Genetic and phenotypic diversity in <Emphasis Type="Italic">Burkholderia</Emphasis>: contributions by prophage and phage-like elements

Background  

Burkholderia species exhibit enormous phenotypic diversity, ranging from the nonpathogenic, soil- and water-inhabiting Burkholderia thailandensis to the virulent, host-adapted mammalian pathogen B. mallei. Genomic diversity is evident within Burkholderia species as well. Individual isolates of Burkholderia pseudomallei and B. thailandensis, for example, carry a variety of strain-specific genomic islands (GIs), including putative pathogenicity and metabolic islands, prophage-like islands, and prophages. These GIs may provide some strains with a competitive advantage in the environment and/or in the host relative to other strains.     

Genome sequencing and analysis of Yersina pestis KIM D27, an avirulent strain exempt from select agent regulation

Yersinia pestis is the causative agent of the plague. Y. pestis KIM 10+ strain was passaged and selected for loss of the 102 kb pgm locus, resulting in an attenuated strain, KIM D27. In this study, whole genome sequencing was performed on KIM D27 in order to identify any additional differences. Initial assemblies of 454 data were highly fragmented, and various bioinformatic tools detected between 15 and 465 SNPs and INDELs when comparing both strains, the vast majority associated with A or T homopolymer sequences. Consequently, Illumina sequencing was performed to improve the quality of the assembly. Hybrid sequence assemblies were performed and a total of 56 validated SNP/INDELs and 5 repeat differences were identified in the D27 strain relative to published KIM 10+ sequence. However, further analysis showed that 55 of these SNP/INDELs and 3 repeats were errors in the KIM 10+ reference sequence. We conclude that both 454 and Illumina sequencing were required to obtain the most accurate and rapid sequence results for Y. pestis KIMD27. SNP and INDELS calls were most accurate when both Newbler and CLC Genomics Workbench were employed. For purposes of obtaining high quality genome sequence differences between strains, any identified differences should be verified in both the new and reference genomes.     

Clonality Despite Sex: The Evolution of Host-Associated Sexual Neighborhoods in the Pathogenic Fungus Penicillium marneffei

Molecular genetic approaches typically detect recombination in microbes regardless of assumed asexuality. However, genetic data have shown the AIDS-associated pathogen Penicillium marneffei to have extensive spatial genetic structure at local and regional scales, and although there has been some genetic evidence that a sexual cycle is possible, this haploid fungus is thought to be genetically, as well as morphologically, asexual in nature because of its highly clonal population structure. Here we use comparative genomics, experimental mixed-genotype infections, and population genetic data to elucidate the role of recombination in natural populations of P. marneffei. Genome wide comparisons reveal that all the genes required for meiosis are present in P. marneffei, mating type genes are arranged in a similar manner to that found in other heterothallic fungi, and there is evidence of a putatively meiosis-specific mutational process. Experiments suggest that recombination between isolates of compatible mating types may occur during mammal infection. Population genetic data from 34 isolates from bamboo rats in India, Thailand and Vietnam, and 273 isolates from humans in China, India, Thailand, and Vietnam show that recombination is most likely to occur across spatially and genetically limited distances in natural populations resulting in highly clonal population structure yet sexually reproducing populations. Predicted distributions of three different spatial genetic clusters within P. marneffei overlap with three different bamboo rat host distributions suggesting that recombination within hosts may act to maintain population barriers within P. marneffei.     

Triallelic SNP-mediated genotyping of regenerated protoplasts of the heterokaryotic fungus Rhizoctonia solani

The aneuploid and heterokaryotic nuclear condition of the soil fungus Rhizoctonia solani have provided challenges in obtaining a complete genome sequence. To better aid in the assembly and annotation process, a protoplast and single nucleotide polymorphism (SNP)-based method was developed to identify regenerated protoplasts with a reduced nuclear genome. Protocol optimization experiments showed that enzymatic digestion of mycelium from a 24 h culture of R. solani increased the proportion of protoplasts with a diameter of ≤7.5 μm and 1-4 nuclei. To determine whether strains regenerated from protoplasts with a reduced number of nuclei were genetically different from the parental strain, triallelic SNPs identified from variance records of the genomic DNA sequence reads of R. solani were used in PCR-based genotyping assays. Results from 16 of the 24 SNP-based PCR assays provided evidence that one of the three alleles was missing in the 11 regenerated protoplast strains, suggesting that these strains represent a reduced genomic complement of the parental strain. The protoplast and triallelic SNP-based method used in this study may be useful in strain development and analysis of other basidiomycete fungi with complex nuclear genomes.