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Investigating H. Influenzae Species Diversity by Comparative Genomic Hybridization

ABSTRACT: Haemophilus influenzae(Hi) is a gram-negative rod shaped bacterium that lives symbiotically in the upper respiratory tract of humans. Capsulated Hi, as well as non-capsulated strains are known to cause a number of significant infections. Recognizing and understanding the links between the phenotypic traits and the genetic background has paramount epidemiological and clinical importance. To date, a panoply of microbiological and molecular biology tools have been developed and utilized by researchers aimed at identifying the evolutionary links among strains and isolates. Comparative genomic hybridization (CGH) has been shown to be a useful tool for screening strains for their genetic content. However, there is a major limitation when CGH is conducted on a microarray based on a single reference genome. CGH results report which genes are present or absent relative to the genome. Hence the information about novel genetic content that the query strain possesses remains obscure. We report here the construction of the first Hi pan-genome microarray representing ca. 4600 features by 70-mers. In addition to those from the Rd strain, new features originate from the unfinished genome sequences present in NCBI database and from our novel gene discovery project efforts using strain HK1212. Genomes of 20strains belonging to different phylogenetic lineages were screened for their gene loss and gain utilizing the species microarray. The results obtained by employing the multistrain species microarray provide comprehensive information about the genomic content of uncharacterized strains. The trees generated by CGH, in general, do not reproduce the phylogeny of a species in terms of vertical evolution, but instead represents the overall relatedness of genomes to one another and provide an assessment about the species genome evolution. Keywords: Comparative Genomic Hybridization

ORGANISM(S): Haemophilus influenzae

PROVIDER: GSE8300 | GEO | 2009/09/09

SECONDARY ACCESSION(S): PRJNA101265

REPOSITORIES: GEO

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Project description:Haemophilus influenzae(Hi) is a gram-negative rod shaped bacterium that lives symbiotically in the upper respiratory tract of humans. Capsulated Hi, as well as non-capsulated strains are known to cause a number of significant infections. Recognizing and understanding the links between the phenotypic traits and the genetic background has paramount epidemiological and clinical importance. To date, a panoply of microbiological and molecular biology tools have been developed and utilized by researchers aimed at identifying the evolutionary links among strains and isolates. Comparative genomic hybridization (CGH) has been shown to be a useful tool for screening strains for their genetic content. However, there is a major limitation when CGH is conducted on a microarray based on a single reference genome. CGH results report which genes are present or absent relative to the genome. Hence the information about novel genetic content that the query strain possesses remains obscure. We report here the construction of the first Hi pan-genome microarray representing ca. 4600 features by 70-mers. In addition to those from the Rd strain, new features originate from the unfinished genome sequences present in NCBI database and from our novel gene discovery project efforts using strain HK1212. Genomes of 20strains belonging to different phylogenetic lineages were screened for their gene loss and gain utilizing the species microarray. The results obtained by employing the multistrain species microarray provide comprehensive information about the genomic content of uncharacterized strains. The trees generated by CGH, in general, do not reproduce the phylogeny of a species in terms of vertical evolution, but instead represents the overall relatedness of genomes to one another and provide an assessment about the species genome evolution. Keywords: Comparative Genomic Hybridization Twenty query strains (denoted HKxxxx) were investigated in this study, with each query strain hybridized against the reference strain, KW20. Two dye-swap experiments were performed, for a total of four hybridizations per query strain. Each 70mer oligo spotted on the H. influenzae species microarray is replicated six times. Positive controls on the array consist of oligos designed from the sequenced reference genome, KW20, and negative controls on the array consist of oligos designed from the thale cress plant, Arabidopsis thaliana.

Project description:The PFGRC has developed a cost effective alternative to complete genome sequencing in order to study the genetic differences between closely related species and/or strains. The comparative genomics approach combines Gene Discovery (GD) and Comparative Genomic Hybridization (CGH) techniques, resulting in the design and production of species microarrays that represent the diversity of a species beyond just the sequenced reference strain(s) used in the initial microarray design. These species arrays may then be used to interrogate hundreds of closely related strains in order to further unravel their evolutionary relationships. Clostridium botulinum produces botulinum neurotoxin (BoNT)and is classified as a “Category A” select agent. BoNT can be classified into seven serotypes designated A-G. There is considerable genetic variation within these serotypes, as demonstrated by the recognition of at least 47 subtypes. The most studied serotype, BoNT/A, has been found in a large and diverse group of clostridia, most of which express the subtype BoNT/A1. The BoNT/A1 producing C. botulinum strain ATCC 3502, used to obtain an initial annotated genome sequence, is not representative of the diverse clostridia group producing BoNT. Nearly 50% of C. botulinum strains producing BoNT/A1 have been shown to also encode unexpressed variants of BoNT/B with a distinct cluster arrangement. This nucleotide cluster is completely absent from the published genome sequence. In addition, a recently identified novel BoNT/A1 strain lacks the gene cluster seen in the genome sequence of ATCC 3502. Furthermore, a strain designated Hall A Hyper differs greatly from the sequenced strain as indicated by its ability to produce higher quantities of BoNT/A1. The genetic and phenotypic basis for this difference in BoNT expression is currently unknown, and the sequences of the BoNT gene and the cluster are identical in both strains. This observation supports the hypothesis that genes outside the toxin cluster are involved in the regulation and maturation of BoNT. The flow of genetic information within this group motivated us to identify novel genes for the purpose of creating a “species” DNA microarray to better understand the ancestral relationships among its members. Based on preliminary genotyping (MLST, and CGH using a single-genome-based array), 20 diverse C. botulinum strains were selected for sequencing. Sequence information obtained from this project, and from other publicly available sources, led to the development of a comprehensive species microarray for C. botulinum group members. The availability of the C. botulinum species DNA microarray has allowed us to carry out a collaborative CGH genotyping project to validate this microarray as well as understand the phylogenomic relationships among members of C. botulinum group.

Project description:The genus Cronobacter (formerly called Enterobacter sakazakii) is composed of five species; C. sakazakii, C. malonaticus, C. turicensis, C. muytjensii, and C. dublinensis. The genus includes opportunistic human pathogens, and the first three species have been associated with neonatal infections. The most severe diseases are caused in neonates and include fatal necrotizing enterocolitis and meningitis. The genetic basis of the diversity within the genus is unknown, and few virulence traits have been identified. We report here the first sequence of a member of this genus, C. sakazakii strain BAA-894. The genome of Cronobacter sakazakii strain BAA-894 comprises a 4.4 Mb chromosome (57% GC content) and two plasmids; 31 Kb (51% GC) and 131 Kb (56% GC). The genome was used to construct a 385,000 probe oligonucleotide tiling DNA microarray covering the whole genome. Comparative genomic hybridization (CGH) was undertaken on five other C. sakazakii strains, and representatives of the four other Cronobacter species. Among 4,382 annotated genes inspected in this study, about 55% of genes were common to all C. sakazakii strains and 43% were common to all Cronobacter strains, with 10 - 17% absence of genes. CGH highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units. Comparative genomic hybridization highlighted 15 clusters of genes in C. sakazakii BAA-894 that were divergent or absent in more than half of the tested strains; six of these are of probable prophage origin. Putative virulence factors were identified in these prophage and in other variable regions. A number of genes unique to Cronobacter species associated with neonatal infections (C. sakazakii, C. malonaticus and C. turicensis) were identified. These included a copper and silver resistance system known to be linked to invasion of the blood-brain barrier by neonatal meningitic strains of Escherichia coli. In addition, genes encoding for multidrug efflux pumps and adhesins were identified that were unique to C. sakazakii strains from outbreaks in neonatal intensive care units.

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Investigating H. Influenzae Species Diversity by Comparative Genomic Hybridization

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