Analysis of ten Brucella genomes reveals evidence for horizontal gene transfer despite a preferred intracellular lifestyle

Abstract

The facultative intracellular bacterial pathogen Brucella infects a wide range of warm-blooded land and marine vertebrates and causes brucellosis. Currently, there are nine recognized Brucella species based on host preferences and phenotypic differences. The availability of 10 different genomes consisting of two chromosomes and representing six of the species allowed for a detailed comparison among themselves and relatives in the order Rhizobiales. Phylogenomic analysis of ortholog families shows limited divergence but distinct radiations, producing four clades as follows: Brucella abortus-Brucella melitensis, Brucella suis-Brucella canis, Brucella ovis, and Brucella ceti. In addition, Brucella phylogeny does not appear to reflect the phylogeny of Brucella species' preferred hosts. About 4.6% of protein-coding genes seem to be pseudogenes, which is a relatively large fraction. Only B. suis 1330 appears to have an intact beta-ketoadipate pathway, responsible for utilization of plant-derived compounds. In contrast, this pathway in the other species is highly pseudogenized and consistent with the "domino theory" of gene death. There are distinct shared anomalous regions (SARs) found in both chromosomes as the result of horizontal gene transfer unique to Brucella and not shared with its closest relative Ochrobactrum, a soil bacterium, suggesting their acquisition occurred in spite of a predominantly intracellular lifestyle. In particular, SAR 2-5 appears to have been acquired by Brucella after it became intracellular. The SARs contain many genes, including those involved in O-polysaccharide synthesis and type IV secretion, which if mutated or absent significantly affect the ability of Brucella to survive intracellularly in the infected host.

FIG. 1.

Mauve alignment of both chromosomes from the nine complete Brucella genomes. A phylogenetic map of the strains derived from the tree shown in Fig. 2 (topology only, not branch lengths) is on the left side (abo, B. abortus; mel, B. melitensis; ovis, B. ovis; canis, B. canis; suis, B. suis). SARs of interest are noted by filled boxes, with the names of those regions directly below them.

FIG. 2.

Phylogenetic trees of 10 Brucella genomes with outgroups (A) and without outgroups (B). The maximum likelihood tree is based on a concatenated alignment of 2,246 protein families. (A) Full tree with outgroup species. (B) Brucella portion, only at a smaller scale. All nodes received 100% bootstrap support except the two very short ones indicated in panel B.

FIG. 3.

AH analysis of B. suis 1330 chromosomes. The 17 shared SARs are annotated in this plot. Genes that were shared among the majority of the OGs are depicted on these peaks, with open circles indicating genes that are present in Brucella and among other members of Rhizobiales. Filled circles indicate genes that are found only among Brucella and share no homology outside this genus. Filled diamonds represent genes that are present in Brucella, absent among other Rhizobiales, but have significant BLASTP hits to genomes that are not members of the Rhizobiales order. The x axis represents the length of the chromosome, and the y axis represents the score range provided by the AH program.

FIG. 4.

Pseudogenization of the β-ketoadipate pathway among the 10 different Brucella genomes and in Ochrobactrum anthropi in illustrated form (A) and tabular form (B). (A) Genes involved in this pathway are in two operons on opposite strands, and the individual genes are represented by arrows. Gene symbols, where available, are provided below the arrows. Hatched arrows indicate genes that are pseudogenized in one or more genomes. (B) Table showing the identification, name, gene symbol, and presence, absence, or pseudogenization of individual genes among the genomes. Checkmarks indicate a normal gene. Open circles denote the absence of this gene, and ψ indicates an apparent pseudogene.