摘要:

Bacterial strains and consortia of bacteria have been isolated for their ability to degrade, under anaerobic conditions, homocyclic monoaromatic compounds, such as phenolic compounds, methylbenzenes, and aminobenzenes. As opposed to aerobic conditions where these compounds are degraded via dihydroxyl intermediates introduced by oxygenases, most of aromatic compounds under anaerobic conditions are metabolized via aromatic acid intermediates, such as nitrobenzoates, hydroxybenzoates, or phenylacetate. These aromatic acids are then transformed to benzoate before the reduction and the cleavage of the benzene ring to aliphatic acid products. One step of these catabolic pathways is the addition of a coenzyme A (CoA) residue to the carboxylic group of the aromatic acids by CoA ligases. This addition would facilitate the enzymatic transformation of the aromatic acids to benzoyl-CoA and the subsequent degradation steps of this latter molecule. Aromatic acid – CoA ligases have been characterized or detected from several bacterial strains that were grown under anaerobic conditions and from an anaerobic syntrophic consortium. They are also involved in the degradation of some aromatic compounds under aerobic conditions. They have molecular masses varying between 48 and 61 kDa, require ATP, Mg2+, and CoASH as cofactors, and have an optimum pH of 8.2–9.3. Amino acid sequence analyses of four aromatic acid–CoA ligases have revealed that they are related to an AMP-binding protein family. Aromatic acid – CoA ligases expressed in anaerobically grown bacterial cells are strictly regulated by the anaerobic conditions and the presence of aromatic acids.Key words: aromatic compounds, coenzyme A ligase, anaerobic microorganisms.

ISSN:0008-4166    

DOI:10.1139/m95-118

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Pentachlorophenol (PCP) dechlorination by a methanogenic consortium was observed when glucose, formate, lactate, or yeast extract was present in the mineral medium as a secondary carbon source. Acetate was not a good substrate to sustain dechlorination. The consortium was able to dechlorinate the different monochlorophenols, although the chlorine in positionorthoandmetawas removed more readily than inparaposition. Dechlorination was most efficient at 37 °C. At 45 °C, the first PCP dechlorination steps were very rapid, but 3,5-dichlorophenol (3,5-DCP) was not further dechlorinated. At 15 and 4 °C, dechlorination was very slow. The dechlorination of PCP to 3-chlorophenol (3-CP) was still observed after the consortium had been subjected to heat treatment (80 °C, 60 min), suggesting that spore-forming bacteria were responsible. The dechlorinating activity of the consortium was significantly reduced by the presence of hydrogen, 2-bromoethanosulfonic acid (BESA), or sulfate but not of nitrate. The dechlorination of 3-CP was completely inhibited by heat treatment or the presence of BESA, suggesting that a syntrophic microorganism would be involved. Vigorous agitation of the consortium stopped the dechlorination, but the presence of DEAE-Sephacel acting as a support was very efficient in restoring the activity, suggesting that association between certain members of the consortium was important.Key words: pentachlorophenol, dechlorination, anaerobic, methanogenesis.

ISSN:0008-4166    

DOI:10.1139/m95-119

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

The recombinant clone pBAW101 (in pBluescript SK–) contains thecelBendoglucanase gene fromRuminococcus flavefaciensFD-1. Subcloning indicated that the endoglucanase activity expressed was present within a 2.4-kb insert (pBAW104). The nucleotide sequence of thecelBgene was determined, and upon analysis, revealed an open reading frame of 1943 nucleotides that encodes a polypeptide of 632 amino acids with a molecular weight of 69 414. A putative Shine–Dalgarno sequence was identified 6 bp upstream from the translation start site. The N-terminal 32 amino acid residues were typical of prokaryotic signal sequences. Hydrophobic cluster analysis (HCA) and DNA alignment of CelB to other published β-glucanase polypeptide sequences in GenBank indicate that CelB belongs in HCA cellulase family 44. Primer extension analyses were performed using RNA isolated fromR.flavefaciensgrown on cellulose and cellobiose, and fromEscherichia colicontaining the plasmid clone pBAW104. Transcription is initiated at different sites inE.coliandR.flavefaciens. In the case ofR.flavefacienstranscription is initiated at a C residue (nucleotides 329), 221 bp upstream from the translation start site. There were no regions resemblingE.coliσ70-like promoter sequences present upstream from this putative transcription initiation site. In contrast, numerous transcription initiation sites were identified when RNA fromE.coliwas used in the primer extension analyses.Key words:Ruminococcus flavefaciens, endoglucanase, transcription, family 44 endoglucanase.

ISSN:0008-4166    

DOI:10.1139/m95-120

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

The activity of fourClavibacter michiganensissubsp.sepedonicusstrains against various cellulose substrates was investigated. Sixty-sevenClavibacter michiganensissubsp.sepedonicusstrains grew well on media amended with carboxymethylcellulose, 64 strains produced zones of hydrolysis. Endoglucanase activity was optimal at 37 °C and pH 6.0 against carboxymethylcellulose incorporated in plate assays. Zymogram and sodium dodecyl sulfate – polyacrylamide gel electrophoresis revealed the presence of a protein band corresponding to the cellulolytic activity in the molecular weight (MW) range of approximately 28 000. Protein bands in the same range were detected in fiveClavibacter michiganensissubsp.sepedonicusstrains. Studies on crude enzyme extracts ofClavibacter michiganensissubsp.sepedonicusstrain N-1-1 revealed thatp-nitrophenyl β-D-cellobioside (pNPC) was hydrolyzed, with optimal activity at 37 °C and pH 7.0.Key words: cellulase, endo-1,4-β-glucanase (EC 3.2.1.4),Corynebacterium sepedonicum,Solanum tuberosum.

ISSN:0008-4166    

DOI:10.1139/m95-121

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

We have used electron spectroscopic imaging to locate the phosphorus in vaccinia DNA in situ in unstained, ultrathin sections of virions. The phosphorus of the DNA backbone appeared to form a halo on the core periphery surrounding a phosphorus-impoverished central element. These results constrain models for how DNA could be packaged into mature vaccinia particles.Key words: vaccinia, electron spectroscopic imaging, DNA.

ISSN:0008-4166    

DOI:10.1139/m95-122

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Investigations were designed to gain fundamental information on the microbial ecology of endophytic bacteria in model dicotyledonous and monocotyledonous hosts. Population dynamics of indigenous endophytic bacteria in cotton (Gossypium hirsutumL. 'DES119') and sweet corn (Zea maysL. 'Silver Queen') stems and roots were studied in a 2-year field trial by quantifying culturable bacteria at intervals during the season on three media: R2A, medium SC, and tryptic soy agar. Population dynamics of endophytic bacteria inside cotton petioles and bolls were also determined in 1 year. Endophytes were recovered from sweet corn roots and stems at seedling emergence at mean population densities of 4 log (colony-forming units per gram fresh weight (cfu/g-fw)) for both seasons, and were present throughout most of the growing season at populations ranging from 4 to 6 log(cfu/g-fw) in 1990 and 4 to 7 log(cfu/g-fw) in 1991. Endophytic bacteria were also present at emergence in cotton roots and stems in 1991 but were not detected until 2 days after emergence in 1990. Endophytic populations in cotton roots ranged from 4 to 6 log(cfu/g-fw) for most of the growing season in 1990 and 1991, while populations in cotton stems fluctuated between 3 and 7 log(cfu/g-fw) during both seasons. In cotton petioles, mean populations generally ranged from 1 to 4 log(cfu/g-fw), while no endophytic bacteria were recovered from bolls (minimum detectable limit = 1.30 log(cfu/g-fw)). The relative contribution of seeds and soil as sources of endophytic bacteria recovered from inside plants was assessed using surface-disinfested seed in a potting mix or on water–agar. With sweet corn, the mean endophytic bacterial population in seedlings grown on water agar was below 2 log(cfu/g-fw), while with cotton the mean was 5 log(cfu/g-fw) 6 days after germination. Internal populations resulting from surface-disinfested seed planted in nonsterile potting mix were 6 log(cfu/g-fw) at 6 days after planting with corn but only 2 log(cfu/g-fw) with cotton. These results indicate that endophytic bacteria are natural inhabitants of internal regions of roots and stems and that the endophytes may arise from both seeds and soils.Key words: cotton, sweet corn, endophytes, colonization.

ISSN:0008-4166    

DOI:10.1139/m95-123

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Bacillus licheniformisATCC 9945A produces γ-poly(glutamic acid) (γ-PGA) when using glutamate, citrate, and glycerol in combination as media carbon sources. Also, various aspects ofB.licheniformiscellular physiology are affected by the concentration of Mn(II) salts in culture media. Thus, the metabolism of carbon sources into γ-PGA as a function of MnSO4 concentration was studied by enriching the media with eitherL-[1,2-13C]glutamic acid (NH2—2CH(1COOH)—(CH2)2-COOH) or [1,5-13C]citric acid (HOO1C—CH2—C(OH)(COOH)—CH2—5COOH) at two media MnSO4concentrations (615 and 0 μM). Enrichment factors (EF) from13C-NMR spectra were calculated from the ratio of peak intensities from13C-enriched γ-PGA divided by the ratio of peak intensities for nonenriched γ-PGA. EF values were than used to determine the percentage of repeat units that were formed from glutamate and citrate. The percentage of repeat units formed from provided glutamate at the 0 and 615 μM Mn(II) media concentrations was 89 ± 14 and 67 ± 11%, respectively. These respective products have 51 ± 9 and 39 ± 11% of their repeat units formed with apparent retention of the glutamate carbon skeleton. Also, enrichment of γ-PGA repeat units at C-1 was found to be lower than C-2 at both Mn(II) levels. Thus, provided glutamate was used to a large extent for polymer formation with both retention of the carbon skeleton as well as decarboxylation at C-1. Provided citrate was also used as a source of carbon to form repeat units. At the 0 and 615 μM media Mn(II) levels, 9 ± 4 and 19 ± 5% of repeat units were formed from citrate. It is believed that citrate is metabolized to γ-PGA by entry into the citric acid cycle and formation of α-ketoglutarate. Analysis of products from cultivations where both glutamate and citrate were13C enriched indicated that citrate and glutamate carbon source metabolism to γ-PGA occurs via independent pathways to common monomer precursors without multiple recycling of these carbon sources through catabolic and anabolic pathways.Key words: γ-poly(glutamic acid), metabolism, manganese,Bacillus licheniformis, nuclear magnetic resonance.

ISSN:0008-4166    

DOI:10.1139/m95-124

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Pseudomonas aeruginosaproduces several extracellular virulence factors including elastase (which is encoded bylasB). Recently, we examined several clinical isolates ofP.aeruginosafor the production of toxin A, elastase, exoenzyme S, and phospholipase C. Although the majority of the isolates produced a high level of elastase, a few isolates produced either very low or no detectable elastase. In this study, we tried to determine the presence of restriction site heterogeneity withinlasBfrom these isolates and the possible correlation between such heterogeneity and the observed variation in elastase production. Chromosomal DNA from the isolates was digested with different restriction enzymes and examined by Southern blot hybridization experiments using twolasBprobes. OnelasBprobe covers 636 bp oflasBstructural gene while the other covers 240 bp of thelasBupstream region. Chromosomal DNA fromP.aeruginosaPAO1 and PA103 was used as controls. Results indicate that chromosomal DNA from all isolates hybridized to bothlasBprobes. Depending on the restriction enzyme used for DNA digestion,lasBfrom 3 to 12% of the isolates showed different patterns of hybridization with thelasBstructural gene probe. However, no difference in the hybridization pattern was seen with thelasBupstream probe. With the exception of one isolate, hybridization of genomic DNA from different isolates (with both probes) produced a single hybridization band. In that isolate, an additional hybridization band was detected. Immunoblotting experiments confirmed that elastase protein is not produced by 6 out of 67 isolates. However,lasBfrom four of these elastase-deficient strains showed no difference in the hybridization pattern with eitherlasBprobe. These results suggest that (i)lasBis present as a single copy in all but one isolate; (ii) with the exception of one, thelasBupstream region from differentP.aeruginosaisolates contains no restriction site polymorphism; (iii) the observed heterogeneity withinlasBstructural genes is limited; and (iv) variations in the hybridization patterns oflasBfrom different isolates do not correlate with the differences between these isolates in elastase production.Key words:Pseudomonas aeruginosa, clinical isolates, DNA hybridization, elastase,lasB.

ISSN:0008-4166    

DOI:10.1139/m95-125

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Carbon and nitrogen metabolism were investigated inAcetobacter diazotrophicusPal 3, a N2-fixing bacterium able to grow at low pH and at high sugar concentration. Enzymatic, respiratory, and uptake studies were performed. The main active pathway for the catabolism of phosphorylated glucose was the pentose phosphate pathway. In addition,A.diazotrophicusdirectly oxidized glucose, gluconate, and ketogluconates through respiratory chain-linked enzymes. Soluble enzymes for the oxidation of glucose and gluconate were also found.Acetobacter diazotrophicushad a complete tricarboxylic acid cycle with a respiratory chain-linked malate dehydrogenase. The ability to grow on two- and three-carbon substrates would be explained by the presence of gluconeogenesis. Lack of bacterial growth on dicarboxylates was explained by the absence of a transport system. Ammonium assimilation proceeded mainly through glutamate dehydrogenase under ammonium excess but also through energy-demanding glutamine synthetase and glutamate synthase under N2-fixing conditions.Acetobacter diazotrophicuswas not able to transport sucrose and its ability to grow on this disaccharide was explained by the presence of an extracellular enzyme with saccharolytic activity.Key words:Acetobacter diazotrophicus, carbon–nitrogen metabolism, extracellular saccharolytic activity, sucrose–succinate uptake.

ISSN:0008-4166    

DOI:10.1139/m95-126

出版商:NRC Research Press    

年代:1995

数据来源: NRC

摘要:

Nearly complete sequences (97–99%) of the 16S rRNA genes were determined for type strains ofClavibacter michiganensissubsp.michiganensis,Clavibacter michiganensissubsp.insidiosus,Clavibacter michiganensissubsp.sepedonicus, andClavibacter michiganensissubsp.nebraskensis. The four subspecies had less than 1% dissimilarity in their 16S rRNA genes. Comparative studies indicated that theC.michiganensissubsp. shared relatively high homology with the 16S rRNA gene ofClavibacter xyli. Further comparison with representatives of other Gram-positive coryneform and related bacteria with high G + C% values showed that this group of bacteria was subdivided into three clusters. One cluster consisted of theClavibacter michiganensissubsp.,Clavibacter xyli,Arthrobacter globiformis,Arthrobacter simplex, andFrankiasp.; another cluster consisted of members of the corynebacteria–mycobacteria–nocardia (CMN) group ofMycobacteriaceaeincludingTsukamurella paurometabolum; andPropionibacterium freudenreichiialone formed a unique cluster, which was remote from other coryneform bacteria analyzed. The three clusters may reflect a systematic rank higher than the genus level among these bacteria.Key words:Clavibacter, coryneform bacteria, phylogeny, 16S rRNA analysis.

ISSN:0008-4166    

DOI:10.1139/m95-127

出版商:NRC Research Press    

年代:1995

数据来源: NRC