Recombination within and between species of the alpha proteobacterium Bartonella infecting rodents

Abstract

Bartonella infections from wild mice and voles (Apodemus flavicollis, Mi. oeconomus, Microtus arvalis and Myodes glareolus) were sampled from a forest and old-field habitats of eastern Poland; a complex network of Bartonella isolates, referrable to B. taylorii, B. grahamii, B. birtlesii and B. doshiae, was identified by the sequencing of a gltA fragment, comparable to previous studies of Bartonella diversity in rodents. Nested clade analysis showed that isolates could be assigned to zero- and one-step clades which correlated with host identity and were probably the result of clonal expansion; however, sequencing of other housekeeping genes (rpoB, ribC, ftsZ, groEl) and the 16S RNA gene revealed a more complex situation with clear evidence of numerous recombinant events in which one or both Bartonella parents could be identified. Recombination within gltA was found to have generated two distinct variant clades, one a hybrid between B. taylorii and B. doshiae, the other between B. taylorii and B. grahamii. These recombinant events characterised the differences between the two-step and higher clades within the total nested cladogram, involved all four species of Bartonella identified in this work and appear to have played a dominant role in the evolution of Bartonella diversity. It is clear, therefore, that housekeeping gene phylogenies are not robust indicators of Bartonella diversity, especially when only a single gene (gltA or 16S RNA) is used. Bartonella clades infecting Microtus were most frequently involved in recombination and were most frequently tip clades within the cladogram. The role of Microtus in influencing the frequency of Bartonella recombination remains unknown.

Figure 1

Phylogeny of a 292 bp fragment of citrate synthase (gltA) generated using minimum evolution algorithm (MEGA 4.1) with 1,000 bootstrap replicates. Species identities based on nearest match to type sequences from GenBank, clade identities based on second-step clades from the nested clade analysis. Two clades (Ur27 and Ur34) could not be assigned to a species with confidence

Figure 2

Cladogram of the Bartonella isolates collected in this work, and relevant haplotypes from Welc-Falęciak et al. [44] based on a 292 bp fragment of citrate synthase showing known (perfect match) recombinant events within the cladogram. “Ur01-Ur37” represents unique variants recovered from Bartonella isolates corresponding to zero-step clades in the terminology of Templeton et al. [41]. Boxes represent one-step clades. ‘Missing’ haplotypes (i.e. predicted but not collected) are indicated by 0. Larger clades corresponding to ‘species’ and other higher clades surrounded by heavy boxes. Distances between clades of more than five base changes not marked because of likelihood that these will be recombinant events. Clades sharing groEl and 16S RNA haplotypes between ‘species’ are marked

Figure 3

Examples of intra-gene recombination events into the gltA gene with polymorphic sites and break points marked on. a Ur35 (B. doshiae) as a recombinant of Ur26 (B. taylorii clade B) and B. doshiae type sequence (Z70017). b Ur27 (species not defined) as a recombinant of Ur20 (B. taylorii clade B) and Ur31 (B. grahamii)

Figure 4

Comparison of phylogenies obtained with the metabolic genes analysed in this work: gltA, ftsZ, ribC, rpoB, groEl and gene coding 16S RNA. Phylogenies generated using maximum likelihood (PhyML) with 100 bootstrap replicates. Species identities boxed, based on nearest match to type sequences from GenBank. Note the lack of support for B. taylorii with groEl