Tony J. Pembroke

Revised Nomenclature for Transposable Genetic Elements

Transposable DNA elements occur naturally in the genomes of nearly all species of prokaryotes. A proposal for a uniform transposable element nomenclature was published prominently in the 1970s but is not, at present, available online even in abstract form, and many of the newly discovered elements have been named without reference to it. We propose here an updated version of the original nomenclature system for all of the various types of prokaryotic, autonomous, transposable elements excluding insertion sequences, for which a nomenclature system already exists. The use of this inclusive and sequential Tn numbering system for transposable elements, as described here, recognizes the ease of interspecies spread of individual elements, and allows for the naming of mosaic elements containing segments from two or more previously described types of transposons or plasmids. It will guard against any future need to rename elements following changes in bacterial nomenclature which occurs constantly with our increased understanding of bacterial phylogenies and taxonomic groupings. It also takes into account the increasing importance of metagenomic sequencing projects and the continued identification of new mobile elements from unknown hosts.

The complete nucleotide sequence and environmental distribution of the cryptic, conjugative, broad-host-range plasmid pIPO2 isolated from bacteria of the wheat rhizosphere

Plasmid pIPO2 is a cryptic, conjugative, broad-host-range plasmid isolated from the wheat rhizosphere. It efficiently self-transfers between alpha, beta and gamma Proteobacteria and has a mobilizing/retromobilizing capacity for IncQ plasmids. The complete nucleotide sequence of pIPO2 is presented on the basis of its mini-Tn5::luxABtet-tagged derivative, pIPO2T. The pIPO2 sequence is 39815 bp long and contains at least 43 complete ORFs. Apart from a suite of ORFs with unknown function, all of the genes carried on pIPO2 are predicted to be involved in plasmid replication, maintenance and conjugative transfer. The overall organization of these genes is different from previously described plasmids, but is similar to the genetic organization seen in pSB102, a conjugative plasmid recently isolated from the bacterial community of the alfalfa rhizosphere. The putative conjugative transfer region of pIPO2 covers 23 kb and contains the genes required for DNA processing (Dtr) and mating pair formation (Mpf). The organization of these transfer genes in pIPO2 is highly similar to the genetic organization seen in the environmental plasmid pSB102 and in pXF51 from the plant pathogen Xylella fastidiosa. Plasmids pSB102 and pXF51 have recently been proposed to form a new family of environmental broad-host-range plasmids. Here it is suggested that pIPO2 is a new member of this family. The proposed Mpf system of pIPO2 shares high amino acid sequence similarity with equivalent VirB proteins from the type IV secretion system of Brucella spp. Sequence information was used to design primers specific for the detection of pIPO2. Environmental DNA from a range of diverse habitats was screened by PCR with these primers. Consistently positive signals for the presence of pIPO2 were obtained from a range of soil-related habitats, including the rhizospheres of young wheat plants, of field-grown oats and of grass (all gramineous plants), as well as from the rhizosphere of tomato plants. These data add to the growing evidence that plasmids carry advantageous genes with as yet undefined functions in plant-associated communities.

Isolation of a novel Thermus thermophilus metal efflux protein that improves Escherichia coli growth under stress conditions.

The mechanisms of metal ion transport in thermophilic organisms are poorly understood. Phage display-based screening of a Thermus thermophilus genomic library in Escherichia coli led to the identification of a novel metal cation efflux protein. The Thermus protein showed extensive sequence and putative structural conservation to Czr and Czc proteins in mesophilic bacterial and mammalian species. Expression of the gene in E. coli led to increased resistance to zinc and cadmium ions, but not to cobalt, in an effect that was apparently caused by increased efflux of metals from the cell. This increased resistance was inducible by zinc and cadmium and, to a lesser extent, by cobalt. Furthermore, E. coli cells containing the Thermus gene exhibited improved cell physiology and delayed cell lysis during recombinant protein production, leading to accumulation of higher levels of recombinant protein. The molecular basis and potential application of the findings are discussed.

Evaluation of the Removal of Indicator Bacteria from Domestic Sludge Processed by Autothermal Thermophilic Aerobic Digestion (ATAD)

The degradation of sludge solids in an insulated reactor during Autothermal Thermophilic Aerobic Digestion (ATAD) processing results in auto-heating, thermal treatment and total solids reduction, however, the ability to eliminate pathogenic organisms has not been analysed under large scale process conditions. We evaluated the ATAD process over a period of one year in a two stage, full scale Irish ATAD plant established in Killarney and treating mixed primary and secondary sludge, by examining the sludge microbiologically at various stages during and following ATAD processing to determine its ability to eliminate indicator organisms. Salmonella spp. (pathogen) and fecal-coliform (indicator) densities were well below the limits used to validate class A biosolids in the final product. Enteric pathogens present at inlet were deactivated during the ATAD process and were not detected in the final product using both traditional microbial culture and molecular phylogenetic techniques. A high DNase activity was detected in the bulk sludge during the thermophilic digestion stage which may be responsible for the rapid turn over of DNA from lysed cells and the removal of mobile DNA. These results offer assurance for the safe use of ATAD sludge as a soil supplement following processing.

Use of PCR-DGGE Based Molecular Methods to Analyse Microbial Community Diversity and Stability during the Thermophilic Stages of an ATAD Wastewater Sludge Treatment Process as an Aid to Performance Monitoring

PCR and PCR-DGGE techniques have been evaluated to monitor biodiversity indexes within an ATAD (autothermal thermophilic aerobic digestion) system treating domestic sludge for land spread, by examining microbial dynamics in response to elevated temperatures during treatment. The ATAD process utilises a thermophilic population to generate heat and operates at elevated pH due to degradation of sludge solids, thus allowing pasteurisation and stabilisation of the sludge. Genera-specific PCR revealed that Archaea, Eukarya and Fungi decline when the temperature reaches 59°C, while the bacterial lineage constitutes the dominant group at this stage. The bacterial community at the thermophilic stage, its similarity index to the feed material, and the species richness present were evaluated by PCR-DGGE. Parameters such as choice of molecular target (16S rDNA or rpoB genes), and electrophoresis condition, were optimised to maximise the resolution of the method for ATAD. Dynamic analysis of microbial communities was best observed utilising PCR-DGGE analysis of the V6-V8 region of 16S rDNA, while rpoB gene profiles were less informative. Unique thermophilic communities were shown to quickly adapt to process changes, and shown to be quite stable during the process. Such techniques may be used as a monitoring technique for process health and efficiency.