bluuebunny/crossref_metadata_2025_split huggingface dataset:
Non-Growth-Associated Demethylation of Dimethylsulfoniopropionate by (Homo)acetogenic Bacteria
Authors: Jansen Michael , Hansen Theo A. 
Published: July 2002

ABSTRACT

The demethylation of the algal osmolyte dimethylsulfoniopropionate (DMSP) to methylthiopropionate (MTPA) by (homo)acetogenic bacteria was studied. Five
Eubacterium limosum
strains (including the type strain),
Sporomusa ovata
DSM 2662
T
,
Sporomusa sphaeroides
DSM 2875
T
, and
Acetobacterium woodii
DSM 1030
T
were shown to demethylate DMSP stoichiometrically to MTPA. The (homo)acetogenic fermentation based on this demethylation did not result in any significant increase in biomass. The analogous demethylation of glycine betaine to dimethylglycine does support growth of acetogens. In batch cultures of
E. limosum
PM31 DMSP and glycine betaine were demethylated simultaneously. In mixed substrates experiments with fructose-DMSP or methanol-DMSP, DMSP was used rapidly but only after exhaustion of the fructose or the methanol. In steady-state fructose-limited chemostat cultures (at a dilution rate of 0.03 h
−1
) with DMSP as a second reservoir substrate, DMSP was biotransformed to MTPA but this did not result in higher biomass values than in cultures without DMSP; cells from such cultures demethylated DMSP at rates of approximately 50 nmol min
−1
mg of protein
−1
, both after growth in the presence of DMSP and after growth in its absence. In cell extracts of glycine betaine-grown strain PM31, DMSP demethylation activities of 21 to 24 nmol min
−1
mg of protein
−1
were detected with tetrahydrofolate as a methyl acceptor; the activities seen with glycine betaine were approximately 10-fold lower. A speculative explanation for the demethylation of DMSP without an obvious benefit for the organism is that the DMSP-demethylating activity is catalyzed by the glycine betaine-demethylating enzyme and that a transport-related factor, in particular a higher energy demand for DMSP transport across the cytoplasmic membrane than for glycine betaine transport, may reduce the overall ATP yield of the fermentation to virtually zero.
Gene Integration and Expression and Extracellular Secretion of
Erwinia chrysanthemi
Endoglucanase CelY (
celY
) and CelZ (
celZ
) in Ethanologenic
Klebsiella oxytoca
P2
Authors: Zhou Shengde , Davis F. C. , Ingram L. O. 
Published: July 2002

ABSTRACT

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of
Klebsiella oxytoca
M5A1 with the native ability to metabolize cellobiose, the need for supplemental β-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes
celY
and
celZ
from
Erwinia chrysanthemi
have been functionally integrated into the chromosome of P2 using surrogate promoters from
Zymomonas mobilis
for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.
EndB, a Multidomain Family 44 Cellulase from
Ruminococcus flavefaciens
17, Binds to Cellulose via a Novel Cellulose-Binding Module and to Another
R. flavefaciens
Protein via a Dockerin Domain
Authors: Rincón Marco T. , McCrae Sheila I. , Kirby James , Scott Karen P. , Flint Harry J. 
Published: July 2002

ABSTRACT

The mechanisms by which cellulolytic enzymes and enzyme complexes in
Ruminococcus
spp. bind to cellulose are not fully understood. The product of the newly isolated cellulase gene
endB
from
Ruminococcus flavefaciens
17 was purified as a His-tagged product after expression in
Escherichia coli
and found to be able to bind directly to crystalline cellulose. The ability to bind cellulose is shown to be associated with a novel cellulose-binding module (CBM) located within a region of 200 amino acids that is unrelated to known protein sequences. EndB (808 amino acids) also contains a catalytic domain belonging to glycoside hydrolase family 44 and a C-terminal dockerin-like domain. Purified EndB is also shown to bind specifically via its dockerin domain to a polypeptide of ca. 130 kDa present among supernatant proteins from Avicel-grown
R. flavefaciens
that attach to cellulose. The protein to which EndB attaches is a strong candidate for the scaffolding component of a cellulosome-like multienzyme complex recently identified in this species (S.-Y. Ding et al., J. Bacteriol. 183:1945–1953, 2001). It is concluded that binding of EndB to cellulose may occur both through its own CBM and potentially also through its involvement in a cellulosome complex.
Recovery and Analysis of Formyltetrahydrofolate Synthetase Gene Sequences from Natural Populations of Acetogenic Bacteria
Authors: Leaphart Adam B. , Lovell Charles R. 
Published: July 2002

ABSTRACT

Primers for PCR amplification of partial (1,102 of 1,680 bp) formyltetrahydrofolate synthetase (FTHFS) gene sequences were developed and tested. Partial FTHFS sequences were successfully amplified from DNA from pure cultures of known acetogens, from other FTHFS-producing organisms, from the roots of the smooth cordgrass,
Spartina alterniflora
, and from fresh horse manure. The amplimers recovered were cloned, their nucleotide sequences were determined, and their translated amino acid sequences were used to construct phylogenetic trees. We found that FTHFS sequences from homoacetogens formed a monophyletic cluster that did not contain sequences from nonhomoacetogens and that FTHFS sequences appear to be informative regarding major physiological features of FTHFS-producing organisms.
Prevalence, Antibiotic Susceptibility, and Diversity of
Escherichia coli
O157:H7 Isolates from a Longitudinal Study of Beef Cattle Feedlots
Authors: Galland John C. , Hyatt Doreene R. , Crupper Scott S. , Acheson David W. 
Published: July 2002

ABSTRACT

Prevalence, antibiotic susceptibility, and genetic diversity were determined for
Escherichia coli
O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of
E. coli
O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were
eaeA
gene positive.
Escherichia coli
O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat
E. coli
infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among
E. coli
isolates identified initially by C/LA as
E. coli
O157:H7. These results demonstrate that better ways of screening and confirming
E. coli
O157:H7 isolates are required for accurate determination of prevalence.
Interaction of the ΦHSIC Virus with Its Host: Lysogeny or Pseudolysogeny?
Authors: Williamson S. J. , McLaughlin M. R. , Paul J. H. 
Published: July 2002

ABSTRACTThe marine phage ΦHSIC has been previously reported to enter into a lysogenic relationship with its host, HSIC, identified asListonella pelagia. This phage produces a variety of plaques on its host, including turbid and haloed plaques, from which lysogens were previously isolated. These lysogens were unstable during long-term storage at −80°C and were lost. When HSIC was reinfected with phage ΦHSIC, pseudolysogen-like interactions between the phage and its host were observed. The cells (termed HSIC-2 or HSIC-2e) produced high viral titers (1011ml−1) in the absence of inoculating phage and yet reached culture densities of nearly 109ml−1. Prophages were not induced by mitomycin C or the polyaromatic hydrocarbon naphthalene in cells harboring such infections. However, such cells were homoimmune to superinfection. Colonies hybridized strongly with a gene probe from a 100-bp fragment of the ΦHSIC genome, while the host did not. Analysis of chromosomal DNA preparations suggested the presence of a chromosomally integrated prophage. Phage adsorption experiments suggested that HSIC-2 was adsorption impaired. Because of the chromosomal prophage integration and homoimmunity, we interpret these results to indicate that ΦHSIC establishes a lysogenic relationship with its host that involves an extremely high level of spontaneous induction. This could be caused by a weak repressor of phage production. Additionally, poor phage adsorption of HSIC-2 compared to the wild type probably helped maintain this pseudolysogen-like relationship. In many ways, pseudolysogenic phage-host interactions may provide a paradigm for phage-host interactions in the marine environment.
Sequence Analysis of Insecticidal Genes from
Xenorhabdus nematophilus
PMFI296
Authors: Morgan J. Alun W. , Sergeant Martin , Ellis Debbie , Ousley Margaret , Jarrett Paul 
Published: July 2002

ABSTRACT

Three strains of
Xenorhabdus nematophilus
showed insecticidal activity when fed to
Pieris brassicae
(cabbage white butterfly) larvae. From one of these strains (
X. nematophilus
PMFI296) a cosmid genome library was prepared in
Escherichia coli
and screened for oral insecticidal activity. Two overlapping cosmid clones were shown to encode insecticidal proteins, which had activity when expressed in
E. coli
(50% lethal concentration [LC
50
] of 2 to 6 μg of total protein/g of diet). The complete sequence of one cosmid (cHRIM1) was obtained. On cHRIM1, five genes (
xptA1, -A2, -B1, -C1
, and
-D1
) showed homology with up to 49% identity to insecticidal toxins identified in
Photorhabdus luminescens
, and also a smaller gene (
chi
) showed homology to a putative chitinase gene (38% identity). Transposon mutagenesis of the cosmid insert indicated that the genes
xptA2, xptD1
, and
chi
were not important for the expression of insecticidal activity toward
P. brassicae
. One gene (
xptA1
) was found to be central for the expression of activity, and the genes
xptB1
and
xptC1
were needed for full activity. The location of these genes together on the chromosome and therefore present on a single cosmid insert probably accounted for the detection of insecticidal activity in this
E. coli
clone. Although multiple genes may be needed for full activity,
E. coli
cells expressing the
xptA1
gene from the bacteriophage lambda
P
L
promoter were shown to have insecticidal activity (LC
50
of 112 μg of total protein/g of diet). This is contrary to the toxin genes identified in
P. luminescens
, which were not insecticidal when expressed individually in
E. coli
. High-level gene expression and the use of a sensitive insect may have aided in the detection of insecticidal activity in the
E. coli
clone expressing
xptA1
. The location of these toxin genes and the chitinase gene and the presence of mobile elements (insertion sequence) and tRNA genes on cHRIM1 indicates that this region of DNA represents a pathogenicity island on the genome of
X. nematophilus
PMFI296.
Energy-Based Dynamic Model for Variable Temperature Batch Fermentation by
Lactococcus lactis
Authors: Dougherty Daniel P. , Breidt, Frederick , McFeeters Roger F. , Lubkin Sharon R. 
Published: July 2002

ABSTRACT

We developed a mechanistic mathematical model for predicting the progression of batch fermentation of cucumber juice by
Lactococcus lactis
under variable environmental conditions. In order to overcome the deficiencies of presently available models, we use a dynamic energy budget approach to model the dependence of growth on present as well as past environmental conditions. When parameter estimates from independent experimental data are used, our model is able to predict the outcomes of three different temperature shift scenarios. Sensitivity analyses elucidate how temperature affects the metabolism and growth of cells through all four stages of fermentation and reveal that there is a qualitative reversal in the factors limiting growth between low and high temperatures. Our model has an applied use as a predictive tool in batch culture growth. It has the added advantage of being able to suggest plausible and testable mechanistic assumptions about the interplay between cellular energetics and the modes of inhibition by temperature and end product accumulation.
Clustering of Clinical Strains of
Helicobacter pylori
Analyzed by Two-Dimensional Gel Electrophoresis
Authors: Enroth Helena , Åkerlund Thomas , Sillén Anna , Engstrand Lars 
Published: July 2002

ABSTRACT

Strain variations of
Helicobacter pylori
have been tested by numerous methods and compared among different patient groups. The aim of this study was to investigate whether
H. pylori
expresses disease-specific proteins that can be detected by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE).
H. pylori
strains isolated from duodenal ulcer, gastric cancer, and gastritis patients were analyzed. Extensive variation in spot patterns was observed between the strains, but a dendrogram analysis revealed that some strains within each disease group clustered together. Eight proteins were sequenced and found in the
H. pylori
genome sequence. 2-D PAGE is a useful method for studies of protein expression and for highlighting the extensive strain variation that
H. pylori
exhibits.
Production of a Mouse Antiserum toBacteroides fragilisEnterotoxin Using a Recombinant Enterotoxin Precursor
Authors: Sandini Silvia , d'Abusco Anna Scotto , La Valle Roberto , Pantosti Annalisa 
Published: July 2002

ABSTRACTThe precursor of theBacteroides fragilismetalloprotease enterotoxin was cloned and expressed inEscherichia coli, which was not able to process the precursor into the biologically active enterotoxin. Mouse antiserum elicited to the recombinant precursor reacted with the purified enterotoxin and with a crude enterotoxin preparation from an enterotoxigenic strain. The antiserum neutralized the cytotoxic activity of the enterotoxin in HT-29 cells.
Type 1 Fimbriation and Its Phase Switching in Diarrheagenic
Escherichia coli
Strains
Authors: Iida Ken-Ichiro , Mizunoe Yoshimitsu , Wai Sun Nyunt , Yoshida Shin-Ichi 
Published: July 2002

ABSTRACT

Type 1 fimbriae can be expressed by most
Escherichia coli
strains and mediate mannose-sensitive (MS) adherence to mammalian epithelial cells. However, the role of type 1 fimbriae in enteric pathogenesis has been unclear. Expression of type 1 fimbriae in
E. coli
is phase variable and is associated with the inversion of a short DNA element (
fim
switch). Forty-six strains of diarrheagenic
E. coli
were examined for the expression of type 1 fimbriae. Only four of these strains were originally type 1 fimbriated. Seventeen strains, originally nonfimbriated, expressed type 1 fimbriae in association with off-to-on inversion of the
fim
switch, after serial passages in static culture. The switching frequencies of these strains, from fimbriate to nonfimbriate, were greater than that of the laboratory strain
E. coli
K-12. None of the 16 strains of serovar O157:H7 or O157:H
−
expressed type 1 fimbriae after serial passages in static culture. The nucleotide sequence analysis of the
fim
switch region revealed that all of the O157:H7 and O157:H
−
strains had a 16-bp deletion in the invertible element, and the
fim
switch was locked in the “off” orientation. The results suggest that expression of type 1 fimbriae may be regulated differently in different
E. coli
pathogens causing enteric infections.
Identification ofAcinetobacterIsolates from Species Belonging to theAcinetobacter calcoaceticus-Acinetobacter baumanniiComplex with Monoclonal Antibodies Specific for O Antigens of Their Lipopolysaccharides
Authors: Pantophlet Ralph , Severin Juliëtte A. , Nemec Alexandr , Brade Lore , Dijkshoorn Lenie , Brade Helmut 
Published: July 2002

ABSTRACTThe unambiguous identification ofAcinetobacterstrains, particularly those belonging to theAcinetobacter calcoaceticus-Acinetobacter baumanniicomplex, is often hindered by their close geno- and phenotypic relationships. In this study, monoclonal antibodies (MAbs) against the O antigens of the lipopolysaccharides from strains belonging to theA. calcoaceticus-A. baumanniicomplex were generated after the immunization of mice with heat-killed bacteria and shown by enzyme immunoassays and Western blotting to be specific for their homologous antigens. Since theA. calcoaceticus-A. baumanniicomplex comprises the most clinically relevant species, the MAbs were subsequently tested in dot and Western blots with proteinase K-treated lysates from a large collection ofAcinetobacterisolates (n= 631) to determine whether the antibodies could be used for the reliable identification of strains from this complex. Reactivity was observed with 273 of the 504 isolates (54%) from theA. calcoaceticus-A. baumanniicomplex which were included in this study. Isolates which reacted positively did so with only one antibody; no reactivity was observed with isolates not belonging to theA. calcoaceticus-A. baumanniicomplex (n= 127). To identify additional putative O serotypes, isolates from theA. calcoaceticus-A. baumanniicomplex which showed no MAb reactivity were subjected to a method that enables the detection of lipid A moieties in lipopolysaccharides with a specific MAb on Western blots following acidic treatment of the membrane. By this method, additional serotypes were indeed identified, thus indicating which strains to select for future immunizations. This study contributes to the completion of a serotype-based identification scheme forAcinetobacterspecies, in particular, those which are presently of the most clinical importance.
Tripeptidyl Peptidase II Promotes Maturation of Caspase-1 inShigella flexneri-Induced Macrophage Apoptosis
Authors: Hilbi Hubert , Puro Robyn J. , Zychlinsky Arturo 
Published: July 2002

ABSTRACTThe invasive enteropathogenic bacteriumShigella flexneriactivates apoptosis in macrophages.Shigella-induced apoptosis requires caspase-1. We demonstrate here that tripeptidyl peptidase II (TPPII), a cytoplasmic, high-molecular-weight protease, participates in the apoptotic pathway triggered byShigella. The TPPII inhibitor Ala-Ala-Phe-chloromethylketone (AAF-cmk) andclasto-lactacystin β-lactone (lactacystin), an inhibitor of both TPPII and the proteasome, protected macrophages fromShigella-induced apoptosis. AAF-cmk was more potent than lactacystin and irreversibly blockedShigella-induced apoptosis by 95% at a concentration of 1 μM. Conversely, peptide aldehyde and peptide vinylsulfone proteasome inhibitors had little effect onShigella-mediated cytotoxicity. Both AAF-cmk and lactacystin prevented the maturation of pro-caspase-1 and its substrate pro-interleukin 1β inShigella-infected macrophages, indicating that TPPII is upstream of caspase-1. Neither of these compounds directly inhibited caspase-1. AAF-cmk and lactacystin did not impair macrophage phagocytosis or the ability ofShigellato escape the macrophage phagosome. TPPII was also found to be involved in apoptosis induced by ATP and the protein kinase inhibitor staurosporine. We propose that TPPII participates in apoptotic pathways.
Differential Host Inflammatory Responses to Viable Versus Antibiotic-Killed Bacteria in Experimental Microbial Sepsis
Authors: Silverstein Richard , Wood John G. , Xue Qiao , Norimatsu Mari , Horn David L. , Morrison David C. 
Published: July 2002

ABSTRACTStaphylococcus aureuskilled during imipenem or ceftazidime chemotherapy in mice elicited an early release of tumor necrosis factor alpha (TNF-α) into the systemic circulation. This response was coincident in time with an increase in leukocyte-endothelium adhesive interactions in the microvasculature. Equivalent responses were not observed without the antibiotic treatment (imipenem or ceftazidime). Protective efficacy of the same antibiotic treatment was markedly diminished ind-galactosamine-treated mice compared to controls; e.g., it dropped from 2,000-fold to 70-fold with 4 mg of imipenem per kg given at the time of challenge. Nevertheless, protection was quantitatively restored upon concurrent administration of neutralizing anti-TNF-α antibody or 4 mg of dexamethasone per kg to these TNF-α-hypersensitive mice. Importantly, protection afforded by dexamethasone was not seen when the animals were challenged with viable organisms but without the concurrent administration of antibiotic. An early TNF-α response could also be demonstrated upon challenge withEscherichia coli, but in this instance, neither the timing nor the magnitude of that response was influenced by treatment with these antibiotics. We conclude from these studies that the inflammatory response to viable versus killed bacteria may differ markedly depending on the particular bacterium, host sensitivity to TNF-α, and possibly the Gram stain classification.
Evaluation of an Isogenic Major Outer Membrane Protein-Deficient Mutant in the Human Model of
Haemophilus ducreyi
Infection
Authors: Throm Robert E. , Al-Tawfiq Jaffar A. , Fortney Kate R. , Katz Barry P. , Hood Antoinette F. , Slaughter Clive A. , Hansen Eric J. , Spinola Stanley M. 
Published: July 2002

ABSTRACT

Haemophilus ducreyi
expresses 2 OmpA homologs, designated MOMP and OmpA2, whose genes are arranged in tandem on the chromosome. Northern blot analysis indicated that
momp
and
ompA2
are transcribed independently. Sequences of the
momp
open reading frame (ORF) lacking the transcriptional start site were amplified by PCR, and an Ω-Km2 cassette was ligated into the ORF. A plasmid containing this construction was electroporated into
H. ducreyi
35000HP, and an isogenic MOMP-deficient mutant (35000HP-SMS2) was generated by allele exchange. In Southern blotting, 35000HP-SMS2 contained one copy of the Ω-Km2 cassette in
momp
. 35000HP and 35000HP-SMS2 had similar outer membrane protein (OMP) and lipooligosaccharide profiles and growth rates except for up-regulation of a putative porin protein in the mutant. Five subjects were inoculated with three doses of live 35000HP-SMS2 on one arm and two doses of live 35000HP and one dose of a heat-killed control on the other arm in a double-blind escalating dose-response trial. Pustules developed at 7 of 10 sites inoculated with 35000HP and at 6 of 15 sites inoculated with 35000HP-SMS2 (
P
= 0.14). 35000HP and 35000HP-SMS2 were recovered at similar rates from daily surface cultures and semiquantitative cultures. The data suggest that expression of MOMP is not required for pustule formation by
H. ducreyi
in the human model of infection.