摘要:

The specific activity of chlorocatechol 1,2-dioxygenase fromAlcaligenes denitrificansBRI 6011 was found to be maximal in the early logarithmic growth phase. The enzyme was purified from cultures at mid-log phase of growth using ammonium sulfate fractionation, and phenyl-Sepharose and DEAE-Sepharose chromatography. The protein gave a single band by SDS polyacrylamide gel electrophoresis with an apparent molecular weight of 33 000, and the temperature and pH optima were 30 °C and 7.5, respectively. Catechol, 3-chlorocatechol (3-CC), 4-CC, 3,4-dichlorocatechol (3,4-DCC), 3,5-DCC, 3,6-DCC, 3-methylcatechol (3-MC), and 4-MC served as substrates for the enzyme. TheVmaxvalues for the dichlorocatechols were similar, while those for the monochlorinated and methylated catechols were higher. TheKmvalues for all the chlorinated catechols were typically below 1 μM, while those for catechol and the methylated catechols were above 10 μM.Key words: chlorocatechol 1,2-dioxygenase,Alcaligenes denitrificans, purification, characterization, chlorobenzoic acid degradation.

ISSN:0008-4166    

DOI:10.1139/m93-001

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

The entomopathogenic fungusBeauveria bassianaproduces two extracellularN-acetylglucosaminidases (NAGase) in liquid medium containing colloidal chitin as the sole source of carbon and nitrogen. To study the regulation of NAGase synthesis,N-acetyl-D-glucosamine (GlcNAc), glucose NH4NO3, or amino acids were added to the colloidal chitin medium and NAGase activity was measured. NAGase synthesis was (i) induced with GlcNAc, and no repression was observed with GlcNAc provided at 2% (w/v); (ii) repressed in the presence of glucose plus NH4NO3; (iii) partially repressed when glucose or NH4NO3was provided; and (iv) repressed to levels that were < 40% of the control levels when glutamic acid, tyrosine, arginine, proline, valine, and histidine were provided to the colloidal chitin medium. Total NAGase activity levels were > 60% of the control activity when alanine, glycine, isoleucine, aspartic acid, and leucine were tested. It appears that synthesis of NAGase is sensitive to cell energy and the carbon and nitrogen requirements.Key words:Beauveria bassiana,N-acetyl-D-glucosaminidase, entomopathogenic fungus, extracellular enzyme, regulation.

ISSN:0008-4166    

DOI:10.1139/m93-002

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

Water samples from the wastewater treatment system of a bleach kraft mill and from the river that supplies the mill were plated on six different media and culturable isolates were screened for substrate utilization patterns, taxonomic characters, plasmid content, and resistance to ampicillin, streptomycin, kanamycin, tetracycline, naldixic acid, mercury, nickel, copper, cobalt, cadmium, and zinc. A cluster analysis of the substrate utilization profiles and taxonomic characters revealed thatPseudomonas,Acinetobacter, andAcidovoraxspp. were common among the culturable isolates from the river, whileAncylobacter aquaticus,Klebsiellaspp., and an unknown group of pleomorphic Gram-negative methylotrophs were common among the culturable isolates from the mill treatment system. Of isolates from the settling pond and aerated lagoon, 78 and 64% carried plasmids, while only 56% of isolates from the river carried plasmids. Plasmids were significantly associated with resistance to cadmium but not with any other resistance characters. Large numbers of plasmid-carryingA.aquaticusstrains and pleomorphic methylotrophs accounted for high plasmid incidence levels in the mill treatment system, and the ability to dechlorinate simple aliphatic substrates was found in these two groups as well as in onePseudomonasstrain.Key words: pulp and paper, wastewater treatment, chlorinated organics, culturable heterotrophs, methylotrophs, plasmids.

ISSN:0008-4166    

DOI:10.1139/m93-003

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

The 28.5-kbp mitochondrial (mt) genome from the entomopathogenic fungusBeauveria bassianawas studied using restriction enzyme analysis, gene probe hybridization, and DNA sequence comparisons. A detailed restriction enzyme map allowed cloning of the entire genome into a number of segments. Hybridization of heterologous gene probes to the mtDNA resulted in the identification of the large ribosomal RNA (lrRNA) and small ribosomal RNA (srRNA) genes. Gene probes derived from several yeasts and fungi failed to identify any additional genes. However, partial DNA sequence analysis revealed thelrRNAandsrRNAgenes as well as four protein-encoding genes: the NADH dehydrogenase subunit 1 (NAD1), NADH dehydrogenase subunit 6 (NAD6), cytochrome oxidase subunit 3 (C03), and ATPase subunit 6 (ATP6) genes. The ATPase subunit 9 (ATP9) gene was not identified by hybridization to mtDNA, but could be detected by hybridization to total cellular DNA. The portions of the genes sequenced were homologous to the equivalent genes from yeast and other filamentous fungi, most notablyAspergillus nidulans. No introns were identified in these regions. The organization of the sequenced region of theB.bassianamt genome more closely resembled that ofA.nidulansthan that ofPodospora anserinaorNeurospora crassa.Key words:Beauveria bassiana, mtDNA, gene mapping, mitochondrial rRNA, mitochondrial organization.

ISSN:0008-4166    

DOI:10.1139/m93-004

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

We have recently described the presence of a high molecular mass multicatalytic proteinase complex (megaproteinase; 28 S, 1300 kDa) inFrankiastrain BR. The complex dissociates into 11 low molecular mass proteinase subunits (40–19 kDa) when subjected to sodium dodecyl sulfate – gelatin – polyacrylamide gel electrophoresis. We show here that the activity of these proteinase subunits strongly increased after cessation of growth in stirred BAP-PCM mineral medium. Subsequent addition of either BAP medium components or sodium propionate alone, as carbon source, to aFrankiaculture at the end of the exponential growth phase was found to prolong growth for 1 additional day, and to delay the increase in activity of the proteinase subunits for 3 days after cessation of growth. Addition of ammonium chloride alone, as nitrogen source, had no effect. On the other hand, whenFrankiacells in the late exponential phase (3 days) were resuspended in a culture filtrate recovered from a 5-day-old culture and supplemented with BAP-PCM medium components, the biomass yield decreased to about 50%. Also, the activity of the proteinase subunits increased as soon as growth ceased. The ability of this culture filtrate to inhibit growth and stimulate the activity of proteinase subunits was partially lost by heating or was largely removed by DEAE-cellulose treatment. Thus, our findings indicate an extracellular control ofFrankiamegaproteinase activity, suggesting that carbon source depletion and probably accumulation of heat-sensitive growth-inhibiting metabolites in the medium are determining factors.Key words:Frankia, proteinases, megaproteinase, proteasome, proteolysis, autolysis.

ISSN:0008-4166    

DOI:10.1139/m93-005

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

Beauveria bassianagrown in a liquid medium containingN-acetyl-D-glucosamine and colloidal chitin produced two distinctN-acetyl-D-glucosaminidases, NAGase 1 and NAGase 2. NAGase 1 had a molecular weight of 97 000 and NAGase 2 was comprised of two subunits, of molecular weights 64 000 and 66 000. The optimal temperature and pH for NAGase 1 were 57 °C and pH 5 and for NAGase 2 they were 37 °C and pH 5. NAGase 1 was more thermostable than NAGase 2. The isoelectric points of NAGase 1 and 2 were ca. pH 9.5 and 5.5, respectively. NAGase 1 and 2 were unaffected by a 10 mM concentration of chloride salts of the ions Ca2+, Mg2+, or Zn2+, by 10 mM EDTA, and by 0.25–1 mM of short chain fatty acids. Dithiothreitol caused some inactivation of NAGase 2 while stimulating activity of NAGase 1. NAGase 1 had aKmof 0.38 mM and aKcat/Kmof 3923.88 M−1∙s−1. NAGase 2 had aKmof 2.095 mM and aKcat/Kmof 411.88 M−1∙s−1whenp-nitrophenyl-β-N-acetylglucosaminide was used as the substrate.Key words:Beauveria bassiana,N-acetyl-D-glucosaminidase, entomopathogenic fungus, extracellular enz

ISSN:0008-4166    

DOI:10.1139/m93-006

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

A method was developed to reduce the numbers of thermophilic bacteria on isolation plates, which in turn facilitated the detection and isolation of thermophilic actinomycetes. The method involves exposing the test material to bacteriophage suspensions prior to inoculation on isolation plates. This method was applied to composted eucalyptus bark samples, which were then inoculated on R8 and 1/2 TSA + 0.2% casein hydrolysate agar plates. The phage susceptibility of thermophilic bacteria provided a selective means of reducing their numbers on isolation plates and hence increased the numbers ofThermomonospora,Saccharopolyspora rectivirgula, and thermophilicStreptomycesspp. on these media in comparison with the numbers recorded from control plates.Key words: bacteriophage, thermophilic bacteria, thermophilic actinomycetes, composted eucalyptus bark.

ISSN:0008-4166    

DOI:10.1139/m93-007

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

The sporocidal properties of peracetic acid, hydrogen peroxide, chlorine, and formaldehyde were comparedin vitrousing a dilution–neutralization micromethod. A combination of peracetic acid and hydrogen peroxide was also tested to assess their interactions. The activities of these agents, which are widely used as disinfectants, were evaluated againstBacillusspore isolates found on stored membranes and collection cultures. Peracetic acid and chlorine exhibited an excellent antimicrobial activity, with a destruction of 105spores/mL after 5 min of contact. Generally the effects of the biocides tested were time dependent. The sporocidal activities of hydrogen peroxide and formaldehyde were the lowest. The combination of peracetic acid and hydrogen peroxide, tested by a checkerboard micromethod, was found to be synergistic. The minimal sporocidal concentration (MSC) was established in terms of time for each biocide. The lowest MSC values for peracetic acid, hydrogen peroxide, chlorine, and formaldehyde were 168–336 ppm (1–2 h of contact), 5625–11250 ppm (5–7 h), 168–336 ppm (2–3 h), and 1875–3750 ppm (5–30 min), respectively. The MSC of a biocide combination of peracetic acid and hydrogen peroxide showed that synergy was maintained with increasing contact time and that the MSC could be reduced by two to eight times when compared with those of the biocides alone. Optimal concentrations and contact times of those chemicals that were promisingin vitrowere then tested for their ability to disinfect ultrafiltration membranes. The sporocidal activities of peroxide compounds and chlorine were confirmed and the synergism between peracetic acid and hydrogen peroxide was also maintained. The biocide combination of peracetic acid and hydrogen peroxide gave successful results; 21 ppm of peracetic acid + 2813 ppm of hydrogen peroxide allowed a total disinfection of hollow fibers in 2–3 h of contact.Key words: peracetic acid, hydrogen peroxide, chlorine, combination, ultrafiltration membranes.

ISSN:0008-4166    

DOI:10.1139/m93-008

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

Changes in the microbial populations, their activities, and the ruminal fermentation were monitored for 50 d following the reintroduction of ciliate protozoa into four defaunated sheep. A protozoal population was reestablished successfully in each recipient, using a washed inoculum containing approximately 103cells, although there were between-animal differences in the rates of recolonization and genus establishment.Entodiniumspp. predominated in the initial stages of the refaunation period and had an apparent maximal generation time of 9–10 h. Bacterial and fungal numbers did not decline following the reintroduction of protozoa and a small transient increase in the numbers of amylolytic and xylanolytic bacteria and fungal zoospores occurred in the early stages of refaunation when the protozoal population was < 105/g ruminal contents, but these subsequently declined as the protozoa established. Although the fibrolytic bacterial population was lowest in period 3 (> 105protozoa/g), thein saccoruminal digestion ofLolium perennehay and polysaccharolytic enzyme activities in the solids-associated populations were either maintained or increased when protozoa were present confirming the important contribution of protozoa to fibre breakdown in the rumen. Significant changes in ruminal microbial activities occurred after protozoal reinoculation but before the rumen had refaunated completely. Arylamidase activities in the liquor-phase population and ruminal ammonia concentrations increased significantly within 48 h of transfaunation; the magnitude of the effects became more pronounced as the protozoal population developed. However, volatile fatty acid formation and ruminal pH were not affected after the reintroduction of protozoa.Key words: rumen, sheep, ciliate protozoa.

ISSN:0008-4166    

DOI:10.1139/m93-009

出版商:NRC Research Press    

年代:1993

数据来源: NRC

摘要:

The secreted endoglucanase (CenA) from the Gram-positive bacteriumCellulomonas fimiand a deletion derivative (ΔCenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of ΔCenA as a reporter molecule inCaulobacter crescentus. Expression ofcenAinC.crescentusyielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of ΔcenAyielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in theC.crescentuscytoplasm. Using the putative cytoplasmic and periplasmic forms of ΔCenA as markers, a simple assay for periplasmic ΔCenA hybrids was developed. This assay indicated that ΔCenA activity was largely independent of cellular location. To facilitate the use of ΔCenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating ΔcenAwas constructed. This vector was used to investigate factors important to the expression of the gene (rsaA) encoding the paracrystalline surface protein (S-layer) of the bacterium. It was found that altering the 5′ untranslated region of thersaAmRNA reduced gene expression by 70%. OnersaA:ΔcenAgene fusion resulting from these experiments that incorporated onlyrsaAtranslation initiation information was further modified to serve as a general reporter for creating transcriptional gene fusions with other promoters. Gene fusions between alkaline phosphatase (phoA) and eithercenAorlacZwere used to supplement information about RsaA secretion derived fromrsaA:phoAgene fusions. It was found that linkage of the N-terminal leader peptide of CenA to PhoA yielded 50–100 times more cell-associated PhoA activity inC.crescentusthan linkage of the RsaA N terminus. Taken together, these experiments indicated that ΔCenA was useful for tagging proteins localized to the cytoplasm, exported to the periplasm, or secreted from the cell, as well as for monitoring events in the cytoplasm such as examining factors important to the level of gene expression. Further, because ΔCenA was active in all cell compartments, it could be used to estimate the efficiency of hybrid protein export-secretion from enzyme activity measurements alone. In short, ΔCenA possessed many of the attributes of an "all-purpose" reporter.Key words: gene fusions, protein secretion, cellulase, alkaline phosphatase,Caulobacter crescentus.

ISSN:0008-4166    

DOI:10.1139/m93-010

出版商:NRC Research Press    

年代:1993

数据来源: NRC