a wide range of pHs (pH 4.4 to 9.2), and its own metabolism shifts the external pH toward either extreme, depending on available nutrients and electron acceptors. Responses to pH values across the growth range were examined through two-dimensional electrophoresis (2-D gels) of the proteome and throughlacgene fusions. Strain W3110 was grown to early log phase in complex broth buffered at pH 4.9, 6.0, 8.0, or 9.1. 2-D gel analysis revealed the pH dependence of 19 proteins not previously known to be pH dependent. At low pH, several acetate-induced proteins were elevated (LuxS, Tpx, and YfiD), whereas acetate-repressed proteins were lowered (Pta, TnaA, DksA, AroK, and MalE). These responses could be mediated by the reuptake of acetate driven by changes in pH. The amplified proton gradient could also be responsible for the acid induction of the tricarboxylic acid (TCA) enzymes SucB and SucC. In addition to the autoinducer LuxS, low pH induced another potential autoinducer component, the LuxH homolog RibB. pH modulated the expression of several periplasmic and outer membrane proteins: acid induced YcdO and YdiY; base induced OmpA, MalE, and YceI; and either acid or base induced OmpX relative to pH 7. Two pH-dependent periplasmic proteins were redox modulators: Tpx (acid-induced) and DsbA (base-induced). The locusalx, induced in extreme base, was identified asygjT, whose product is a putative membrane-bound redox modulator. The cytoplasmic superoxide stress protein SodB was induced by acid, possibly in response to increased iron solubility. High pH induced amino acid metabolic enzymes (TnaA and CysK) as well aslacfusions to the genes encoding AstD and GabT. These enzymes participate in arginine and glutamate catabolic pathways that channel carbon into acids instead of producing alkaline amines. Overall, these data are consistent with a model in whichE. colimodulates multiple transporters and pathways of amino acid consumption so as to minimize the shift of its external pH toward either acidic or alkaline extreme.
Characterization of an Unusual Mycobacterium: a Possible Missing Link between
Mycobacterium marinum
and
Mycobacterium ulcerans
Authors: Chemlal K. , Huys G. , Laval F. , Vincent V. , Savage C. , Gutierrez C. , Laneelle M.-A. , Swings J. , Meyers W. M. , Daffe M. , Portaels F. 
Published: July 2002

ABSTRACT

In an attempt to characterize an unusual mycobacterial isolate from a 44-year-old patient living in France, we applied phenotypic characterizations and various previously described molecular methods for the taxonomic classification of mycobacteria. The results of the investigations were compared to those obtained in a previous study with a set of temporally and geographically diverse
Mycobacterium ulcerans
(
n
= 29) and
Mycobacterium marinum
(
n
= 29) isolates (K. Chemlal, G. Huys, P.-A. Fonteyne, V. Vincent, A. G. Lopez, L. Rigouts, J. Swings, W. M. Meyers, and F. Portaels, J. Clin. Microbiol. 39:3272-3278, 2001). The isolate, designated ITM 00-1026 (IPP 2000-372), is closely related to
M. marinum
according to its phenotypic properties, lipid pattern, and partial 16S rRNA sequence. Moreover, fingerprinting by amplified fragment length polymorphism (AFLP) analysis unequivocally classified this strain as a member of the species
M. marinum
, although it lacked two species-specific AFLP marker bands. However, PCR and restriction fragment length polymorphism analysis based on
M. ulcerans
-specific insertion sequence IS
2404
showed the presence of this element in a low copy number in isolate ITM 00-1026. In conclusion, the designation of this isolate as a transitional species further supports the recent claim by Stinear et al. (T. Stinear, G. Jenkin, P. D. Johnson, and J. K. Davies, J. Bacteriol. 182:6322-6330, 2000) that
M. ulcerans
represents a relatively recent phylogenetic derivative of
M. marinum
resulting from the systematic acquisition of foreign DNA fragments.
Multiplex, Quantitative, Real-Time PCR Assay for Cytomegalovirus and Human DNA
Authors: Sanchez Jason L. , Storch Gregory A. 
Published: July 2002

ABSTRACTWe created a multiplex, quantitative, real-time PCR assay that amplifies cytomegalovirus (CMV) and human DNA in the same reaction tube, allowing for a viral load determination that is normalized to measured human DNA. The assay targets a conserved region of the CMV DNA polymerase gene that is not affected by known drug resistance mutations. All 36 strains of CMV detected by culture or qualitative PCR in a population of lung transplant recipients were detected. The assay detected 1 to 10 copies of CMV plasmid DNA. The analytic sensitivity was not affected by the presence of DNA from 106human cells but was reduced approximately 10-fold by alkaline lysates of leukocyte preparations. CMV quantitation was linear over a range of 101to 106copies. The intraassay and interassay coefficients of variation were 29 and 40%. Human DNA was regularly detected in patient plasma samples, and the amount was increased by storage of blood at room temperature before plasma separation and by plasma separation techniques that allowed leukocyte contamination. Applied to whole blood, the assay provides a measurement of CMV DNA in relation to cellular content without a need for cell counting procedures. Applied to plasma, the assay can reveal artifactual increases in plasma CMV levels resulting from leukocyte contamination. Further study of the utility of this assay to monitor patient populations at risk for CMV disease is warranted.
Detection and Genotyping of Human Group A Rotaviruses by Oligonucleotide Microarray Hybridization
Authors: Chizhikov V. , Wagner M. , Ivshina A. , Hoshino Y. , Kapikian A. Z. , Chumakov K. 
Published: July 2002

ABSTRACT
A rapid and reliable method for the identification of five clinically relevant G genotypes (G1 to G4 and G9) of human rotaviruses based on oligonucleotide microarray hybridization has been developed. The genotype-specific oligonucleotides immobilized on the surface of glass slides were selected to bind to the multiple target regions within the VP7 gene that are highly conserved among individual rotavirus genotypes. Rotavirus cDNA was amplified in a PCR with primers common to all group A rotaviruses. A second round of nested PCR amplification was performed in the presence of indodicarbocyanine-dCTP and another pair of degenerate primers also broadly specific for all genotypes. The use of one primer containing 5′-biotin allowed us to prepare fluorescently labeled single-stranded hybridization probe by binding of another strand to magnetic beads. The identification of rotavirus genotype was based on hybridization with several individual genotype-specific oligonucleotides. This approach combines the high sensitivity of PCR with the selectivity of DNA-DNA hybridization. The specificity of oligonucleotide microchip hybridization was evaluated by testing 20 coded rotavirus isolates from different geographic areas for which genotypes were previously determined by conventional methods. Analysis of the coded specimens showed that this microarray-based method is capable of unambiguous identification of all rotavirus strains. Because of the presence of random mutations, each individual virus isolate produced a unique hybridization pattern capable of distinguishing different isolates of the same genotype and, therefore, subgenotype differentiation. This strain information indicates one of several advantages that microarray technology has over conventional PCR techniques.
Histologic and Genotypic Characterization of a Novel
Mycobacterium
Species Found in Three Cats
Authors: Appleyard Greg D. , Clark Edward G. 
Published: July 2002

ABSTRACT

Three cases of feline atypical mycobacteriosis from different geographical regions in North America were characterized by large clusters of filamentous bacteria visible on hematoxylin-and-eosin-stained tissue sections. PCR amplification demonstrated the presence of
Mycobacterium
-specific nucleic acid in samples of skin lesions from these cases. PCR-assisted cloning and DNA sequence analysis of a 541-bp length of the
Mycobacterium
16S rRNA gene generated DNA sequences which were >95% identical, suggesting that the three isolates were closely related. Two of the sequences were 99% identical and may represent the same species. Alignment with comparable 16S rRNA gene sequences from 66
Mycobacterium
species and partially characterized isolates highlighted similarities (>94%) with
Mycobacterium bohemicum
,
Mycobacterium haemophilum
,
Mycobacterium ulcerans
,
Mycobacterium avium
subsp.
avium
, and isolate IWGMT 90242. Parsimony analysis of sequence data suggested relatedness to
M. leprae
. Significant molecular genetic and pathobiological differences between these three similar isolates and other known species of mycobacteria suggested that the organisms may not have been described previously and that these cases may represent a new form of mycobacterial disease in cats. We suggest the term “
Mycobacterium visibilis
” to describe the organism from which the two nearly identical sequences were obtained.
Seroprevalence ofBabesiaInfections in Humans Exposed to Ticks in Midwestern Germany
Authors: Hunfeld Klaus-Peter , Lambert Annette , Kampen Helge , Albert Sabine , Epe Christian , Brade Volker , Tenter Astrid M. 
Published: July 2002

ABSTRACTBabesiosis is considered to be an emerging tick-borne disease in humans worldwide. However, most studies on the epidemiology of human babesiosis to date have been carried out in North America, and there is little knowledge on the prevalence of infection and frequency of disease in other areas. The aim of this study was to investigate the prevalence ofBabesiainfections in a human population in Germany. A total of 467 sera collected between May and October 1999 from individuals living in the Rhein-Main area were tested for the presence of immunoglobulin G (IgG) and IgM antibodies to antigens ofBabesia microtiandBabesia divergensby indirect fluorescent-antibody (IFA) tests. These sera were derived from 84 Lyme borreliosis patients suffering from erythema migrans, 60 asymptomatic individuals with positive borreliosis serology, and 81 individuals with a history of tick bite. Cutoff values for discrimination between seronegative and seropositive results in the IFA tests were determined using sera from 120 healthy blood donors and 122 patients suffering from conditions other than tick-borne diseases (malaria,n= 40; toxoplasmosis,n= 22; syphilis,n= 20; Epstein-Barr virus infection,n= 20; and presence of antinuclear antibodies,n= 20). The overall specificities of the IFA tests forB. microtiandB. divergenswere estimated to be ≥97.5%. Positive IgG reactivity againstB. microtiantigen (titer, ≥1:64) orB. divergensantigen (titer, ≥1:128) was detected significantly more often (P< 0.05) in the group of patients exposed to ticks (26 of 225 individuals; 11.5%) than in the group of healthy blood donors (2 of 120 individuals; 1.7%). IgG antibody titers of ≥1:256 against at least one of the babesial antigens were found significantly more often (P< 0.05) in patients exposed to ticks (9 of 225) than in the control groups (1 of 242). In the human population investigated here, the overall seroprevalences forB. microtiandB. divergenswere 5.4% (25 of 467) and 3.6% (17 of 467), respectively. The results obtained here provide evidence for concurrent infections withBorrelia burgdorferiandBabesiaspecies in humans exposed to ticks in midwestern Germany. They also suggest that infections withBabesiaspecies in the German human population are more frequent than believed previously and should be considered in the differential diagnosis of febrile illness occurring after exposure to ticks or blood transfusions, in particular in immunocompromised patients.
Clostridium difficile
Genotyping Based on
slpA
Variable Region in S-Layer Gene Sequence: an Alternative to Serotyping
Authors: Karjalainen Tuomo , Saumier Nicolas , Barc Marie-Claude , Delmée Michel , Collignon Anne 
Published: July 2002

ABSTRACT

Recent investigations of
Clostridium difficile
cell wall components have revealed the presence of an S-layer encoded by the
slpA
gene. The aim of this study was to determine whether
slpA
genotyping can be used as an alternative to serotyping. The variable regions of
slpA
were amplified by PCR from serogroup reference strains and various clinical isolates chosen randomly. Amplified products were analyzed after restriction enzyme digestion and DNA sequencing. The sequences of the variable region of the SlpA protein were found to be strictly identical within a given serogroup but divergent between serogroups
.
These preliminary results suggest that PCR-restriction fragment length polymorphism, in conjunction with DNA sequencing of the
slpA
variable region, could constitute an alternative typing method for determining
C. difficile
serotypes.
Genetic Diversity of Clinical and Environmental Strains of
Salmonella enterica
Serotype Weltevreden Isolated in Malaysia
Authors: Thong K. L. , Goh Y. L. , Radu S. , Noorzaleha S. , Yasin R. , Koh Y. T. , Lim V. K. E. , Rusul G. , Puthucheary S. D. 
Published: July 2002

ABSTRACT

The incidence of food-borne salmonellosis due to
Salmonella enterica
serotype Weltevreden is reported to be on the increase in Malaysia. The pulsed-field gel electrophoresis (PFGE) subtyping method was used to assess the extent of genetic diversity and clonality of
Salmonella
serotype Weltevreden strains from humans and the environment. PFGE of
Xba
I-digested chromosomal DNA from 95 strains of
Salmonella
serotype Weltevreden gave 39 distinct profiles with a wide range of Dice coefficients (0.27 to 1.00), indicating that PFGE is very discriminative and that multiple clones of
Salmonella
serotype Weltevreden exist among clinical and environmental isolates. Strains of one dominant pulsotype (pulsotype X1/X2) appeared to be endemic in this region, as they were consistently recovered from humans with salmonellosis between 1996 and 2001 and from raw vegetables. In addition, the sharing of similar PFGE profiles among isolates from humans, vegetables, and beef provides indirect evidence of the possible transmission of salmonellosis from contaminated raw vegetables and meat to humans. Furthermore, the recurrence of PFGE profile X21 among isolates found in samples of vegetables from one wet market indicated the persistence of this clone. The environment in the wet markets may represent a major source of cross-contamination of vegetables with
Salmonella
serotype Weltevreden. Antibiotic sensitivity tests showed that the clinical isolates of
Salmonella
serotype Weltevreden remained drug sensitive but that the vegetable isolates were resistant to at least two antibiotics. To the best of our knowledge, this is the first study to compare clinical and environmental isolates of
Salmonella
serotype Weltevreden in Malaysia.
Molecular Peculiarities of the
lytA
Gene Isolated from Clinical Pneumococcal Strains That Are Bile Insoluble
Authors: Obregón Virginia , García Pedro , García Ernesto , Fenoll Asunción , López Rubens , García José L. 
Published: July 2002

ABSTRACT

The autolytic LytA amidase from 12 bile (deoxycholate)-insoluble streptococcal isolates (formerly classified as atypical
Streptococcus pneumoniae
) showing different antibiotic resistance patterns was studied. These atypical strains, which autolyze at the end of the stationary phase of growth, contain highly divergent
lytA
alleles (pairwise evolutionary distances of about 20%) compared to the
lytA
alleles of typical pneumococci. The atypical LytA amidases exhibit a peculiar deletion of two amino acids responsible for cell wall anchoring in the carboxy-terminal domain and have a reduced specific activity. These enzymes were inhibited by 1% deoxycholate but were activated by 1% Triton X-100, a detergent that could be used as an alternative diagnostic test for this kind of strain. Preparation of functional chimeric enzymes, PCR mutagenesis, and gene replacements demonstrated that the characteristic bile insolubility of these atypical strains was due to their peculiar carboxy-terminal domain and that the 2-amino-acid deletion was responsible for the inhibitory effect of deoxycholate. However, the deletion alone did not affect the specific activity of LytA. A detailed characterization of the genes encoding the 16S rRNA and SodA together with multilocus sequence typing indicated that the strains studied here are not a single clone and, although they cannot be strictly classified as typical pneumococci, they represent a quite diverse pool of organisms closely related to
S. pneumoniae
. The clinical importance of these findings is underlined by the role of the
lytA
gene in shaping the course of pneumococcal diseases. This study can also contribute to solving diagnostic problems and to understanding the evolution and pathogenic potential of species of the
Streptococcus mitis
group.