Identification and potential enzyme capacity of flavobacteria isolated from the rhizosphere of barley (Hordeum vulgare L.).

The diversity of 99 flavobacterium-like isolates from a barley rhizosphere is described. They were identified on 1/10 strength tryptic soy agar by their yellowish colour and a flexirubin reaction after exposure to 10% KOH.16S rDNA partial sequencing identified the majority (70%) of isolates as Flavobacterium. Twelve percent of the isolates belonged to other genera in the phylum Bacteroidetes. Finally 17% of the isolates did not belong to the phylum Bacteroidetes. Most of the Flavobacterium isolates were affiliated to various aquatic, validly named species and likely represent a bulk of undescribed soil Flavobacterium species found in especially high numbers in the rhizosphere of young plant roots. Most Flavobacterium isolates showed gliding motility on CY agar and VY/2 agar, whereas none of the other isolates shared this feature. A high percentage of Flavobacterium strains produced enzymes involved in polysaccharide and protein digestion as well as extracellular phosphatases, compared with strains related to other genera in the phylum Bacteroidetes.

Occurrence and pathogenic potential of Bacillus cereus group bacteria in a sandy loam.

The major part (94%) of the Bacillus cereus-like isolates from a Danish sandy loam are psychrotolerant Bacillus weihenstephanensis according to their ability to grow at temperatures below 7 degrees C and/or two PCR-based methods, while the remaining 6% are B. cereus. The Bacillus mycoides-like isolates could also be divided into psychrotolerant and mesophilic isolates. The psychrotolerant isolates of B. mycoides could be discriminated from the mesophilic by the two PCR-based methods used to characterize B. weihenstephanensis. It is likely that the mesophilic B. mycoides strains are synonymous with Bacillus pseudomycoides, while psychrotolerant B. weihenstephanensis, like B. mycoides, are B. mycoides senso stricto. B. cereus is known to produce a number of factors, which are involved in its ability to cause gastrointestinal and somatic diseases. All the B. cereus-like and B. mycoides like isolates from the sandy loam were investigated by PCR for the presence of 12 genes encoding toxins. Genes for the enterotoxins (hemolysin BL and nonhemolytic enterotoxin) and the two of the enzymes (cereolysin AB) were present in the major part of the isolates, while genes for phospolipase C and hemolysin III were present in fewer isolates, especially among B. mycoides like isolates. Genes for cytotoxin K and the hemolysin II were only present in isolates affiliated to B. cereus. Most of the mesophilic B. mycoides isolates did not possess the genes for the nonhemolytic enterotoxin and the cereolysin AB. The presence of multiple genes coding for virulence factors in all the isolates from the B. cereus group suggests that all the isolates from the sandy loam are potential pathogens.

Transfer and expression of the mosquitocidal plasmid pBtoxis in Bacillus cereus group strains.

The toxicity of Bacillus thuringiensis subsp. israelensis to dipteran larvae (mosquitoes and black flies) depends on the presence of the pBtoxis plasmid. In this paper, two antibiotic resistance tagged pBtoxis were transferred by conjugation to other Bacillus cereus group strains. Among 15 potential recipients, only a lepidopteran active B. thuringiensis subspecies kurstaki and a B. cereus strain received the plasmid pBtoxis with a low transfer rate of about 10(-8) transconjugants/recipient. The resulting B. thuringiensis subspecies kurstaki transconjugant was active to both lepidopteran and dipteran targets and the B. cereus transconjugant was active against dipteran insects. Phase contrast microscopy showed that the B. cereus transconjugants could produce only round crystalline inclusion bodies while B. thuringiensis subspecies kurstaki transconjugant could produce both round and bipyramidal crystals during sporulation. SDS-PAGE revealed that all the major mosquitocidal proteins from pBtoxis could express in the two transconjugants, including Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa and Cyt1Aa. However, none of the experiment showed any indications of mobilising abilities of pBtoxis. The limited number of strains, which could receive and maintain pBtoxis using a conjugational helper plasmid, indicates a very narrow host range of the B. thuringiensis subsp. israelensis pBtoxis plasmid.

Isolation and taxonomic affiliation of N-heterocyclic aromatic hydrocarbon-transforming bacteria.

The azaarenes (nitrogen-containing heterocyclic aromatic hydrocarbons) are products of incomplete combustion processes and thus are widely distributed with tar and oil products in the environment. Despite their adverse organoleptic, toxic, and carcinogenic characteristics, the biodegradability and fate of multi-ring azaarenes have received little attention. This work demonstrates the presence of genetically diverse azaarene-degrading bacteria in coal tar-contaminated soils. Thirty-eight bacterial strains able to transform the three-ring azaarenes, 5,6- and 7,8-benzoquinoline, phenanthridine, phenazine, or acridine, were isolated. Only seven of these strains grew in liquid medium on the specific azaarene compounds on which they were isolated using plates; and the rest transformed the azaarenes without growth. Taxonomic characterization by 16S ribosomal DNA sequencing revealed that our enrichment technique provided a diversity of 18 different azaarene-transforming bacterial species. Only a few strains were able to mineralize the homocyclic analogue, phenanthrene. Several of the isolates, e.g., Dyadobacter fermentans, Methylopila capsulata, and Agrobacterium tumefaciens, were related to genera relatively unknown with respect to the biodegradation of xenobiotic compounds. These strains can provide further information on the fate of azaarenes in the environment.