bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA
Authors: Liu W T , Marsh T L , Cheng H , Forney L J
Published: January 2020

A quantitative molecular technique was developed for rapid analysis of microbial community diversity in various environments. The technique employed PCR in which one of the two primers used was fluorescently labeled at the 5' end and was used to amplify a selected region of bacterial genes encoding 16S rRNA from total community DNA. The PCR product was digested with restriction enzymes, and the fluorescently labeled terminal restriction fragment was precisely measured by using an automated DNA sequencer. Computer-simulated analysis of terminal restriction fragment length polymorphisms (T-RFLP) for 1,002 eubacterial sequences showed that with proper selection of PCR primers and restriction enzymes, 686 sequences could be PCR amplified and classified into 233 unique terminal restriction fragment lengths or "ribotypes." Using T-RFLP, we were able to distinguish all bacterial strains in a model bacterial community, and the pattern was consistent with the predicted outcome. Analysis of complex bacterial communities with T-RFLP revealed high species diversity in activated sludge, bioreactor sludge, aquifer sand, and termite guts; as many as 72 unique ribotypes were found in these communities, with 36 ribotypes observed in the termite guts. The community T-RFLP patterns were numerically analyzed and hierarchically clustered. The pattern derived from termite guts was found to be distinctly different from the patterns derived from the other three communities. Overall, our results demonstrated that T-RFLP is a powerful tool for assessing the diversity of complex bacterial communities and for rapidly comparing the community structure and diversity of different ecosystems.
Specific detection and confirmation of Campylobacter jejuni by DNA hybridization and PCR
Authors: Ng L K , Kingombe C I , Yan W , Taylor D E , Hiratsuka K , Malik N , Garcia M M
Published: January 2020

Conventional detection and confirmation methods for Campylobacter jejuni are lengthy and tedious. A rapid hybridization protocol in which a 1,475-bp chromogen-labelled DNA probe (pDT1720) and Campylobacter strains filtered and grown on 0.22-micron-pore-size hydrophobic grid membrane filters (HGMFs) are used was developed. Among the environmental and clinical isolates of C. jejuni, Campylobacter coli, Campylobacter jejuni subsp. doylei, Campylobacter lari, and Arcobacter nitrofigilis and a panel of 310 unrelated bacterial strains tested, only C. jejuni and C. jejuni subsp. doylei isolates hybridized with the probe under stringent conditions. The specificity of the probe was confirmed when the protocol was applied to spiked skim milk and chicken rinse samples. Based on the nucleotide sequence of pDT1720, a pair of oligonucleotide primers was designed for PCR amplification of DNA from Campylobacter spp. and other food pathogens grown overnight in selective Mueller-Hinton broth with cefoperazone and growth supplements. All C. jejuni strains tested, including DNase-producing strains and C. jejuni subsp. doylei, produced a specific 402-bp amplicon, as confirmed by restriction and Southern blot analysis. The detection range of the assay was as low as 3 CFU per PCR to as high as 10(5) CFU per PCR for pure cultures. Overnight enrichment of chicken rinse samples spiked initially with as little as approximately 10 CFU/ml produced amplicons after the PCR. No amplicon was detected with any of the other bacterial strains tested or from the chicken background microflora. Since C. jejuni is responsible for 99% of Campylobacter contamination in poultry, PCR and HGMF hybridization were performed on naturally contaminated chicken rinse samples, and the results were compared with the results of conventional cultural isolation on Preston agar. All samples confirmed to be culture positive for C. jejuni were also identified by DNA hybridization and PCR amplification, thus confirming that these DNA-based technologies are suitable alternatives to time-consuming conventional detection methods. DNA hybridization, besides being sensitive, also has the potential to be used in direct enumeration of C. jejuni organisms in chicken samples.
Small-subunit rRNA genes and in situ hybridization with oligonucleotides specific for the bacterial symbionts in the larvae of the bryozoan Bugula neritina and proposal of "Candidatus endobugula sertula"
Authors: Haygood M G , Davidson S K
Published: January 2020

Larvae of the bryozoan Bugula neritina harbor bacterial symbionts. These symbionts were identified as a novel species of gamma-proteobacterium, based on ribosomal small-subunit rRNA gene sequences. In situ hybridization with oligonucleotides specific for the symbiont confirmed the origin of the sequence. The taxonomic status "Candidatus Endobugula sertula" is proposed for the larval symbiont.
Methane and trichloroethylene oxidation by an estuarine methanotroph, Methylobacter sp. strain BB5.1
Authors: Smith K S , Costello A M , Lidstrom M E
Published: January 2020

An estuarine methanotroph was isolated from sediment enrichments and designated Methylobacter sp. strain BB5.1. In cells grown on medium with added copper, oxidation of methane and trichloroethylene occurred with similar Ks values, but the Vmax for trichloroethylene oxidation was only 0.1% of the methane oxidation Vmax. Cells grown on low-copper medium did not oxidize trichloroethylene and showed a variable rate of methane oxidation.
Dissimilatory nitrate reduction in anaerobic sediments leading to river nitrite accumulation
Authors: Kelso B , Smith R V , Laughlin R J , Lennox S D
Published: January 2020

Recent studies on Northern Ireland rivers have shown that summer nitrite (NO(inf2)(sup-)) concentrations greatly exceed the European Union guideline of 3 (mu)g of N liter(sup-1) for rivers supporting salmonid fisheries. In fast-flowing aerobic small streams, NO(inf2)(sup-) is thought to originate from nitrification, due to the retardation of Nitrobacter strains by the presence of free ammonia. Multiple regression analyses of NO(inf2)(sup-) concentrations against water quality variables of the six major rivers of the Lough Neagh catchment in Northern Ireland, however, suggested that the high NO(inf2)(sup-) concentrations found in the summer under warm, slow-flow conditions may result from the reduction of NO(inf3)(sup-). This hypothesis was supported by field observations of weekly changes in N species. Here, reduction of NO(inf3)(sup-) was observed to occur simultaneously with elevation of NO(inf2)(sup-) levels and subsequently NH(inf4)(sup+) levels, indicating that dissimilatory NO(inf3)(sup-) reduction to NH(inf4)(sup+) (DNRA) performed by fermentative bacteria (e.g., Aeromonas and Vibrio spp.) is responsible for NO(inf2)(sup-) accumulation in these large rivers. Mechanistic studies in which (sup15)N-labelled NO(inf3)(sup-) in sediment extracts was used provided further support for this hypothesis. Maximal concentrations of NO(inf2)(sup-) accumulation (up to 1.4 mg of N liter(sup-1)) were found in sediments deeper than 6 cm associated with a high concentration of metabolizable carbon and anaerobic conditions. The (sup15)N enrichment of the NO(inf2)(sup-) was comparable to that of the NO(inf3)(sup-) pool, indicating that the NO(inf2)(sup-) was predominantly NO(inf3)(sup-) derived. There is evidence which suggests that the high NO(inf2)(sup-) concentrations observed arose from the inhibition of the DNRA NO(inf2)(sup-) reductase system by NO(inf3)(sup-).
Genetic evidence for nonrandom sorting of mitochondria in the basidiomycete Agrocybe aegerita
Authors: Barroso G , Labarère J
Published: January 2020

We studied mitochondrial transmission in the homobasidiomycete Agrocybe aegerita during plasmogamy, vegetative growth, and basidiocarp differentiation. Plasmogamy between homokaryons from progeny of three wild-type strains resulted in bidirectional nuclear migration, and the dikaryotization speed was dependent on the nuclear genotype of the recipient homokaryon. Little mitochondrial migration accompanied the nuclear migration. A total of 75% of the dikaryons from the fusion lines had both parental mitochondrial haplotypes (mixed dikaryons), and 25% had only a single haplotype (homoplasmic dikaryons); with some matings, there was a strong bias in favor of one parental haplotype. We demonstrated the heteroplasmic nature of mixed dikaryons by (i) isolating and subculturing apical cells in micromanipulation experiments and (ii) identifying recombinant mitochondrial genomes. This heteroplasmy is consistent with the previously reported suggestion that there is recombination between mitochondrial alleles in A. aegerita. Conversion of heteroplasmons into homoplasmons occurred (i) during long-term storage, (ii) in mycelia regenerated from isolated apical cells, and (iii) during basidiocarp differentiation. Homokaryons that readily accepted foreign nuclei were the most efficient homokaryons in maintaining their mitochondrial haplotype during plasmogamy, long-term storage, and basidiocarp differentiation. This suggests that the mechanism responsible for the nonrandom retention or elimination of a given haplotype may be related to the nuclear genotype or the mitochondrial haplotype or both.
Two cellulases, CelA and CelC, from the polycentric anaerobic fungus Orpinomyces strain PC-2 contain N-terminal docking domains for a cellulase-hemicellulase complex
Authors: Li X L , Chen H , Ljungdahl L G
Published: January 2020

Two cDNAs encoding two cellulases, CelA and CelC, were isolated from a cDNA library of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 constructed in Escherichia coli. Nucleotide sequencing revealed that the celA cDNA (1,558 bp) and celC cDNA (1,628 bp) had open reading frames encoding polypeptides of 459 (CelA) and 449 (CelC) amino acids, respectively. The two cDNAs were 76.9 and 67.7% identical at the nucleotide and amino acid levels, respectively. Analysis of the deduced amino acid sequences showed that starting from the N termini, both CelA and CelC had signal peptides, which were followed by noncatalytic repeated peptide domains (NCRPD) containing two repeated sequences of 33 to 40 amino acid residues functioning as docking domains. The NCRPDs and the catalytic domains were separated by linker sequences. The NCRPDs were homologous to those found in several hydrolases of anaerobic fungi, whereas the catalytic domains were homologous to the catalytic domains of fungal cellobiohydrolases and bacterial endoglucanases. The linker sequence of CelA contained predominantly glutamine and proline residues, while that of CelC contained mainly threonine residues. CelA and CelC did not have a typical cellulose binding domain (CBD). CelA and CelC expressed in E. coli rapidly decreased the viscosity of carboxymethyl cellulose (CMC), indicating that there was endoglucanase activity. In addition, they produced cellobiose from CMC, acid-swollen cellulose, and cellotetraose, suggesting that they had cellobiohydrolase activity. The optimal activity conditions with CMC as the substrate were pH 4.3 to 6.8 and 50 degrees C for CelA and pH 4.6 to 7.0 and 40 degrees C for CelC. Despite the lack of a CBD, CelC displayed a high affinity for microcrystalline cellulose, whereas CelA did not.
Detection of stable pre-rRNA in toxigenic Pseudo-nitzschia species
Authors: Cangelosi G A , Hamlin A M , Marin R , Scholin C A
Published: January 2020

Nucleotide sequence analysis of ribosomal DNA (rDNA) spacer regions is useful for taxonomic comparisons of closely related microorganisms. These regions have been less useful for routine microbial identification and detection, partly because rRNA precursors (pre-rRNAs) in microbial cells are assumed to be too labile to be detectable by high-throughput probe hybridization methods. We characterized the sequence diversity and physiological stability of pre-rRNA in the toxigenic marine diatoms Pseudo-nitzschia australis, P. multiseries, and P. pungens. As with nucleotide sequences of the first internal transcribed spacer (ITS1) reported previously, sequences of ITS2 and the 5' external transcribed spacer (ETS1) exhibited considerable divergence among these species, including large insertions-deletions detectable by PCR-based spacer length analysis. In slot blot hybridization assays on RNA extracted from lysates of Pseudo-nitzschia cells, oligonucleotide probes directed to pre-rRNA spacers generated much stronger signals than did complementary probes directed to the coding strands of the rDNAs, indicating that the pre-rRNA-targeted probes detected multicopy transcripts. A group of probes directed to a discrete 90-base region within the ITS1 pre-rRNA gave no detectable signal, suggesting that this region is degraded early in the rRNA maturation pathway. Other pre-rRNA regions were always detectable and, in marked contrast to prokaryotic systems analyzed in this manner, were stable and abundant in both actively dividing and nondividing cells. Long, multilabeled RNA probes, which would exhibit considerable cross-reactivity if directed to mature rRNA sequences, detected species-specific pre-rRNA sequences from as few as 1,000 cells. Pre-rRNA is a potentially useful molecular target for detecting and identifying Pseudo-nitzschia species and possibly other unicellular eukaryotes as well.
Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species
Authors: Peters M , Heinaru E , Talpsep E , Wand H , Stottmeister U , Heinaru A , Nurk A
Published: January 2020

Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation.
Comparative study of the toxic actions of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane and 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene on the growth and respiratory activity of a microorganism used as a model
Authors: Donato M M , Jurado A S , Antunes-Madeira M C , Madeira V M
Published: January 2020

A strain of Bacillus stearothermophilus was used as a model for a comparative study of the toxic effect of 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane and 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene. Bacterial growth, the O2 consumption rate, and respiration-related enzymatic activities provided quantitative data in agreement with results reported for other systems. The use of this bacterium for screening for chemical toxicity is discussed.
A simple, efficient method for the separation of humic substances and DNA from environmental samples
Authors: Jackson C R , Harper J P , Willoughby D , Roden E E , Churchill P F
Published: January 2020

Three different gels (Sepharose 4B, Sephadex G-200, and Sephadex G-50) were evaluated as a means of removing humic contaminants from DNA extracts of environmental samples. Sepharose 4B gave superior separation of DNA from humics, and DNA purified in this way showed consistently greater amplification than DNA purified by the other materials.
Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium
Authors: Wang S L , Chang W T
Published: January 2020

Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187. The molecular weights of FI and FII were 30,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 60,000 and 30,000, respectively, by gel filtration. The pIs for FI and FII were 5.2 and 4.8, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FI were pH 8, 50 degrees C, pH 6 to 9, and 50 degrees C; those of FII were pH 7, 40 degrees C, pH 5 to 10, and 60 degrees C. The activities of both enzymes were activated by Cu2+; strongly inhibited by Mn2+, Mg2+, and Zn2+; and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol. Both chitinases showed lysozyme activity. The purified enzymes had antibacterial and cell lysis activities with many kinds of bacteria. This is the first report of a bifunctional chitinase/lysozyme from a prokaryote.
Isolation and characterization of a bacteriocin (Butyrivibriocin AR10) from the ruminal anaerobe Butyrivibrio fibrisolvens AR10: evidence in support of the widespread occurrence of bacteriocin-like activity among ruminal isolates of B. fibrisolvens
Authors: Kalmokoff M L , Teather R M
Published: January 2020

Forty-nine isolates of Butyrivibrio fibrisolvens and a single isolate of Butyrivibrio crossotus were screened for the production of inhibitors by a deferred plating procedure. Twenty-five isolates produced factors which, to various degrees, inhibited the growth of the other Butyrivibrio isolates. None of the inhibitory activity was due to bacteriophages. The inhibitory products from 18 of the producing strains were sensitive to protease digestion. Differences in the ranges of activity among the Butyrivibrio isolates and protease sensitivity profiles suggest that a number of different inhibitory compounds are produced. These findings suggest that the production of bacteriocin-like inhibitors may be a widespread characteristic throughout the genus Butyrivibrio. The bacteriocin-like activity from one isolate, B. fibrisolvens AR10, was purified and confirmed to reside in a single peptide. Crude bacteriocin extracts were prepared by ammonium sulfate and methanol precipitation of spent culture supernatants, followed by dialysis and high-speed centrifugation. The active component was isolated from the semicrude extract by reverse-phase chromatography. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirmed that the peptide was purified to homogeneity, having an estimated molecular mass of approximately 4,000 Da. The N terminus of the peptide was blocked. A cyanogen bromide cleavage fragment of the native peptide yielded a sequence of 20 amino acids [(M)GIQLAPAXYQDIVNXVAAG]. No homology with previously reported bacteriocins was found. Butyrivibriocin AR10 represents the first bacteriocin isolated from a ruminal anaerobe.
Composition and role of extracellular polymers in methanogenic granules
Authors: Veiga M C , Jain M K , Wu W , Hollingsworth R I , Zeikus J G
Published: January 2020

Methanobacterium formicicum and Methanosarcina mazeii are two prevalent species isolated from an anaerobic granular consortium grown on a fatty acid mixture. The extracellular polysaccharides (EPS) were extracted from Methanobacterium formicicum and Methanosarcina mazeii and from the methanogenic granules to examine their role in granular development. The EPS made up approximately 20 to 14% of the extracellular polymer extracted from the granules, Methanobacterium formicicum, and Methanosarcina mazeii. The EPS produced by Methanobacterium formicicum was composed mainly of rhamnose, mannose, galactose, glucose, and amino sugars, while that produced by Methanosarcina mazeii contained ribose, galactose, glucose, and glucosamine. The same sugars were also present in the EPS produced by the granules. These results indicate that the two methanogens, especially Methanobacterium formicicum, contributed significantly to the production of the extracellular polymer of the anaerobic granules. Growth temperature, substrates (formate and H(inf2)-CO(inf2)), and the key nutrients (nitrogen and phosphate concentrations) affected polymer production by Methanobacterium formicicum.
Use of rRNA gene restriction patterns to evaluate lactic acid bacterium contamination of vacuum-packaged sliced cooked whole-meat product in a meat processing plant
Authors: Björkroth K J , Korkeala H J
Published: January 2020

Molecular typing was applied to an in-plant lactic acid bacterium (LAB) contamination analysis of a vacuum-packaged sliced cooked whole-meat product. A total of 982 LAB isolates from the raw mass, product, and the environment at different production stages were screened by restriction endonuclease (EcoRI and HindIII) analysis. rRNA gene restriction patterns were further determined for different strains obtained from each source. These patterns were used for recognizing the spoilage-causing LAB strains from the product on the sell-by day and tracing the sources and sites of spoilage LAB contamination during the manufacture. LAB typing resulted in 71 different ribotypes, of which 27 were associated with contamination routes. Raw material was distinguished as the source of the major spoilage strains. Contamination of the product surfaces after cooking was shown to be airborne. The removal of the product from the cooking forms was localized as a major site of airborne LAB contamination. Food handlers and some surfaces in contact with the product during the manufacture were also contaminated with the spoilage strains. Some LAB strains were also able to resist cooking in the core of the product bar. These strains may have an effect on the product shelf life by contaminating the slicing machine. The air in the slicing department and adjacent cold room contained very few LAB. Surface-mediated contamination was detected during the slicing and packaging stages. Food handlers also carried strains later found in the packaged product. Molecular typing provided useful information revealing the LAB contamination sources and sites of this product. The production line will be reorganized in accordance with these results to reduce spoilage LAB contamination.
Monoclonal antibodies for use in detection of Bacillus and Clostridium spores
Authors: Quinlan J J , Foegeding P M
Published: January 2020

Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed. Two antibodies (B48 and B183) were selected for their reactivity with B. cereus T spores, two (C33 and C225) were selected for their reactivity with C. sporogenes spores, and one (D89) was selected for its reactivity with both B. cereus and C sporogenes spores. The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89). The antibodies reacted with spores of B. cereus T, Bacillus subtilis subsp. globigii, Bacillus megaterium, Bacillus stearothermophilus, C. sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans. Antibody D89 also reacted with vegetative cells of B. cereus and C. sporogenes. Analysis of B. cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa. Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B. cereus T spore. Antibody D89 reacted with the exosporium and outer cortex of C. sporogenes spores in immunocytochemical localization studies but did not react with extracts of C. sporogenes or B. cereus spores in Western blotting. Some C. sporogenes antigens were not stable during long-term storage at -20 degrees C. Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.
An enzyme-linked immunosorbent assay for detection of Vibrio vulnificus biotype 2: development and field studies
Authors: Biosca E G , Marco-Noales E , Amaro C , Alcaide E
Published: January 2020

Vibrio vulnificus biotype 2 is a primary eel pathogen which constitutes a lipopolysaccharide (LPS)-based homogeneous O serogroup within the species. In the present work, we have developed an enzyme-linked immunosorbent assay (ELISA) based on the specificity of LPS for the detection of this pathogen. The ELISA specificity was confirmed after testing 36 biotype 2 strains from laboratory cultures and environmental samples, 31 clinical and environmental biotype 1 isolates, and several strains of Vibrio, Aeromonas, and Yersinia species, including the fish pathogens V. anguillarum, V. furnissii, A. hydrophila, and Y. ruckerii. The detection limits for biotype 2 cells were around 10(4) to 10(5) cells/well, and the immunoassay was also able to detect cells in the nonculturable state. Artificially infected eels and environmental samples were analyzed, and the immunodetection was confirmed by cultural methods (isolation on selective and nonselective media before and after broth enrichment). With this methodology, V. vulnificus biotype 2 was successfully detected in infected eels and asymptomatic carriers, which suggests that eels can act as a reservoir for this pathogen.
Competition for cellobiose among three predominant ruminal cellulolytic bacteria under substrate-excess and substrate-limited conditions
Authors: Shi Y , Weimer P J
Published: January 2020

The ruminal cellulolytic bacteria Ruminococcus flavefaciens FD-1 and Fibrobacter succinogenes S85 coexisted in substrate-excess coculture with about equal population size, but R. flavefaciens outcompeted F. succinogenes for cellobiose in the substrate-limited cocultures whether the two strains were coinoculated or a steady-state culture of F. succinogenes was challenged by R. flavefaciens. This outcome of competition between these two strains is due to a classical pure and simple competition mechanism based on affinity for cellobiose. Although the population size of F. succinogenes was much higher (> 70%) than that of another cellulolytic species, Ruminococcus albus 7 in substrate-excess coculture, F. succinogenes was replaced by a population of R. albus in the substrate-limited coculture in both coinoculation and challenge experiments. R albus outcompeted F. succinogenes, apparently due to selection in the chemostat of a population of R. albus with a higher affinity for cellobiose. R. albus also outcompeted R. flavefaciens under substrate-limited conditions.
In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1
Authors: Yan Z , Fujiwara S , Kohda K , Takagi M , Imanaka T
Published: January 2020

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.
Cloning of novel enterotoxin genes from Bacillus cereus and Bacillus thuringiensis
Authors: Asano S I , Nukumizu Y , Bando H , Iizuka T , Yamamoto T
Published: January 2020

A novel enterotoxin gene was cloned from Bacillus cereus FM1, and its nucleotide sequence was determined. Previously, a 45-kDa protein causing characteristic enterotoxin symptoms in higher animals had been isolated (K. Shinagawa, p. 181-193, in A. E. Pohland et al., ed., Microbial Toxins in Foods and Feeds, 1990) from the same B. cereus strain, but no report of cloning of the enterotoxin gene has been published. In the present study, a specific antibody to the purified enterotoxin was produced and used to screen the genomic library of B. cereus FM1 made with the lambda gt11 vector. An immunologically positive clone was found to contain the full protein-coding region and some 5' and 3' flanking regions. The deduced amino acid sequence of the cloned gene indicated that the protein is rich in beta structures and contains some unusual sequences, such as consecutive Asn residues. In order to clone enterotoxin genes from Bacillus thuringiensis, two PCR primers were synthesized based on the nucleotide sequence of the B. cereus gene. These primers were designed to amplify the full protein-coding region. PCR conducted with DNA preparations from the B. thuringiensis subsp. sotto and B. thuringiensis subsp. israelensis strains successfully amplified a segment of DNA with a size almost identical to that of the protein-coding region of the B. cereus enterotoxin. Nucleotide sequences of the amplified DNA segments showed that these B. thuringiensis strains contain an enterotoxin gene very similar to that of B. cereus. Further PCR screening of additional B. thuringiensis strains with four primer pairs in one reaction revealed that some additional B. thuringiensis strains contain enterotoxin-like genes.
Influence of Exposure to Single versus Multiple Toxins of Bacillus thuringiensis subsp. israelensis on Development of Resistance in the Mosquito Culex quinquefasciatus (Diptera: Culicidae)
Authors: Georghiou G P , Wirth M C
Published: January 2020

The impending widespread use of transgenic crop plants encoding a single insecticidal toxin protein of Bacillus thuringiensis has focused attention on the perceived risk of rapid selection of resistance in target insects. We have used Bacillus thuringiensis subsp. israelensis toxins as a model system and determined the speed and magnitude of evolution of resistance in colonies of the mosquito Culex quinquefasciatus during selection for 28 consecutive generations with single or multiple toxins. The parental strain was synthesized by combining approximately 500 larvae from each of 19 field collections obtained from the states of California, Oregon, Louisiana, and Tennessee. At least 10,000 larvae were selected in each generation of each line at an average mortality level of 84%. The susceptibilities of the parental and selected lines were compared in parallel tests in every third generation by using fresh suspensions of toxin powders. The normal toxin complement of B. thuringiensis subsp. israelensis consists of four toxins, CryIVA, CryIVB, CryIVD, and CytA. Resistance became evident first in the line that was selected with a single toxin (CryIVD), attaining the highest level (resistance ratio [RR], >913 at 95% lethal concentration) by generation F(inf28) when the study was completed. Resistance evolved more slowly and to a lower level (RR, >122 by F(inf25)) in the line selected with two toxins (CryIVA+CryIVB) and lower still (RR, 91 by F(inf28)) in the line selected with three toxins (CryIVA+CryIVB+ CryIVD). Resistance was remarkably low (RR, 3.2) in the line selected with all four toxins. The results reveal the importance of the full complement of toxins found in natural populations of B. thuringiensis subsp. israelensis as an effective approach to resistance management.
Group-specific small-subunit rRNA hybridization probes to characterize filamentous foaming in activated sludge systems
Authors: de los Reyes F L , Ritter W , Raskin L
Published: January 2020

Foaming in activated sludge systems is characterized by the formation of a thick, chocolate brown-colored scum that floats on the surface of aeration basins and secondary clarifiers. These viscous foams have been associated with the presence of filamentous mycolic acid-containing actinomycetes. To aid in evaluating the microbial representation in foam, we developed and characterized group-, genus-, and species-specific oligonucleotide probes targeting the small subunit rRNA of the Mycobacterium complex, Gordona spp., and Gordona (Nocardia) amarae, respectively. The use of a universal base analog, 5-nitroindole, in oligonucleotide probe design was evaluated by comparing the characteristics of two different versions of the Mycobacterium complex probe. The temperature of dissociation of each probe was determined. Probe specificity studies with a diverse collection of 67 target and nontarget rRNAs demonstrated the specificity of the probes to the target groups. Whole-cell hybridizations with fluorescein- and rhodamine-labeled probes were performed with pure cultures of various members of the Mycobacterium complex as well as with environmental samples from a full-scale activated sludge plant which experienced foaming. Quantitative membrane hybridizations with activated sludge and anaerobic digester foam showed that 15.0 to 18.3% of the total small-subunit rRNAs could be attributed to members of the Mycobacterium complex, of which a vast majority consisted of Gordona rRNA. Several G. amarae strains made up only a very small percentage of the Gordona strains present. We demonstrated that group-specific rRNA probes are useful tools for the in situ monitoring and identification of filamentous bacteria in activated sludge systems.
Assessment of fecal bacteria with bile acid 7 alpha-dehydroxylating activity for the presence of bai-like genes
Authors: Doerner K C , Takamine F , LaVoie C P , Mallonee D H , Hylemon P B
Published: January 2020

Eubacterium sp. strain VPI 12708 has several bile acid-inducible (bai) genes which encode enzymes in the bile acid 7 alpha-dehydroxylation (7 alpha DeOH) pathway. Twelve 7 alpha DeOH-positive intestinal bacterial strains were assayed for 7 alpha DeOH activity, and 13 strains were tested for hybridization with bai genes. Cholic acid 7 alpha DeOH activity varied greatly (> 100-fold) among these strains. Southern blot experiments showed that DNA prepared from 7 of 13 strains hybridized with at least one of the bai genes from Eubacterium sp. strain VPI 12708.
Frequency of Antibiotic-Producing Pseudomonas spp. in Natural Environments
Authors: Raaijmakers J M , Weller D M , Thomashow L S
Published: January 2020

The antibiotics phenazine-1-carboxylic acid (PCA) and 2,4-diacetylphloroglucinol (Phl) are major determinants of biological control of soilborne plant pathogens by various strains of fluorescent Pseudomonas spp. In this study, we described primers and probes that enable specific and efficient detection of a wide variety of fluorescent Pseudomonas strains that produce various phenazine antibiotics or Phl. PCR analysis and Southern hybridization demonstrated that specific genes within the biosynthetic loci for Phl and PCA are conserved among various Pseudomonas strains of worldwide origin. The frequency of Phl- and PCA-producing fluorescent pseudomonads was determined on roots of wheat grown in three soils suppressive to take-all disease of wheat and four soils conducive to take-all by colony hybridization followed by PCR. Phenazine-producing strains were not detected on roots from any of the soils. However, Phl-producing fluorescent pseudomonads were isolated from all three take-all-suppressive soils at densities ranging from approximately 5 x 10(sup5) to 2 x 10(sup6) CFU per g of root. In the complementary conducive soils, Phl-producing pseudomonads were not detected or were detected at densities at least 40-fold lower than those in the suppressive soils. We speculate that fluorescent Pseudomonas spp. that produce Phl play an important role in the natural suppressiveness of these soils to take-all disease of wheat.
Nitrate-reducing bacteria on rat tongues
Authors: Li H , Duncan C , Townend J , Killham K , Smith L M , Johnston P , Dykhuizen R , Kelly D , Golden M , Benjamin N , Leifert C
Published: January 2020

Nitrite-producing bacteria (NPB) were isolated from tongues of laboratory rats. The most commonly found nitrite-producing organism was Staphylococcus sciuri, followed by Staphylococcus intermedius, Pasteurella spp., and finally Streptococcus spp. Both morphometric quantification of bacteria on tongue sections and enumeration of culturable bacteria (CFU) showed an increase in the density of bacteria towards the posterior tongue. Up to 65% of bacteria were located in the deep clefts on the posterior tongue. The proportion of culturable NPB in the total culturable microbial population increased from 6% (10(5) CFU cm-2) on the anterior tongue to 65% (10(7) CFU cm-2) on the posterior tongue. Different species compositions of NPB were found on different tongue sections with S. intermedius populations decreasing and S. sciuri and Pasteurella populations increasing towards the posterior tongue. Nitrite production was sensitive to oxygen, and significant nitrite production was only detected on the posterior tongue where the majority of bacteria are situated in deep clefts in the tongue surface. This study suggests the importance of bacteria in nitrite production, from nitrate, on the tongue. Nitrite produced on the tongue may subsequently form nitric oxide in the acidic environment of the stomach. Because of the antimicrobial properties of nitric oxide, a key role for nitrate-reducing tongue bacteria in host animal defense against food-borne pathogens in proposed.
Escherichia coli mutants resistant to inactivation by high hydrostatic pressure
Authors: Hauben K J , Bartlett D H , Soontjens C C , Cornelis K , Wuytack E Y , Michiels C W
Published: January 2020

Alternating cycles of exposure to high pressure and outgrowth of surviving populations were used to select for highly pressure-resistant mutants of Escherichia coli MG1655. Three barotolerant mutants (LMM1010, LMM1020, and LMM1030) were isolated independently by using outgrowth temperatures of 30, 37, and 42 degrees C, respectively. Survival of these mutants after pressure treatment for 15 min at ambient temperature was 40 to 85% at 220 MPa and 0.5 to 1.5% at 800 MPa, while survival of the parent strain, MG1655, decreased from 15% at 220 MPa to 2 x 10(-8)% at 700 MPa. Heat resistance of mutants LMM1020 and LMM1030 was also altered, as evident by higher D values at 58 and 60 degrees C and reduced z values compared to those for the parent strain. D and z values for mutant LMM1010 were not significantly different from those for the parent strain. Pressure sensitivity of the mutants increased from 10 to 50 degrees C, as opposed to the parent strain, which showed a minimum around 40 degrees C. The ability of the mutants to grow at moderately elevated pressure (50 MPa) was reduced at temperatures above 37 degrees C, indicating that resistance to pressure inactivation is unrelated to barotolerant growth. The development of high levels of barotolerance as demonstrated in this work should cause concern about the safety of high-pressure food processing.
Role of the histidine kinase, EnvZ, in the production of outer membrane proteins in the symbiotic-pathogenic bacterium Xenorhabdus nematophilus
Authors: Forst S A , Tabatabai N
Published: January 2020

We show that inactivation of envZ, the gene encoding the histidine kinase sensor protein, EnvZ, of Xenorhabdus nematophilus, affected the production of several outer membrane proteins (Opns). X. nematophilus produced five major Opns during exponential growth. Insertional inactivation of envZ led to a decrease in the production of OpnP, the OmpF-like pore-forming protein which constitutes approximately 50% of the total outer membrane protein in X. nematophilus. OpnA production was also reduced, while the remaining Opns were produced normally. During the transition to stationary phase, three new outer membrane proteins, OpnB, OpnS, and OpnX, were induced in the wild-type strain. The envZ-minus strain, ANT1, did not produce OpnB and OpnX, while OpnS was induced at markedly reduced levels. These results suggest that EnvZ was required for the high-level production of OpnP during exponential growth and may be involved in the production of OpnB, OpnS, and OpnX during stationary-phase growth. We also show that ANT1 was more pathogenic than the wild-type strain when as few as five cells were injected into the hemolymph of the larval stage of the tobacco hornworm (Manduca sexta). The larvae died before significant numbers of bacteria were detectable in the hemolymph. These results are discussed in relation to the role of EnvZ in the life cycle of X. nematophilus.
Poliovirus-1 inactivation and interaction with biofilm: a pilot-scale study
Authors: Quignon F , Sardin M , Kiene L , Schwartzbrod L
Published: January 2020

A pilot-scale study was initiated to examine the behavior of viruses pulse injected into a distribution system. The influence of a free-chlorine residual and that of virus preadsorption to clay particles was evaluated by tracing the viruses both in the water flow and after elution from the biofilm. These experiments demonstrated, first, that virus preadsorption on 40 mg of Na-montmorillonite per liter increased the residence time of the viruses within the pilot plant by roughly three times and, second, that preadsorption to clay did not prevent viruses from being inactivated by chlorine. Moreover, with no clay added, a greater amount of viruses was recovered from the biofilm than from the water flow (by a factor of 2 or 10 in the absence or presence of chlorine, respectively), indicating a tendency for virus accumulation within biofilms.
Development of polyclonal antibodies for detection of aflatoxigenic molds involving culture filtrate and chimeric proteins expressed in Escherichia coli
Authors: Shapira R , Paster N , Menasherov M , Eyal O , Mett A , Meiron T , Kuttin E , Salomon R
Published: January 2020

Polyclonal antibodies (PAb) were raised against an aflatoxigenic strain of Aspergillus parasiticus by using two different sources for antibody elicitation: (i) filtrate of a culture on which the fungus had been grown (ii) and two chimeric proteins, expressed in Escherichia coli as separate products, of the genes ver-1 and apa-2, which are involved in aflatoxin biosynthesis. The gene products were amplified by PCR, and each was cloned into the E. coli expression vector pGEX2T. Upon induction, the bacteria overexpressed 38- and 33-kDa chimeric proteins corresponding to the N-terminal domains of the genes ver-1 and apa-2, respectively. The chimeric proteins were isolated and affinity purified for use as antigens. The specificity of the raised antibodies was examined by enzyme-linked immunosorbent assay (ELISA). The PAbs raised against the culture filtrate reacted with all the species of Aspergillus and Penicillium tested but not with Fusarium species or corn gain. However, the PAbs elicited against the chimeric proteins were highly specific, showing significantly higher ELISA absorbance values (A405) against A. parasiticus and A. flavus than against the other fungi tested and the corn grain. The approach of utilizing gene products associated with aflatoxin biosynthesis for antibody production therefore appears to be feasible. Such a multiantibody system combined with the PCR technique, could provide a useful tool for the rapid, sensitive, and accurate detection of aflatoxin producers present in grains and foods.
Polymorphic Chromosomes Bearing the Tox2 Locus in Cochliobolus carbonum Behave as Homologs during Meiosis
Authors: Canada S R , Dunkle L D
Published: January 2020

The HTS1 gene in the Tox2 locus of the fungal pathogen Cochliobolus carbonum race 1 is required for synthesis of a host-selective phytotoxin and for increased virulence on susceptible genotypes of maize. The locus is present in race 1 isolates but absent from isolates of the other races, which do not produce the toxin. By pulsed-field gel electrophoresis and Southern analysis with HTS1 sequences and chromosome-specific markers, the HTS1 gene was detected on a 4-Mb chromosome in one group of isolates and on a 2.3-Mb chromosome in another group, which lacked the 4-Mb chromosome. A chromosome-specific marker from C. heterostrophus hybridized to a 2.3-Mb chromosome in non-toxin-producing isolates and in toxin-producing isolates, including those with a 4-Mb chromosome. A marker from C. carbonum hybridized to the 4-Mb chromosome, but in isolates lacking the 4-Mb chromosome, this marker hybridized to a smaller, 2.0-Mb chromosome. Thus, the Tox2 locus is on different chromosomes in different groups of race 1 isolates. Single ascospore progeny from crosses between isolates having HTS1 on different chromosomes were analyzed for toxin-producing ability, virulence, and the presence and chromosomal location of HTS1. All progeny produced HC toxin in culture, incited race 1-type lesions on susceptible maize genotypes, and contained HTS1 sequences, as determined by PCR amplification with gene-specific primers. Analysis of the chromosomal complements of several progeny indicated that they all had only one Tox2-containing chromosome. Thus, despite their differences in size, these chromosomes behave as homologs during meiosis and may have arisen by a translocation.
Regulation of cellulase gene expression in the filamentous fungus Trichoderma reesei
Authors: Ilmén M , Saloheimo A , Onnela M L , Penttilä M E
Published: January 2020

Basic features of regulation of expression of the genes encoding the cellulases of the filamentous fungus Trichoderma reesei QM9414, the genes cbh1 and cbh2 encoding cellobiohydrolases and the genes egl1, egl2 and egl5 encoding endoglucanases, were studied at the mRNA level. The cellulase genes were coordinately expressed under all conditions studied, with the steady-state mRNA levels of cbh1 being the highest. Solka floc cellulose and the disaccharide sophorose induced expression to almost the same level. Moderate expression was observed when cellobiose or lactose was used as the carbon source. It was found that glycerol and sorbitol do not promote expression but, unlike glucose, do not inhibit it either, because the addition of 1 to 2 mM sophorose to glycerol or sorbitol cultures provokes high cellulase expression levels. These carbon sources thus provide a useful means to study cellulase regulation without significantly affecting the growth of the fungus. RNA slot blot experiments showed that no expression could be observed on glucose-containing medium and that high glucose levels abolish the inducing effect of sophorose. The results clearly show that distinct and clear-cut mechanisms of induction and glucose repression regulate cellulase expression in an actively growing fungus. However, derepression of cellulase expression occurs without apparent addition of an inducer once glucose has been depleted from the medium. This expression seems not to arise simply from starvation, since the lack of carbon or nitrogen as such is not sufficient to trigger significant expression.