Mutation of PEB4 alters the outer membrane protein profile of Campylobacter jejuni

Although anaerobic bioremediation of chlorinated organic contaminants in the environment often requires exogenous supply of hydrogen as an electron donor, little is known about the ability of hydrogen-producing bacteria to grow in the presence of chlorinated solvents. In this study, 18 Clostridium strains including nine uncharacterized isolates originating from chlorinated solvent contaminated groundwater were tested to determine their ability to fermentatively produce hydrogen in the presence of three common chlorinated aliphatic groundwater contaminants: 1,2-dichloroethane (DCA), 1,1,2-trichloroethane (TCA), and tetrachloroethene (PCE). All strains produced hydrogen in the presence of at least 7.4 mM DCA, 2.4 mM TCA, and 0.31 mM PCE. Some strains produced hydrogen in media containing concentrations as high as 29.7 mM DCA, 9.8 mM TCA, and 1.1 mM PCE. None of the strains biotransformed chlorinated solvents under the conditions tested. Results demonstrate that many Clostridium species are chlorinated solvent tolerant, producing hydrogen even in the presence of high concentrations of DCA, TCA, and PCE. These findings have important implications for bioremediation of contaminated soil and groundwater.     

Isolation and sources of 'blown pack' spoilage clostridia in beef abattoirs

Aims:  (i) To evaluate methods for isolation and molecular detection of blown pack spoilage (BPS) clostridia and (ii) to survey beef abattoirs for sources and distributions of Clostridium estertheticum and Cl. gasigenes .
Methods and Results:  Molecular detection and conventional isolation methods were used to detect and recover BPS associated clostridia ( Cl. estertheticum and Cl. gasigenes ), from four commercial Irish beef abattoirs and their environments, during a one year study. DNA-based methods detected 218 Cl. estertheticum and 300 Cl. gasigenes , from 1680 samples, whereas culture-methods only yielded 17 Cl. estertheticum and 176 Cl. gasigenes isolates. BPS Clostridia were frequently detected in beef abattoirs and their environments, especially at areas prior to hide removal. The study noted a higher percentage of positive samples during the month of May (38·6%).
Conclusions:  (i) DNA-based techniques are the most reliable ways to determine the presence of these organisms in various samples and (ii) hides and faeces are the main reservoirs of BPS clostridia in the abattoirs.
Significance and Impact of the Study:  This paper provides useful information to detect BPS organisms, as well as to develop a science-based control strategy of the problem.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h, respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l⁻¹ was produced without by-products from 500 g d-psicose l⁻¹ after 6 h.     

Cellulolytic, fermentative, and methanogenic guilds in benthic periphyton mats from the Florida Everglades

Phosphorus enrichment caused by runoff from agricultural areas has resulted in ecosystem-level changes in the northern Florida Everglades, including a loss of periphyton mats from nutrient-impacted areas. The potential for methanogenesis resulting from the anaerobic decomposition of cellulose and fermentation products, and the microorganisms responsible for these processes, were studied in mats from a region not impacted by nutrient enrichment. Methane was produced from periphyton incubated with cellulose, propionate, butyrate, and formate, with an accumulation of fatty acids in incubations. The accumulation of fatty acids may have been caused by the inhibition of syntrophic oxidation, a potentially significant route for methane production in soils. Sequence analysis of 16S rRNA genes characteristic of Clostridium, the primary genus responsible for anaerobic decomposition and fermentation in soils of the area, indicated that Clostridium Cluster I assemblages present in the mat differed from those in the soils of the area. Significantly, sequences characteristic of the Clostridium group that dominates the soils of the area, group XIV, were not detected in the mat. These results indicate that benthic periphyton is probably a significant source of methane in the Everglades, and the responsible microorganisms differ significantly from those in the soils of the area.     

Functional characterization and mutation analysis of family 11, Carbohydrate-Binding Module (<Emphasis Type="Italic">Ct</Emphasis>CBM11) of cellulosomal bifunctional cellulase from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

The non-catalytic, family 11 carbohydrate binding module (CtCBM11) belonging to a bifunctional cellulosomal cellulase from Clostridium thermocellum was hyper-expressed in E. coli and functionally characterized. Affinity electrophoresis of CtCBM11 on nondenaturing PAGE containing cellulosic polysaccharides showed binding with β-glucan, lichenan, hydroxyethyl cellulose and carboxymethyl cellulose. In order to elucidate the involvement of conserved aromatic residues Tyr 22, Trp 65 and Tyr 129 in the polysaccharide binding, site-directed mutagenesis was carried out and the residues were changed to alanine. The results of affinity electrophoresis and binding adsorption isotherms showed that of the three mutants Y22A, W65A and Y129A of CtCBM11, two mutants Y22A and Y129A showed no or reduced binding affinity with polysaccharides. These results showed that tyrosine residue 22 and 129 are involved in the polysaccharide binding. These residues are present in the putative binding cleft and play a critical role in the recognition of all the ligands recognized by the protein.     

Proteomic profile changes in membranes of ethanol-tolerant <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, has potential for commercial exploitation in converting fibrous biomass to ethanol. However, ethanol concentrations above 1% (w/v) are inhibitory to growth and fermentation, and this limits industrial application of the organism. Recent work with ethanol-adapted strains suggested that protein changes occurred during ethanol adaptation, particularly in the membrane proteome. A two-stage Bicine-doubled sodium dodecyl sulfate-polyacrylamide gel electrophoresis protocol was designed to separate membrane proteins and circumvent problems associated with membrane protein analysis using traditional gel-based proteomics approaches. Wild-type and ethanol-adapted C. thermocellum membranes displayed similar spot diversity and approximately 60% of proteins identified from purified membrane fractions were observed to be differentially expressed in the two strains. A majority (73%) of differentially expressed proteins were down-regulated in the ethanol-adapted strain. Based on putative identifications, a significant proportion of these down-regulated proteins were involved with carbohydrate transport and metabolism. Approximately one-third of the up-regulated proteins in the ethanol-adapted species were associated with chemotaxis and signal transduction. Overall, the results suggested that membrane-associated proteins in the ethanol-adapted strain are either being synthesized in lower quantities or not properly incorporated into the cell membrane.     

Probiotic properties of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Bifidobacterium</Emphasis> strains isolated from porcine gastrointestinal tract

One strain of Lactobacillus salivarius, two strains of Lactobacillus reuteri and Lactobacillus amylovorus, and two strains of Bifidobacterium thermacidophilum with antagonistic effect against Clostridium perfringens were isolated from porcine gastrointestinal tract. Isolates were assayed for their ability to survive in synthetic gastric juice at pH 2.5 and were examined for their ability to grow on agar plate containing porcine bile extract. There was a large variation in the survival of the isolates in gastric juice and growth in the medium containing 0.3% (w/v) bile. L. salivarius G11 and L. amylovorus S6 adhered to the HT-29 epithelial cell line. Cell-free supernatant of L. amylovorus S6 showed higher antagonistic activity as effective as the antibiotics such as neomycin, chlortetracycline, and oxytetracycline against bacterial pathogens including C. perfringens, Salmonella typhimurium, Staphylococcus aureus, Vibrio cholerae, Edwardsiella tarda, and Aeromonas salmonicida subsp. salmonicida.     

The effect of peroxyacetic acid-based sanitizer, heat and ultrasonic waves on the survival of Clostridium estertheticum spores in vitro

AIM: To determine the effect of selected physical and chemical treatments on the survival of 'blown pack'-causing Clostridium estertheticum. METHODS AND RESULTS: The study investigated the survival of the spores of 'blown pack'-causing C. estertheticum following the four treatments, which include: heat alone, ultrasound followed by heat treatment, peroxyacetic acid (POAA)-based sanitizer followed by heat treatment and POAA sanitizer followed by heat treatment in the presence of 20% animal fat. No C. estertheticum survivors were recovered in spore preparations that underwent either of the two treatments with the sanitizer, resulting in the inactivation of 4 to 5 log CFU ml(-1) of spores. Similarly, no survivors were detected in spore preparations that were treated with the sanitizer for 5 min at room temperature without further heat treatment. When using heat alone and ultrasound followed by heat treatment, complete spore inactivation did not occur for spores heated at times and temperature combinations other than 240 s at 100 degrees C. CONCLUSIONS: POAA sanitizer used with or without heat is capable of in vitro inactivation of at least 4 log CFU ml(-1)C. estertheticum spores. SIGNIFICANCE AND IMPACT OF THE STUDY: The data generated in the study provide background information for controlling 'blown pack'-causing clostridia on dressed carcasses and in meat plant environment.     

Identification of structural genes for Clostridium botulinum type C neurotoxin-converting phage particles

The structural genes for strain C-Stockholm (c-st) phage particles, a representative type C toxin-converting phage of Clostridium botulinum, have been determined. First, by determining the N-terminal amino acid sequences of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) bands of c-st phage particles, it became clear that four proteins, 14, 25, 32 and 42 kDa, are the products of the ORFs, cst166, cst165, cst160 and cst164, respectively, of the c-st phage genome. The Western blot analyses reacting these phage bands with an antiphage serum prepared previously indicated that the products of cst165 and cst160 are the main proteins of the phage particles. Then, six candidates for the phage structural proteins, including cst165 and cst160 gene products, were prepared as recombinant proteins. Also, the protein corresponding to the cst164 gene product was excised from SDS-PAGE gels. The antibodies against these seven proteins were prepared in rabbits, and finally, the reaction of these antibodies to the c-st phage particles was analyzed by electron microscopy. It was concluded that a sheath protein and a head protein of the c-st phage are the products of genes cst160 and cst165, respectively, and that these two proteins are conserved in the other three converting phages, but not in the nonconverting phage.     

Complete activity profile of Clostridium acetobutylicum [FeFe]-hydrogenase and kinetic parameters for endogenous redox partners

In Clostridium acetobutylicum, [FeFe]-hydrogenase is involved in hydrogen production in vivo by transferring electrons from physiological electron donors, ferredoxin and flavodoxin, to protons. In this report, by modifications of the purification procedure, the specific activity of the enzyme has been improved and its complete catalytic profile in hydrogen evolution, hydrogen uptake, proton/deuterium exchange and para-H2/ortho-H2 conversion has been determined. The major ferredoxin expressed in the solvent-producing C. acetobutylicum cells was purified and identified as encoded by ORF CAC0303. Clostridium acetobutylicum recombinant holoflavodoxin CAC0587 was also purified. The kinetic parameters of C. acetobutylicum [FeFe]-hydrogenase for both physiological partners, ferredoxin CAC0303 and flavodoxin CAC0587, are reported for hydrogen uptake and hydrogen evolution activities.