bluuebunny/crossref_metadata_2025_split huggingface dataset:
Duplicate Copies of Genes Encoding Methanesulfonate Monooxygenase in
Marinosulfonomonas methylotropha
Strain TR3 and Detection of Methanesulfonate Utilizers in the Environment
Authors: Baxter Nardia J. , Scanlan Julie , De Marco Paolo , Wood Ann P. , Murrell J. Colin 
Published: July 2002

ABSTRACT

Marinosulfonomonas methylotropha
strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from
M. methylotropha
strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp
Hin
dIII fragment contained
msmA, msmB
, and
msmC
, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp
Hin
dIII fragment contained duplicate copies of
msmA
and
msmB
, as well as
msmD
, encoding the reductase component of methanesulfonate. Both sets of
msmA
and
msmB
genes were virtually identical, and the derived
msmA
and
msmB
sequences of
M. methylotropha
strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer
Methylosulfonomonas methylovora
strain M2 were found to be 82 and 69% identical. The
msmA
gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of
msmA
which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding
msmA
genes from newly isolated
Hyphomicrobium, Methylobacterium
, and
Pedomicrobium
species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these
msmA
gene fragments, compared to
msmA
sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.
Determination of Subunit Composition of
Clostridium cellulovorans
Cellulosomes That Degrade Plant Cell Walls
Authors: Murashima Koichiro , Kosugi Akihiko , Doi Roy H. 
Published: July 2002

ABSTRACT

Clostridium cellulovorans
produces a cellulase enzyme complex (cellulosome). In this study, we isolated two plant cell wall-degrading cellulosomal fractions from culture supernatant of
C. cellulovorans
and determined their subunit compositions and enzymatic activities. One of the cellulosomal fractions showed fourfold-higher plant cell wall-degrading activity than the other. Both cellulosomal fractions contained the same nine subunits (the scaffolding protein CbpA, endoglucanases EngE and EngK, cellobiohydrolase ExgS, xylanase XynA, mannanase ManA, and three unknown proteins), although the relative amounts of the subunits differed. Since only cellobiose was released from plant cell walls by the cellulosomal fractions, cellobiohydrolases were considered to be key enzymes for plant cell wall degradation.
Detection of Microbial Growth on Polycyclic Aromatic Hydrocarbons in Microtiter Plates by Using the Respiration Indicator WST-1
Authors: Johnsen Anders R. , Bendixen Karen , Karlson Ulrich 
Published: July 2002

ABSTRACT
We have developed a microtiter plate method for screening a large number of bacterial isolates for the ability to grow on different crystalline polycyclic aromatic hydrocarbons (PAHs). Growth on PAHs cannot easily be determined with standard growth assays because of the very low aqueous solubility and bioavailability of the PAHs. Our microtiter plate assay utilizes a new water-soluble respiration indicator, WST-1 {4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate}, in combination with easily degradable carbon sources. PAH-mineralizing strains were grown on PAHs in microtiter plates for 7 to 10 days. The tetrazolium dye WST-1 was added after incubation. Dehydrogenases in growing cells reduced WST-1 to a water-soluble colored formazan, and the intensity of the color was a measure of the respiration rate. Addition of easily degradable carbon to the wells along with WST-1 resulted in a 3- to 40-fold increase in the absorbance of positive wells within 90 min, which made it possible to detect growth on fluorene, phenanthrene, anthracene, fluoranthene, and pyrene. Addition of the electron transport blocker sodium azide unexpectedly decreased formazan formation. The method was adapted for most-probable-number enumeration of PAH degraders in soil.
Bacteriophage Resistance of a Δ
thyA
Mutant of
Lactococcus lactis
Blocked in DNA Replication
Authors: Pedersen Martin B. , Jensen Peter R. , Janzen Thomas , Nilsson Dan 
Published: July 2002

ABSTRACT

The
thyA
gene, which encodes thymidylate synthase (TS), of
Lactococcus lactis
CHCC373 was sequenced, including the upstream and downstream regions. We then deleted part of
thyA
by gene replacement. The resulting strain, MBP71 Δ
thyA
, was devoid of TS activity, and in media without thymidine, such as milk, there was no detectable dTTP pool in the cells. Hence, DNA replication was abolished, and acidification by MBP71 was completely unaffected by the presence of nine different phages tested at a multiplicity of infection (MOI) of 0.1. Nonreplicating MBP71 must be inoculated at a higher level than CHCC373 to achieve a certain pH within a specified time. For a pH of 5.2 to be reached in 6 h, the inoculation level of MBP71 must be 17-fold higher than for CHCC373. However, by adding a limiting amount of thymidine this could be lowered to just 5-fold the normal amount, while acidification was unaffected with MBP71 up to an MOI of 0.01. It was found that nonreplicating MBP71 produced largely the same products as CHCC373, though the acetaldehyde production of the former was higher.
Humoral Immune Response in Chromoblastomycosis during and after Therapy
Authors: Esterre P. , Jahevitra M. , Andriantsimahavandy A. 
Published: July 2002

ABSTRACT

A longitudinal study was carried out in Madagascar, the most important focus of chromoblastomycosis (P. Esterre, A. Andriantsimahavandy, E. Ramarcel, and J. L. Pecarrere, Am. J. Trop. Med. Hyg. 55:45–47, 1996), to investigate natural immunity to this disease. Sequential blood samples were obtained before, during, and at the end of a successful therapeutic trial with terbinafine, a new antifungal drug. Using enzyme-linked immunosorbent assay and immunoblot methods, detailed analyses of antibody concentration and antigen mapping were conducted for 136 serum samples and tentatively correlated to epidemiological and pathobiological data. Two different cytoplasmic antigens, corresponding to the two fungal species involved (
Fonsecaea pedrosoi
and
Cladophialophora carrionii
), were used to analyze the distribution of different classes of immunoglobulins. This was done with respect to the origin of the isolates, clinical and pathobiological. Although strong individual variations were noticed, some major antigens (one of 18.5 kDa specific for
F. pedrosoi
and two of 23.5 and 33 kDa, respectively, specific for
C. carrionii
) corresponded to high antibody prevalence and concentration. As some antigenic components were also detected by immunoglobulin M (IgM) and IgA antibodies, the role that these specific antibodies could play in the immune response is discussed.
Serological Differentiation of Murine Typhus and Epidemic Typhus Using Cross-Adsorption and Western Blotting
Authors: La Scola Bernard , Rydkina Lena , Ndihokubwayo Jean-Bosco , Vene Sirkka , Raoult Didier 
Published: July 2002

ABSTRACTDifferentiation of murine typhus due toRickettsia typhiand epidemic typhus due toRickettsia prowazekiiis critical epidemiologically but difficult serologically. Using serological, epidemiological, and clinical criteria, we selected sera from 264 patients with epidemic typhus and from 44 patients with murine typhus among the 29,188 tested sera in our bank. These sera cross-reacted extensively in indirect fluorescent antibody assays (IFAs) againstR. typhiandR. prowazekii, as 42% of the sera from patients with epidemic typhus and 34% of the sera from patients with murine typhus exhibited immunoglobulin M (IgM) and/or IgG titers against the homologous antigen (R. prowazekiiandR. typhi, respectively) that were more than one dilution higher than those against the heterologous antigen. Serum cross-adsorption studies and Western blotting were performed on sera from 12 selected patients, 5 with murine typhus, 5 with epidemic typhus, and 2 suffering from typhus of undetermined etiology. Differences in IFA titers againstR. typhiandR. prowazekiiallowed the identification of the etiological agent in 8 of 12 patients. Western blot studies enabled the identification of the etiological agent in six patients. When the results of IFA and Western blot studies were considered in combination, identification of the etiological agent was possible for 10 of 12 patients. Serum cross-adsorption studies enabled the differentiation of the etiological agent in all patients. Our study indicates that when used together, Western blotting and IFA are useful serological tools to differentiate betweenR. prowazekiiandR. typhiexposures. While a cross-adsorption study is the definitive technique to differentiate between infections with these agents, it was necessary in only 2 of 12 cases (16.7%), and the high costs of such a study limit its use.
Host Cell Responses to Genotypically SimilarHelicobacter pyloriIsolates from United States and Japan
Authors: Ando Takafumi , Peek Richard M. , Lee Yong-Chan , Krishna Uma , Kusugami Kazuo , Blaser Martin J. 
Published: July 2002

ABSTRACTAssociations ofHelicobacter pylorigenotypes with disease differ between Western countries and Asia. Therefore, we directly compared histopathological and in vitro responses to clinical isolates with similar genotypes. Sixty-threecagA+vacAs1/m1H. pyloriisolates (United States,n= 24; Japan,n= 39) and eightcagA-negativevacAs2/m2 strains were incubated with AGS cells, and supernatants were assayed for interleukin-8 (IL-8) and for DNA fragmentation. CagA tyrosine phosphorylation in AGS cells and the sequence of the putative HP0638 (oipA) signal sequence region were determined for 22 representative strains. HP0638 and/orcagisland mutant strains were created and examined in IL-8 and CagA tyrosine phosphorylation assays. Levels of IL-8 induction and DNA fragmentation were similar in the U.S. and JapanesecagA+vacAs1/m1 isolates. All 10 of the isolates with the highest IL-8 induction and 8 of the 10 isolates with the lowest IL-8 induction had an in-frameoipAopen reading frame, and all 10 of the isolates with the highest IL-8 induction and 7 of the 10 isolates with the lowest IL-8 induction induced CagA tyrosine phosphorylation in AGS cells. Eight isolates from gastric ulcer patients induced significantly more apoptosis in vitro, and more severe gastritis and atrophy in vivo, than other Japanese isolates. Disruption of HP0638 did not affect IL-8 induction or CagA tyrosine phosphorylation. Thus,H. pylori cagA+vacAs1/m1 isolates from the United States and Japan induce similar IL-8 and apoptosis levels. Inactivation of HP0638 does not alter epithelial responses mediated by thecagisland in vitro. Assessment of apoptosis in vitro identified a group ofH. pyloriisolates that induce more severe gastric inflammation and atrophy.
Mycolic Acid Analysis by High-Performance Liquid Chromatography for Identification ofMycobacteriumSpecies
Authors: Butler W. Ray , Guthertz Linda S. 
Published: July 2002

SUMMARYMycobacterium tuberculosis is the etiologic agent of tuberculosis and can be accurately detected by laboratories using commercial genetic tests. Nontuberculosis mycobacteria (NTM) causing other mycobacterioses can be difficult to identify. The identification processes are confounded by an increasing diversity of newly characterized NTM species. The ubiquitous nature of NTM, combined with their potential to be opportunistic pathogens in immunocompromised as well as nonimmunodeficient patients, further complicates the problem of their identification. Since clinical case management varies depending on the etiologic agent, laboratories must identify the species in a timely manner. However, only a few identification methods can detect the species diversity within the Mycobacterium genus. Over the last decade, high-performance liquid chromatography analysis of the mycolic acids has become an accepted method for identification of mycobacteria. In this review, we assess its development and usefulness as an identification technique for Mycobacterium species.
Regulation of Cathelicidin Gene Expression: Induction by Lipopolysaccharide, Interleukin-6, Retinoic Acid, and
Salmonella enterica
Serovar Typhimurium Infection
Authors: Wu Hua , Zhang Guolong , Minton J. Ernest , Ross Christopher R. , Blecha Frank 
Published: July 2002

ABSTRACT

Cathelicidins are a family of antimicrobial peptides prominent in the host defense mechanisms of several mammalian species. In addition to their antimicrobial activities, these peptides have been implicated in wound healing, angiogenesis, and other innate immune mechanisms. To investigate the regulatory mechanisms of cathelicidin gene expression, we conducted in vitro experiments evaluating the bone marrow cell expression of two porcine cathelicidins, PR-39 and protegrin, and cloned and evaluated the promoter sequence of PR-39. In addition, we evaluated in vivo kinetics of cathelicidin gene expression in pigs during an infection with
Salmonella enterica
serovar Typhimurium. Lipopolysaccharide (LPS) increased PR-39 and protegrin mRNA expression, which was ameliorated by polymyxin B. Concentrations of PR-39 in supernatants from bone marrow cell cultures were increased 10-fold after LPS stimulation. Similarly, interleukin-6 (IL-6) and all-trans retinoic acid (RA) markedly induced cathelicidin gene expression. To verify the transcriptional activation of the PR-39 gene by these agents, we made a PR-39 promoter-luciferase construct containing the full-length PR-39 promoter driving luciferase gene expression and transiently transfected PK-15 epithelial cells. RA and IL-6 increased luciferase activity in PK-15 cells transfected with the PR-39 promoter-luciferase reporter. Similarly,
Salmonella
-challenged pigs showed increased expression of PR-39 and protegrin mRNA in bone marrow cells at 6 and 24 h postchallenge. Taken together, these findings show that bacterial products (LPS), IL-6, RA, and
Salmonella
infection enhance the expression of the cathelicidins, PR-39 and protegrin, in bone marrow progenitor cells, and we suggest that extrinsic modulation of this innate host defense mechanism may be possible.
Campylobacter fetus sap
Inversion Occurs in the Absence of RecA Function
Authors: Ray Kevin C. , Tu Zheng-Chao , Grogono-Thomas Rosemary , Newell Diane G. , Thompson Stuart A. , Blaser Martin J. 
Published: July 2002

ABSTRACT

Phase variation of
Campylobacter fetus
surface layer proteins (SLPs) occurs by inversion of a 6.2-kb DNA segment containing the unique
sap
promoter, permitting expression of a single SLP-encoding gene. Previous work has shown that the
C. fetus sap
inversion system is RecA dependent. When we challenged a pregnant ewe with a
recA
mutant of wild-type
C. fetus
(strain 97-211) that expressed the 97-kDa SLP, 15 of the 16 ovine-passaged isolates expressed the 97-kDa protein. However, one strain (97-209) expressed a 127-kDa SLP, suggesting that chromosomal rearrangement may have occurred to enable SLP switching. Lack of RecA function in strains 97-211 and 97-209 was confirmed by their sensitivity to the DNA-damaging agent methyl methanesulfonate. Southern hybridization and PCR of these strains indicated that the
aphA
insertion into
recA
was stably present. However, Southern hybridizations demonstrated that in strain 97-209 inversion had occurred in the
sap
locus. PCR data confirmed inversion of the 6.2-kb DNA element and indicated that in these
recA
mutants the
sap
inversion frequency is reduced by 2 to 3 log
10
units compared to that in the wild type. Thus, although the major
sap
inversion pathway in
C. fetus
is RecA dependent, alternative lower-frequency, RecA-independent inversion mechanisms exist.
IS
1414
, an
Escherichia coli
Insertion Sequence with a Heat-Stable Enterotoxin Gene Embedded in a Transposase-Like Gene
Authors: McVeigh Annette , Fasano Alessio , Scott Daniel A. , Jelacic Sandra , Moseley Steve L. , Robertson Donald C. , Savarino Stephen J. 
Published: July 2002

ABSTRACT

Enteroaggregative
Escherichia coli
(EAEC) heat-stable enterotoxin 1 (EAST1) was originally discovered in EAEC but has also been associated with enterotoxigenic
E. coli
(ETEC). Multiple genomic restriction fragments from each of three ETEC strains of human origin showed homology with an EAST1 gene probe. A single hybridizing fragment was detected on the plasmid of ETEC strain 27D that also encodes heat-stable enterotoxin Ib and colonization factor antigen I. We isolated and characterized this fragment, showing that it (i) carries an allele of
astA
nearly identical to that originally reported from EAEC 17-2 and (ii) expressed enterotoxic activity. Sequence analysis of the toxin coding region revealed that
astA
is completely embedded within a 1,209-bp open reading frame (ORF1), whose coding sequence is on the same strand but in the −1 reading frame in reference to the toxin gene. In vitro expression of the predicted
M
r
-∼46,000 protein product of ORF1 was demonstrated. ORF1 is highly similar to transposase genes of IS
285
from
Yersinia pestis
, IS
1356
from
Burkholderia cepacia
, and IS
Rm3
from
Rhizobium meliloti
. It is bounded by 30-bp imperfect inverted repeat sequences and flanked by 8-bp direct repeats. Based on these structural features, pathognomonic of a regular insertion sequence, this element was designated IS
1414
. Preliminary experiments to show IS
1414
translocation were unsuccessful. Overlapping genes of the type suggested by the IS
1414
core region have heretofore not been described in bacteria. It seems to offer a most efficient mechanism for intragenomic and horizontal dissemination of EAST1.
Identification and Immunogenicity of Group AStreptococcusCulture Supernatant Proteins
Authors: Lei Benfang , Mackie Stacy , Lukomski Slawomir , Musser James M. 
Published: July 2002

ABSTRACTExtracellular proteins made by group AStreptococcus(GAS) play critical roles in the pathogenesis of human infections caused by this bacterium. Although many extracellular GAS proteins have been identified and characterized, there has been no systematic analysis of culture supernatant proteins. Proteins present in the culture supernatant of strains of serotype M1 (MGAS 5005) and M3 (MGAS 315) mutants lacking production of the major extracellular cysteine protease were separated by two-dimensional gel electrophoresis and identified by amino-terminal amino acid sequencing and interrogation of available databases, including a serotype M1 genome sequence. In the aggregate, amino-terminal amino acid sequence data for 66 protein spots were generated, 53 unique sequences were obtained, and 44 distinct proteins were identified. Sixteen of the 44 proteins had apparent secretion signal sequences and 27 proteins did not. Eight of the 16 proteins with apparent secretion signal sequences have not been previously described for GAS. Antibodies against most of the apparently secreted proteins were present in sera from mice infected subcutaneously with MGAS 5005 or MGAS 315. Humans with documented GAS infections (pharyngitis, acute rheumatic fever, and severe invasive disease) also had serum antibodies reacting with many of the apparently secreted proteins, indicating that they were synthesized in the course of GAS-human interaction. The genes encoding four of the eight previously undescribed and apparently secreted culture supernatant proteins were cloned, and the proteins were overexpressed inEscherichia coli. Western blot analysis with these recombinant proteins and sera from GAS-infected mice and humans confirmed the immunogenicity of these proteins. Taken together, the data provide new information about the molecular aspects of GAS-host interactions.
Urease as a Virulence Factor in Experimental Cryptococcosis
Authors: Cox Gary M. , Mukherjee Jean , Cole Garry T. , Casadevall Arturo , Perfect John R. 
Published: July 2002

ABSTRACT

Urease catalyzes the hydrolysis of urea to ammonia and carbamate and has been found to be an important pathogenic factor for certain bacteria.
Cryptococcus neoformans
is a significant human pathogenic fungus that produces large amounts of urease; thus we wanted to investigate the importance of urease in the pathogenesis of cryptococcosis. We cloned and sequenced the genomic locus containing the single-copy
C. neoformans
urease gene (
URE1
) and used this to disrupt the native
URE1
in the serotype A strain H99. The
ure1
mutant strains were found to have in vitro growth characteristics, phenoloxidase activity, and capsule size similar to those of the wild type. Comparison of a
ure1
mutant with H99 after intracisternal inoculation into corticosteroid-treated rabbits revealed no significant differences in colony counts recovered from the cerebrospinal fluid. However, when these two strains were compared in both the murine intravenous and inhalational infection models, there were significant differences in survival. Mice infected with a
ure1
strain lived longer than mice infected with H99 in both models. The
ure1
strain was restored to urease positivity by complementation with
URE1
, and two resulting transformants were significantly more pathogenic than the
ure1
strain. Our results suggest that urease activity is involved in the pathogenesis of cryptococcosis but that the importance may be species and/or infection site specific.
Interleukin-4 Receptor Alpha-Deficient BALB/c Mice Show an Unimpaired T Helper 2 Polarization in Response toLeishmania majorInfection
Authors: Mohrs Markus , Holscher Christoph , Brombacher Frank 
Published: July 2002

ABSTRACTWe recently generated interleukin-4 (IL-4) receptor alpha-deficient (IL-4Rα−/−) BALB/c mice and showed evidence for a protective role of IL-13-mediated functions in leishmaniasis. In this study, we investigated the IL-4 expression and T helper 2 (Th2) development inLeishmania major-infected IL-4Rα−/−mice. Here we show that the early burst of IL-4 expression observed inL. major-infected BALB/c mice is independent of IL-4Rα-mediated functions. Subsequently, we confirmed an impaired Th2 development in vitro. Unexpectedly, duringL. majorinfection, isolated CD4+IL-4Rα−/−T cells expressed high IL-4- but low gamma interferon (IFN-γ)-specific mRNA, comparable to Th2-polarized BALB/c CD4+cells and in contrast to Th1-polarized C57BL/6 CD4+cells. Since antigen-specific restimulated popliteal lymph node cells (PLN) of IL-4Rα−/−mice also responded with high IL-4 but low IFN-γ production, comparable to Th2-polarized cells from wild-type BALB/c mice and in contrast to Th1-polarized C57BL/6 cells, these results suggested an unimpaired Th2 polarization during an established infection withL. major. To further define the observed IL-4 receptor-independent Th2 cell phenotype, we determined an independent Th2 marker, the IL-12 receptor beta-2 (IL-12Rβ2)-specific transcript levels of CD4+T cells. Confirming Th2 polarization inL. major-infected IL-4Rα−/−mice, comparable IL-12Rβ2 message levels between CD4+T cells from infected IL-4Rα−/−mice and Th2 cells from BALB/c mice were found, whereas Th1-polarized C57BL/6 cells showed strikingly increased IL-12Rβ2 expression levels. These results indicate that signals mediated by the IL-4Rα are not necessary to induce and sustain an efficient IL-4 expression and Th2 polarization inL. major-infected BALB/c mice and suggest that IL-4Rα-independent mechanisms underlie the default Th2 development inL. major-infected BALB/c mice.