Bacterial pathogen evolution: breaking news

The immense social and economic impact of bacterial pathogens, from drug-resistant infections in hospitals to the devastation of agricultural resources, has resulted in major investment to understand the causes and conse- quences of pathogen evolution. Recent genome se- quencing projects have provided insight into the evolution of bacterial genome structures; revealing the impact of mobile DNA on genome restructuring and pathogenicity. Sequencing of multiple genomes of relat- ed strains has enabled the delineation of pathogen evo- lution and facilitated the tracking of bacterial pathogens globally. Other recent theoretical and empirical studies have shown that pathogen evolution is significantly influenced by ecological factors, such as the distribution of hosts within the environment and the effects of co- infection. We suggest that the time is ripe for experi- mentalists to use genomics in conjunction with evolu- tionary ecology experiments to further understanding of how bacterial pathogens evolve.

A maize bacterial artificial chromosome (BAC) library from the European flint inbred line F2

We report here the construction and characterisation of a BAC library from the maize flint inbred line F2, widely used in European maize breeding programs. The library contains 86,858 clones with an average insert size of approximately 90 kb, giving approximately 3.2-times genome coverage. High-efficiency BAC cloning was achieved through the use of a single size selection for the high-molecular-weight genomic DNA, and co-transformation of the ligation with yeast tRNA to optimise transformation efficiency. Characterisation of the library showed that less than 0.5% of the clones contained no inserts, while 5.52% of clones consisted of chloroplast DNA. The library was gridded onto 29 nylon filters in a double-spotted 8 × 8 array, and screened by hybridisation with a number of single-copy and gene-family probes. A 3-dimensional DNA pooling scheme was used to allow rapid PCR screening of the library based on primer pairs from simple sequence repeat (SSR) and expressed sequence tag (EST) markers. Positive clones were obtained in all hybridisation and PCR screens carried out so far. Six BAC clones, which hybridised to a portion of the cloned Rp1-D rust resistance gene, were further characterised and found to form contigs covering most of this complex resistance locus.

Determination of the impact of continuous defoliation of Lolium perenne and Trifolium repens on bacterial and fungal community structure in rhizosphere soil

To determine the effects of defoliation on microbial community structure, rhizosphere soil samples were taken pre-, and post-defoliation from the root tip and mature root regions of Trifolium repens L. and Lolium perenne L. Microbial DNA isolated from samples was used to generate polymerase chain reaction-denaturing gradient gel electrophoresis molecular profiles of bacterial and fungal communities. Bacterial plate counts were also obtained. Neither plant species nor defoliation affected the bacterial and fungal community structures in both the root tip and mature root regions, but there were significant differences in the bacterial and fungal community profiles between the two root regions for each plant. Prior to defoliation, there was no difference between plants for bacterial plate counts of soils from the root tip regions; however, counts were greater in the mature root region of L. perenne than T. repens. Bacterial plate counts for T. repens were higher in the root tip than the mature root region. After defoliation, there was no effect of plant type, position along the root or defoliation status on bacterial plate counts, although there were significant increases in bacterial plate counts with time. The results indicate that a general effect existed during maturation in the root regions of each plant, which had a greater impact on microbial community structure than either plant type or the effect of defoliation. In addition there were no generic consequences with regard to microbial populations in the rhizosphere as a response to plant defoliation.

Use of an artificial root to examine the influence of 8-hydroxyquinoline on soil microbial activity and bacterial community structure

Invasive plant species have been shown to alter the microbial community composition of the soils they invade and it is suggested that this below-ground perturbation of potential pathogens, decomposers or symbionts may feedback positively to allow invasive success. Whether these perturbations are mediated through specific components of root exudation are not understood. We focussed on 8-hydroxyquinoline, a putative allelochemical of Centaurea diffusa (diffuse knapweed) and used an artificial root system to differentiate the effects of 8-hydroxyquinoline against a background of total rhizodeposition as mimicked through supply of a synthetic exudate solution. In soil proximal (0-10 cm) to the artificial root, synthetic exudates had a highly significant (P < 0.001) influence on dehydrogenase, fluorescein diacetate hydrolysis and urease activity. in addition, 8-hydroxyquinoline was significant (p = 0.003) as a main effect on dehydrogenase activity and interacted with synthetic exudates to affect urease activity (p = 0.09). Hierarchical cluster analysis of 16S rDNA-based DGGE band patterns also identified a primary affect of synthetic exudates and a secondary affect of 8-hydroxyquinoline on bacterial community structure. Thus, we show that the artificial rhizosphere produced by the synthetic exudates was the predominant effect, but, that the influence of the 8-hydroxyquinoline signal on the activity and structure of soil microbial communities could also be detected. (C) 2009 Elsevier Ltd. All rights reserved.

Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation.

Formulation and delivery of the bacterial antagonist Bacillus subtilis for management of lentil vascular wilt caused by Fusarium oxysporum f. sp lentis

Different formulations of Bacillus subtilis were prepared using standard laboratory protocols. Bacillus subtilis survived in glucose and talc powders at 8.6 and 7.8 log(10) CFU/g, respectively, for 1 year of storage at room temperature compared with 3.5 log(10) CFU/g on a peat formulation. Glasshouse experiments using soil and seed treatments were conducted to test the efficacy of B. subtilis for protecting lentil against the wilt disease caused by Fusariumoxysporum f. sp. lentis. Seed treatments with formulations of B. subtilis on glucose, talc and peat significantly enhanced its biocontrol activity against Fusarium compared with a treatment in which spores were applied directly to seed. The formulations decreased disease severity by reducing colonization of plants by the pathogen, promoting their growth and increased the dry weight of lentil plants. Of these treatments the glucose and talc-based powder formulations were more effective than the peat formulation and the spore application without a carrier. It was shown that the B. subtilis spores applied with glucose were viable for longer than those applied with other carriers. Seed treatment with these formulated spores is an effective delivery system that can provide a conducive environment for B. subtilis to suppress vascular wilt disease on lentil and has the potential for utilization in commercial field application.

Factors affecting biological control effectiveness of Pasteuria penetrans in Meloidogyne javanica and the bacterial development in the nematode body

The invasion and infectivity of Meloidogyne javanica juveniles (J2) encumbered with spore of Pasteuria Penetrans were influenced by the temperature and the time J2 were in the soil before exposure to roots. The percentage of infected females decreased as the time juveniles spent in soil increased. When spore encumbered J2 were maintained at 30 degrees C the decrease in infection was greater than that at 18 degrees C. The thermal time requirements and the base temperature for P. penetrans development were estimated. The rate of development followed an exponential curve between 21 and 36 degrees C and the base temperature for development was estimated by extrapolation to be 18.5 degrees C. The effect of integrating a nematode resistant tomato cultivar with the biocontrol agent P. penetrans also was investigated. The ability of the biocontrol agent to reduce numbers of root-knot nematodes was dependent on the densities of the nematode and P. penetrans spores in the soil.

Antagonistic potential of bacterial isolates associated with entomopathogenic nematodes against tomato wilt caused by Fusarium oxysporum f.sp., Lycopersici under greenhouse conditions

Three concentrations of Xenorhabdus nematophila and Xenorhabdus spp., (4x10(5,) 4x10(6,) 4x10(7) cells/ml) were evaluated in the laboratory and in pot experiments to test their antagonistic effects on Fusarium oxysporum f.sp., lycopersici. All concentrations effectively inhibited its growth on agar plates. In soil under greenhouse conditions treatments with each bacterium at 4x10(7) cells/ml reduced the disease incidence of tomato by up to 40.38 and 47.54% respectively and there were significant increases of plant biomass by 198 and 211% respectively. The rhizosphere population of Fusarium oxysporum f.sp., lycopersici was reduced by 97%. The Xenorhabdus spp., was comparatively more effective than X. nematophila.

Susceptibility of different insect pupae to the bacterial symbiont, Xenorhabdus nematophila, isolated from the entomopathogenic nematode Steinernema carpocapsae

Cells of the bacterial symbiont Xenorhabdus nematophila from the entomopathogenic nematode, Steinernema carpocapsae entered the pupae of Plutella xylostella after 15 minutes treatment with suspensions containing the bacterial cells. Secretions of Xenorhabdus nematophila, in either broth or water, were found lethal to the pupae of P. xylostella when applied in moist sand. The bacterial symbiont Xenorhabdus nematophila was found lethal to the pupae of greater wax moth (Galleria mellonella), beet armyworm (Spodoptera exigua), diamondback moth (Plutella xylostella) and black vine weevil (Otiorhynchus sulcatus) in the absence of the nematode vector and the cells of X. nematophila entered the haemocoele of the pupae.

A novel parallel approach to the likelihood-based estimation of admixture in population genetics

Inferring population admixture from genetic data and quantifying it is a difficult but crucial task in evolutionary and conservation biology. Unfortunately state-of-the-art probabilistic approaches are computationally demanding. Effectively exploiting the computational power of modern multiprocessor systems can thus have a positive impact to Monte Carlo-based simulation of admixture modeling. A novel parallel approach is briefly described and promising results on its message passing interface (MPI)-based C implementation are reported.

Bacterial evolution by genomic island transfer occurs via DNA transformation in planta

Our understanding of the evolution of microbial pathogens has been advanced by the discovery of "islands" of DNA that differ from core genomes and contain determinants of virulence [1, 2]. The acquisition of genomic islands (GIs) by horizontal gene transfer (HGT) is thought to have played a major role in microbial evolution. There are, however, few practical demonstrations of the acquisition of genes that control virulence, and, significantly, all have been achieved outside the animal or plant host. Loss of a GI from the bean pathogen Pseudomonas syringae pv. phaseolicola (Pph) is driven by exposure to the stress imposed by the plant's resistance response [3]. Here, we show that the complete episomal island, which carries pathogenicity genes including the effector avrPphB, transfers between strains of Pph by transformation in planta and inserts at a specific att site in the genome of the recipient. Our results show that the evolution of bacterial pathogens by HGT may be achieved via transformation, the simplest mechanism of DNA exchange. This process is activated by exposure to plant defenses, when the pathogen is in greatest need of acquiring new genetic traits to alleviate the antimicrobial stress imposed by plant innate immunity [4].