Screening microorganisms for chitin hydrolysis and production of ethanol from amino sugars

Seventy-two strains of bacteria representing 39 genera and one yeast (Candida albicans) were screened for ability to hydrolyze chitin. Chitin hydrolysis was determined by a clear zone surrounding colonies growing on the surface of chitin agar. Species with the largest clear zone to colony size (CZ/CS) ratio were further compared for chitinolysis by assaying the level of reducing sugar produced in broth culture. Three yeasts and one bacterial strain known to produce ethanol from glucose were compared for their abilities to produce ethanol from amino sugars. Of the 72 strains screened, 23 produced CZ/CS ratios ranging from 0·38 to 2·5. The highest ratios were observed for strains in the genera: Bacillus and Serratia, followed by Micrococcus, Aeromonas, Vibrio, Clostridium and Plesiomonas. The other species examined produced ratios of less than 1 or were unable to hydrolyze chitin.Hansenula anomala, Pachysolen tannophilus, Saccharomyces cerevisiae, and Zymomonas mobilis were compared for their abilities to grow on and produce ethanol from glucose, glucosamine, and N-acetylglucosamine (NAG). Saccharomyces cerevisiae and H. anomala produced ethanol only from glucose. Pachysolen tannophilus and Z. mobilis produced ethanol from glucose, glucosamine and NAG. The highest concentration of ethanol produced from amino sugar was 598 μg ml⁻¹ from 10 mg ml⁻¹ glucosamine by Z. mobilis. This level was achieved only when yeast extract was included in the medium. Saccharomyces cerevisiae did not grow on glucosamine and Z. mobilis did not grow well on NAG.     

Novel tellurite-amended media and specific chromosomal and Ti plasmid probes for direct analysis of soil populations of Agrobacterium biovars 1 and 2

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K(2)TeO(3) was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K(2)TeO(3) concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K(2)TeO(3)-amended medium. The bona fide agrobacterium colonies growing on media amended with K(2)TeO(3) were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10(3) to 10(4) agrobacteria. g of dry soil(-1) in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.     

A modified procedure for the detection of microbial producers of extracellular proteolytic enzymes

Summary A simple procedure for the detection of microbial producers of proteolytic enzymes using dyed gelatin microcarrier particles incorporated into appropriate nutrient agar is described. Extracellular proteinases produced by the tested microbial strains hydrolyzed the substrate and clear dyed zones around and under the colonies were formed.     

Biochemical and genetical aspects of the taxonomy of Rhizobium japonicum

Summary The major distinguishing characteristics of fifty diverse strains ofR. japonicum were surveyed. Nodulation onGlycine max, slow growth rate in yeast extract-mannitol broth, alkaline reaction with no serum zone in litmus milk, and lack of an acid reaction in either rhamnose or xylose media rather consistently separatedR. japonicum from other rhizobium species. The degree of relatedness between strains ofR. japonicum was measured using antibiotic resistance, immunological reactions and DNA homology. Generally the strains were resistant to erythromycin, polymyxin B, chloramphenicol, and sensitive to novobiocin. This response pattern differred from control strains consisting ofAgrobacterium tumefaciens, R. meliloti, andR. phaseoli. Response to six other antibiotics was quite random. Serological data show that none of 38 strains shared all somatic antigens. There was a relatively homogeneous group consisting of about ? of the strains tested. Two other, smaller diffuse clusters of strains shared some common antigens, while about one-half of the strains tested reacted only with their own antisera or perhaps reacted with antiserum of one other strain. Comparisons of the theoretical DNA homology between 26 strains showed three statistically significant but overlapping clusters. However, the degree of homology between strains at the extremes of these clusters is only about 70 per cent. Antibiotic response, serological reactions, and DNA homology did not correlate well but demonstrate considerable heterogeneity within this species. Lipid analysis of 5 strains showed the major lipids present to be phospholipids including large amounts of phosphatidylcholine and phosphatidylethanolamine. Such large levels of phosphatidylcholine have previously been found only in the pseudomonads although smaller amounts have been reported in Agrobacterium. Several workers have devised taxonomic schemes based on lipid composition and have thus linked Agrobacterium to the Pseudomonadales. The findings reported here tend to linkR. japonicum to both Agrobacterium and the Pseudomonadales. Glucose and gluconate catabolism were investigated both by the radio-respirometric method and by assaying for key enzymes of the major energy-producing pathways. The results show glucose is metabolized solely by the Entner-Doudoroff Pathway in the one strain studied. The Embden Meyerhoff Parnas and pentose-phosphate pathways were absent. While an inducible Entner-Doudoroff Pathway has been found in many gram-negative Eubacteriales, glucose metabolism solely by this pathway has been shown in the Pseudomonadales, linkingR. japonicum to this order. Gluconate catabolism occurred via the Entner-Doudoroff Pathway but an additional pathway was participating. Evidence was found that this pathway is the ketogluconate pathway previously reported only in the Pseudomonadales. Amino acid analysis patterns and distribution of label from radioactive aspartic acid indicate that glutamate is synthesized in the one strain studied, partly via a pathway previously reported inAcetobacter suboxydans. These investigations indicate thatR. japonicum is a recognizable species, albeit an extremely heterogeneous one. Metabolic data suggest a relatively close genetic relationship between this species and the Pseudomonadales. There is evidence thatR. japonicum may be more closely related to Agrobacterium than to the ‘fast growing’ species of Rhizobium, but because the data indicate thatR. japonicum is an authentic species, combination of these organisms and Agrobacterium is not presently warranted.     

Comparative study of the phospholipase activity of pathogenic and saprophytic Leptospira cultured on a serum-lecithin agar

The phospholipase activity of leptospires cultivated on serum-lecithin agar has been studied. Two zones of changes in the medium have been found to appear around the colonies of saprophytic Leptospira strains: transparent (5.25 +/- 2.09 mm wide) and turbid (6.90 +/- +/- 1.46 mm wide), which is linked with the production of phospholipases A and C. Only a single clear zone is formed around the colonies of pathogenic strains due to the production of phospholipase A. At the same time virulent Leptospira strains show greater phospholipase activity (the zones are 6.0 +/- 1.2 mm wide) than avirulent strains (the zones are 1.6 +/- +/- 0.04 mm wide).     

Identification of Agrobacterium spp. present within Brassica napus seed by TaqMan PCR--implications for GM screening procedures

A fluorogenic probe (fliG-P), designed within a chromosomal DNA sequence, was used in a TaqMan PCR assay to identify Agrobacterium spp. The TaqMan assay detected 58 of 59 Agrobacterium strains tested, but did not detect 13 other Rhizobiaceae strains. Seedlings were grown from seven lots of surface-sterilised Brassica napus seed. Seedlings from these samples were placed in phosphate buffer and the resulting suspensions used to inoculate broth media selective for Agrobacterium biovars 1 and 2. Lysed broths (after 48 h incubation) were used as template in the fliG TaqMan PCR to detect Agrobacterium sp. in one of the seed samples. Individual Agrobacterium strains were isolated from this sample and tested by three Ti-plasmid conventional PCR assays. None of the strains possessed a plasmid. This is the first report of Agrobacterium sp. present within the seed of B. napus, a crop routinely screened for genetically modified DNA contamination using PCR assays with Agrobacterium sequences as targets.     

Bacterial Interference with L-Forms

During the course of studying the effect of normal nasal flora on the growth of L-forms, a clear zone of inhibition was observed around colonies of many coagulase-negative staphylococci. Subsequent investigation demonstrated that Staphylococcus aureus and some S. albus strains produce a substance which is capable of markedly inhibiting the growth of stable staphylococcal and streptococcal L-forms. This interfering substance is separable from the staphylococcal organism and is diffusible through 1.5% agar, but not through a dialysis membrane. It is heat-stable.     

A STUDY OF SOIL BACTERIA DISSOLVING CERTAIN MINERAL PHOSPHATE FERTILIZERS AND RELATED COMPOUNDS

SUMMARY: Over a hundred isolates which produced haloes around their colonies on dilution plates containing calcium carbonate or dicalcium phosphate have been obtained in pure culture from the root region of the oat plant. Of these, more than 50% were pleomorphic, and this group had the highest proportion of isolates which could produce clear zones on agar media containing either calcium carbonate, dicalcium phosphate, tricalcium phosphate, freshly precipitated hydroxyapatite or basic slag. None of the isolates showed dissolving ability on agar media containing gafsa rock phosphate, variscite, strengite or taranakite. However, when an analytical method was used, 82% of the isolates tested proved able to release phosphate from gafsa rock phosphate, though to a much lesser extent than from dicalcium phosphate. None of the isolates tested by this method released phosphate from variscite, strengite or taranakite.
The nature of the organic acids produced from glucose by 26 of the isolates was also investigated. The majority produced mainly lactic acid, but a few also gave an acid with chromatographic properties similar to those of 2-keto-gluconic acid.     

Novel Tellurite-Amended Media and Specific Chromosomal and Ti Plasmid Probes for Direct Analysis of Soil Populations of Agrobacterium Biovars 1 and 2

Ecology and biodiversity studies of Agrobacterium spp. require tools such as selective media and DNA probes. Tellurite was tested as a selective agent and a supplement of previously described media for agrobacteria. The known biodiversity within the genus was taken into account when the selectivity of K₂TeO₃ was analyzed and its potential for isolating Agrobacterium spp. directly from soil was evaluated. A K₂TeO₃ concentration of 60 ppm was found to favor the growth of agrobacteria and restrict the development of other bacteria. Morphotypic analyses were used to define agrobacterial colony types, which were readily distinguished from other colonies. The typical agrobacterial morphotype allowed direct determination of the densities of agrobacterial populations from various environments on K₂TeO₃-amended medium. The bona fide agrobacterium colonies growing on media amended with K₂TeO₃ were confirmed to be Agrobacterium colonies by using 16S ribosomal DNA (rDNA) probes. Specific 16S rDNA probes were designed for Agrobacterium biovar 1 and related species (Agrobacterium rubi and Agrobacterium fici) and for Agrobacterium biovar 2. Specific pathogenic probes from different Ti plasmid regions were used to determine the pathogenic status of agrobacterial colonies. Various morphotype colonies from bulk soil suspensions were characterized by colony blot hybridization with 16S rDNA and pathogenic probes. All the Agrobacterium-like colonies obtained from soil suspensions on amended media were found to be bona fide agrobacteria. Direct colony counting of agrobacterial populations could be done. We found 10³ to 10⁴ agrobacteria · g of dry soil⁻¹ in a silt loam bulk soil cultivated with maize. All of the strains isolated were nonpathogenic bona fide Agrobacterium biovar 1 strains.     

Variation in polygalacturonase production among Aspergillus flavus isolates.

Pectinase production by Aspergillus flavus was determined by measuring clear zones formed around colonies stained with ruthenium red. Several isolates produced red zones instead of clear zones. Red zones were reproduced with pectinesterase and correlated with absence of specific polygalacturonases. Of 87 isolates tested, 15 produced red zones.     

Variation in polygalacturonase production among Aspergillus flavus isolates

Pectinase production by Aspergillus flavus was determined by measuring clear zones formed around colonies stained with ruthenium red. Several isolates produced red zones instead of clear zones. Red zones were reproduced with pectinesterase and correlated with absence of specific polygalacturonases. Of 87 isolates tested, 15 produced red zones.     

Distribution of chitinase and chitobiase in bacillus

Sixty strains representing 29 taxospecies ofBacillus were assayed for their ability to hydrolyze colloidal chitin. A qualitative estimation of chitinolysis was made from the clear zone produced around colonies in the conventional agar plate method and chitobiase activity by use of the fluorescence of 4-methylumbelliferyl-N-acetyl--d-glucosaminide.Strains positive in the chitin-agar plate method were assayed for production of reducing sugar in liquid culture. Seventeen of 52 strains representing 10 species ofBacillus were chitinolytic. The most chitinolytic species ofBacillus were:B. chitinosporus, B. pulvifaciens, B. alvei, B. Macerans, andB. licheniformis. Seventy-eight percent ofBacillus isolates from chitinenriched soil (AU Y91B1, AU-X (unidentified), and AU B₂–B₈) were chitinolytic. Twenty-three strains representing 15 species gave a positive test for chitobiase. Many strains negative for endochitinase gave a strong positive reaction (4+) for chitobiase.     

Universal PCR primers for detection of phytopathogenic Agrobacterium strains.

Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants.     

Structure and characterization of the crown gall opines heliopine, vitopine and ridéopine

The crown gall opines heliopine from tumors induced by octopine type Agrobacterium tumefaciens strains A6, A136(pTiB6-806), E9, A652 and 1590-1 and vitopine from tumor induced by grapevine strains S4 and T2 are identical to synthetic N2-(1'R-carboxyethyl)-L-glutamine. Tumors produced by strains S4 and T2 do not contain octopine or lysopine, but they do contain heliopine and the new opine ridéopine identified as N-(4'-aminobutyl)-D-glutamic acid. Grapevine strains S4 and T2 grow normally on tumor heliopine or synthetic heliopine and on tumor and synthetic ridéopine as well as on ridéopine lactam as sole carbon source. While octopine strains A6 and A136(pTiB6-806) do not grow on heliopine, mutant colonies do appear after a few weeks. Heliopine catabolism by octopine strains is not induced by octopine.     

Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum.

We studied the general mechanism for regulation of glucoamylase and pullulanase synthesis in Clostridium thermohydrosulfuricum. These amylases were expressed only when the organism was grown on maltose or other carbohydrates containing maltose units. Amylase synthesis was more severely repressed by glucose than by xylose. Catabolite repression-resistant mutants were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained glucose-starch agar plates. Amylases were produced in both wild-type and mutant strains when starch was added to cells growing on xylose but not when starch was added to cells growing on glucose. In both wild-type and mutant strains, glucoamylase and pullulanase were produced at high levels in starch-limited chemostats but not in glucose- or xylose-limited chemostats. Therefore, we concluded that amylase synthesis in C. thermohydrosulfuricum was inducible and subject to catabolite repression. The mutants produced about twofold more glucoamylase and pullulanase, and they were catabolite repression resistant for production of glucose isomerase, lactase, and isomaltase. The mutants displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production, and starch consumption.     

Observations on brilliant green agar with H2S indicator.

Several formulations of brilliant green agar with an added H2S indicator were evaluated. Results were optimum with variations of a basic formula consisting of 40 g of tryptic soy agar (Difco), 8 g of lactose, 8 g of sucrose, 80 mg of phenol red, 1 g of sulfanilamide, 1.5 g of ferric ammonium citrate, 5 g of sodium thiosulfate pentahydrate, and 7 mg of brilliant green dye per liter. Brilliant green dye was added after sterilization of the other components This formulation supported good growth of all of 39 strains of Salmonella tested. Normal biochemical types formed pink colonies with black centers, and an H2S-negative S. choleraesuis formed pink colonies without black centers. Of other bacteria tested, only Enterobacter, Klebsiella, and a few Citrobacter strains showed significant growth in 24 h. When lactose was omitted from the formulation, a lactose-fermenting strain formed pink colonies with black centers, and differentiation of Salmonella from the Enterobacter-Klebsiella groups was equally good. Addition of xylose (4.0 g) and L-lysine hydrochloride (5.4 g) to the above formulation improved differentiation between Salmonella and the few Citrobacter strains that grew and produced more intense blackening in Salmonella colonies. Addition of an H2S indicator to brilliant green agar formulations aided in identification of Salmonella colonies, especially in mixtures with other bacteria. These media were judged to give better differentiation of salmonellae from other bacteria than Hektoen agar with added novobiocin (10 mg/liter).     

Observations on brilliant green agar with H2S indicator.

Several formulations of brilliant green agar with an added H2S indicator were evaluated. Results were optimum with variations of a basic formula consisting of 40 g of tryptic soy agar (Difco), 8 g of lactose, 8 g of sucrose, 80 mg of phenol red, 1 g of sulfanilamide, 1.5 g of ferric ammonium citrate, 5 g of sodium thiosulfate pentahydrate, and 7 mg of brilliant green dye per liter. Brilliant green dye was added after sterilization of the other components This formulation supported good growth of all of 39 strains of Salmonella tested. Normal biochemical types formed pink colonies with black centers, and an H2S-negative S. choleraesuis formed pink colonies without black centers. Of other bacteria tested, only Enterobacter, Klebsiella, and a few Citrobacter strains showed significant growth in 24 h. When lactose was omitted from the formulation, a lactose-fermenting strain formed pink colonies with black centers, and differentiation of Salmonella from the Enterobacter-Klebsiella groups was equally good. Addition of xylose (4.0 g) and L-lysine hydrochloride (5.4 g) to the above formulation improved differentiation between Salmonella and the few Citrobacter strains that grew and produced more intense blackening in Salmonella colonies. Addition of an H2S indicator to brilliant green agar formulations aided in identification of Salmonella colonies, especially in mixtures with other bacteria. These media were judged to give better differentiation of salmonellae from other bacteria than Hektoen agar with added novobiocin (10 mg/liter).     

The evaluation of amylolytic activity of strains of coagulase negative staphylococci

Amylase activity of 30 strains of Staphylococcus spp. was determined by Tryptic Soy Agar on supplemented with 1.0% starch as the substrate. After incubation (time incubation 24 h or 168 h), the plates were flooded with Lugol solution. A clear zone around the colonies indicated amylase activity. The 23 (76.7%) strains CNS demonstrated the amylase activity. It was observed that 17 (80.9%) strains of S. epidermidis, and 6 (66.7%) strains non-S. epidermidis, starch hydrolyzed. Amylase production depends of time incubation (frequently 168 h) and growth atmosphere (frequently oxygen atmosphere)     

Cell-associated oligosaccharides of Bradyrhizobium spp.

We report the initial characterization of the cell-associated oligosaccharides produced by four Bradyrhizobium strains: Bradyrhizobium japonicum USDA 110, USDA 94, and ATCC 10324 and Bradyrhizobium sp. strain 32H1. The cell-associated oligosaccharides of these strains were found to be composed solely of glucose and were predominantly smaller than the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species. Linkage studies and nuclear magnetic resonance analyses demonstrated that the bradyrhizobial glucans are linked primarily by beta-1,6 and beta-1,3 glycosidic bonds. Thus, the bradyrhizobia appear to synthesize cell-associated oligosaccharides of structural character substantially different from that of the cyclic beta-1,2-glucans produced by Agrobacterium and Rhizobium species.     

Influence of nutrient media on the characteristics of the exopolysaccharide produced by three mucoid Pseudomonas aeruginosa strains

Mucoid strains of Pseudomonas aeruginosa of the 'gelatinous' (strain PM1) and 'mucoid' (strains PM3 and PM11) types (Wahba and Darrell, 1965), from cystic fibrosis patients were grown on different nutrient media, in liquid and on solid matrix, and their ability to synthesize uronic acid-containing exopolysaccharide of varying molecular sizes was assessed. Strain PM1 produced the exopolysaccharide in all liquid media tested. However, the exopolysaccharide was always polydispersed when citrate was present but monodispersed and of high molecular weight (HMW) in its absence. Strain PM1 also formed non-mucoid colonies on some solid media and on those media no exopolysaccharide was produced. On media, on which the organism was always mucoid monodispersed, HMW exopolysaccharide was recovered. Strains PM3 and PM1 produced monodispersed, HMW exopolysaccharide in liquid MacConkey's and V-8 media, but polydispersed or no exopolysaccharide in ll other liquid media tested. On MacConkey's agar these strains were mucoid initially but appeared non-mucoid as the cultures aged. This colonial change was accompanied by a quantitative and qualitative change in the exopolysaccharide. In media on which these strains produced only non-mucoid colonies little or no exopolysaccharide was recovered. Crude enzyme preparations from all three strains indicate that enzyme(s) capable of depolymerizing the indigenous exopolysaccharide exist in each organism.