DNA/DNA hybridization to microarrays reveals gene-specific differences between closely related microbial genomes

Abstract

DNA microarrays constructed with full length ORFs from Shewanella oneidensis, MR-1, were hybridized with genomic DNA from nine other Shewanella species and Escherichia coli K-12. This approach enabled visualization of relationships between organisms by comparing individual ORF hybridizations to 164 genes and is further amenable to high-density high-throughput analyses of complete microbial genomes. Conserved genes (arcA and ATP synthase) were identified among all species investigated. The mtr operon, which is involved in iron reduction, was poorly conserved among other known metal-reducing Shewanella species. Results were most informative for closely related organisms with small subunit rRNA sequence similarities greater than 93% and gyrB sequence similarities greater than 80%. At this level of relatedness, the similarity between hybridization profiles was strongly correlated with sequence divergence in the gyrB gene. Results revealed that two strains of S. oneidensis (MR-1 and DLM7) were nearly identical, with only 3% of the ORFs hybridizing poorly, in contrast to hybridizations with Shewanella putrefaciens, formerly considered to be the same species as MR-1, in which 63% of the ORFs hybridized poorly (log ratios below -0.75). Genomic hybridizations showed that genes in operons had consistent levels of hybridization across an operon in comparison to a randomly sampled data set, suggesting that similar applications will be informative for identification of horizontally acquired genes. The full value of microbial genomic hybridizations lies in providing the ability to understand and display specific differences between closely related organisms providing a window into understanding microheterogeneity, bacterial speciation, and taxonomic relationships.

Figure 1

Hierarchical cluster analysis of Shewanella DNA/DNA microarray hybridizations. Columns represent the mean log ratios for dual-labeled DNA/DNA hybridizations for: (1) E. coli, (2) S. woodyi, (3) env. isolate PV-4, (4) S. oneidensis, MR-4, (5) S. putrefaciens (strain 95), (6) env. isolate W3 18–1, (7) Shewanella sp. (strain 184), (8) Shewanella baltica (strain 63), (9) Shewanella sp. (strain CL256/73), (10) S. oneidensis (strain DLM7), and (11) S. oneidensis (strain MR-1). Black represents log ratios of 0, and bright green represents log ratios approaching −1.5. (A) Conserved cluster of genes. Hybridizations in columns 4–11 are all strains in the halotolerant branch of the Shewanella genus. (B) Variant cluster of genes. Note that S. oneidensis strains DLM7 and MR-1 are very similar, even in the most variable cluster of genes surveyed.

Figure 2

Phylogenetic tree showing strains of Shewanella based on (A) SSU rRNA and (B) gyrB nucleotide sequence. Relationships calculated by using maximum-likelihood analysis. Species in bold were used in this study. Numbers at nodes represent bootstrap values (of 100 replicates).

Figure 3

Relationship between percent similarity in nucleotide sequences for the SSU rRNA (circles) and gyrB (triangles) genes (calculated as percent similarity of test strain relative to the S. oneidensis, MR-1 strain) and percent similarity (31) determined for microarray hybridization profiles between test strains and S. oneidensis, MR-1. Note that the data were not included for E. coli and for env. isolate PV-4, the two most distantly related organisms tested in this study, as the relationship falls off at greater distances.