Phylogenetic identification and in situ detection of individual microbial cells without cultivation.

The frequent discrepancy between direct microscopic counts and numbers of culturable bacteria from environmental samples is just one of several indications that we currently know only a minor part of the diversity of microorganisms in nature. A combination of direct retrieval of rRNA sequences and whole-cell oligonucleotide probing can be used to detect specific rRNA sequences of uncultured bacteria in natural samples and to microscopically identify individual cells. Studies have been performed with microbial assemblages of various complexities ranging from simple two-component bacterial endosymbiotic associations to multispecies enrichments containing magnetotactic bacteria to highly complex marine and soil communities. Phylogenetic analysis of the retrieved rRNA sequence of an uncultured microorganism reveals its closest culturable relatives and may, together with information on the physicochemical conditions of its natural habitat, facilitate more directed cultivation attempts. For the analysis of complex communities such as multispecies biofilms and activated-sludge flocs, a different approach has proven advantageous. Sets of probes specific to different taxonomic levels are applied consecutively beginning with the more general and ending with the more specific (a hierarchical top-to-bottom approach), thereby generating increasingly precise information on the structure of the community. Not only do rRNA-targeted whole-cell hybridizations yield data on cell morphology, specific cell counts, and in situ distributions of defined phylogenetic groups, but also the strength of the hybridization signal reflects the cellular rRNA content of individual cells. From the signal strength conferred by a specific probe, in situ growth rates and activities of individual cells might be estimated for known species. In many ecosystems, low cellular rRNA content and/or limited cell permeability, combined with background fluorescence, hinders in situ identification of autochthonous populations. Approaches to circumvent these problems are discussed in detail

Isolation of DD carboxypeptidase from Streptomyces albus G culture filtrates

Streptomyces albus G secretes a soluble DD carboxypeptidase whose catalytic activities are similar to those of the particulate DD carboxypeptidase from Escherichia coli. Both enzymes hydrolyze the C-terminal D-alanyl-D-alanine linkage of UDP-N-acetylmuramyl-L-alanyl-γ-D-glutamyl-(L)-meso-diaminopimelyl-(L)-D-alanyl-D-aIanine and the enzyme-peptide interactions have identical Michaelis constants. Like the E. coli enzyme, the Streptomyces DD carboxypeptidase exhibits endopeptidase activities. The Streptomyces enzyme is lytic for those walls in which the peptidoglycan interpeptide bonds are mediated through C-terminal D-alanyl-D linkages. There is no strict requirement for a specific structure of the C-terminal D-amino acid residue. The tripeptide Nα , Nє -bisacetyl-L-lysyl-D-alanyl-D-alanine is an excellent substrate for the Streptomyces DD carboxypeptidase

Use of green fluorescent protein for online, single cell detection of bacteria introduced into activated sludge microcosms

A derivative of broad host-range plasmid RP4 was tagged with the green fluorescent protein (GFP) and this construct was used to mark various Gram-negative bacteria. Video recording of combined phase contrast and epifluorescence microscopy was employed to track the fate of individual marked cells after introduction into a sludge microcosm. Whether the introduced bacteria were laboratory strains or activated sludge isolates. they were found to be rapidly eliminated from the microcosms as a result of intense predation by protozoa. Furthermore, a method was developed that allows detection of GFP-tagged bacteria in fixed samples that are simultaneously used for in situ hybridisation with a eukaryote-specific rRNA-targeted oligonucleotide probe for visualisation of protozoa

Inactivation of gltB Abolishes Expression of the Assimilatory Nitrate Reductase Gene (nasB) in Pseudomonas putida KT2442

By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase, luxAB, to genes of Pseudomonas putida KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking nasB suggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction of nasB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of the gltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods

Phylogenetic Diversity In The Genus Bacillus As Seen By 16S rRNA Sequencing Studies.

Comparative sequence analysis of 16S ribosomal (r)RNAs or DNAs of Bacillus alvei, B. laterosporus, B. macerans, B. macquariensis, B. polymyxa and B. stearothermophilus revealed the phylogenetic diversity of the genus Bacillus. Based on the presently available data set of 16S rRNA sequences from bacilli and relatives at least four major Bacillus clusters can be defined: a Bacillus subtilis cluster including B. stearothermophilus, a B. brevis cluster including B. laterosporus, a B. alvei cluster including B. macerans, B. maquariensis and B. polymyxa and a B. cycloheptanicus branch