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. 2009 Jul;5(7):e1000546.
doi: 10.1371/journal.pgen.1000546. Epub 2009 Jul 3.

Run-off Replication of Host-Adaptability Genes Is Associated With Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella Grahamii

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Free PMC article

Run-off Replication of Host-Adaptability Genes Is Associated With Gene Transfer Agents in the Genome of Mouse-Infecting Bartonella Grahamii

Eva C Berglund et al. PLoS Genet. .
Free PMC article

Abstract

The genus Bartonella comprises facultative intracellular bacteria adapted to mammals, including previously recognized and emerging human pathogens. We report the 2,341,328 bp genome sequence of Bartonella grahamii, one of the most prevalent Bartonella species in wild rodents. Comparative genomics revealed that rodent-associated Bartonella species have higher copy numbers of genes for putative host-adaptability factors than the related human-specific pathogens. Many of these gene clusters are located in a highly dynamic region of 461 kb. Using hybridization to a microarray designed for the B. grahamii genome, we observed a massive, putatively phage-derived run-off replication of this region. We also identified a novel gene transfer agent, which packages the bacterial genome, with an over-representation of the amplified DNA, in 14 kb pieces. This is the first observation associating the products of run-off replication with a gene transfer agent. Because of the high concentration of gene clusters for host-adaptation proteins in the amplified region, and since the genes encoding the gene transfer agent and the phage origin are well conserved in Bartonella, we hypothesize that these systems are driven by selection. We propose that the coupling of run-off replication with gene transfer agents promotes diversification and rapid spread of host-adaptability factors, facilitating host shifts in Bartonella.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Comparison of the structures of the Bartonella genomes.
A schematic illustration of the phylogenetic relationship of the five sequenced Bartonella species is shown to the left of a linear representation of their genomes. The total size of each genome is shown within parenthesis. Genes are color-coded based on phylogenetic classifications and annotation. Grey lines between genes indicate orthology. Bt: B. tribocorum, Bg: B. grahamii, Bh: B. henselae, Bq: B. quintana, Bb: B. bacilliformis.
Figure 2
Figure 2. Gene classifications in the Bartonella genomes.
Diagram showing the number of genes that are predicted as vertically inherited (from closely related alpha-proteobacterial species), imported (horizontally transferred to the ancestor of Bartonella or more recently), Bartonella-specific (only present in Bartonella), and orphans (only present in one species) in the sequenced Bartonella genomes.
Figure 3
Figure 3. Sequence similarities to genes located on auxiliary replicons in other rhizobiales species.
The innermost circle represents the genome of B. grahamii, with prophages in yellow, genomic islands in magenta and the chromosomal high plasticity zone in black. Numbers within the circle represent the genomic islands BgGI 1–16. The remaining circles show on which replicon the top Blast hit of each B. grahamii gene is located in each of Brucella suis (Bs; inner circle), Ochrobactrum anthropi (Oa; middle circle), and Agrobacterium tumefaciens (At; outer circle). The genome positions are shown on the outside, with 100 kb between each line.
Figure 4
Figure 4. Comparison of the chromosomal high plasticity zone in the Bartonella genomes.
Comparative gene map of the chromosomal high plasticity zone in the five sequenced Bartonella genomes. The locations of T4SS (virB/vbh) and T5SS (autotransporters, fha/hec) are shown. Species abbreviations are as in the legend to Figure 1. The total size of the region in each species is shown within parentheses. Due to the rearrangement around the replication origin in B. bacilliformis, the chromosomal high plasticity zone is divided into two parts, located on different sides of the origin.
Figure 5
Figure 5. Phylogenetic analysis of genes for type V secretion systems.
Phylogenetic trees of (A) autotransporters and (B) filamentous hemagglutinin. Bartonella genes are named with locus_tag (BARBAKC583 abbreviated as BB) and color-coded according to species: B. grahamii in red, B. tribocorum in blue, B. henselae in green, B. quintana in purple and B. bacilliformis in brown. After each B. grahamii gene, the genomic island in which the gene is located is indicated with numbers in circles. Branch lengths are according to maximum likelihood analysis and numbers represent bootstrap support values.
Figure 6
Figure 6. Phage clusters in B. grahamii.
(A) Circular overview of the B. grahamii as4aup genome. The locations of phage clusters (yellow), genomic islands (magenta), the chromosomal high plasticity zone (black) and the gene with similarity to S. thermophilus phage Sfi18 (blue) are shown. Numbers outside the circle represent the phage clusters I-IV. Numbers within the circle represent the genomic islands BgGI 1–16. Detailed comparative gene maps are shown for (B) prophage Ia, (C) phage cluster II, and (D) phage cluster III. The color legend shown in (D) also applies to (B) and (C). Genes identified in mass spectrometry are marked with black dots. The putative origin of replication identified in the microarray analysis is located close to the helicase in (D).
Figure 7
Figure 7. Bacteriophage particles from B. grahamii.
Transmission electron microscopy of bacteriophage particles isolated from B. grahamii strain af165up. Round to icosahedral heads of 50–70 nm were observed both with and without tail, whereas there were few loose tails. The white bar is 100 nm.
Figure 8
Figure 8. Bacteriophage DNA from B. grahamii.
Agarose gel electrophoresis of B. grahamii bacteriophage DNA. Lane 1, Marker (Low Range PFG Marker, New England Biolabs); lane 2, phage DNA from B. grahamii af165up; lane 3, phage DNA from B. grahamii as4aup; lane 4, cellular DNA from B. grahamii af165up; lane 5, cellular DNA from B. grahamii as4aup; lane 6, marker. The sizes of the relevant bands of the marker are shown to the left.
Figure 9
Figure 9. DNA content of B. grahamii bacteriophage particles.
Results from microarray hybridizations of phage DNA versus cellular DNA of the same strain. The x-axis represents the genome of B. grahamii as4aup and the y-axis the hybridization signal (log2-ratio of phage DNA and cellular DNA). To exclude possible misinterpretation of repeated probes, probes with more than one exact match in the genome were not plotted unless located in the prophage regions. (A) Total phage DNA from B. grahamii as4aup. (B) Total phage DNA from B. grahamii af165up. Phage DNA extracted from (C) the 45 kb band and (D) the 14 kb band in agarose electrophoreses from B. grahamii af165up. Above the x-axis in all graphs is a representation of the genome of B. grahamii as4aup with the same color-coding as in Figure 1. In particular, phage genes are yellow. Grey dots in (C) and (D) show the result of the hybridization of total phage DNA from B. grahamii af165up (Figure 9B).
Figure 10
Figure 10. Growth curve of B. grahamii af165up.
Growth curve of B. grahamii strain af165up in supplemented Schneider's medium. Bacterial growth was determined by measuring the OD600 in triplicates (grey line) and by quantifying the number of viable bacteria, expressed as CFU/ml (black line), at 24-h intervals. Samples were collected at the mid-logarithmic growth phase (day 2), the stationary phase (day 5), and at the end of the death phase (day 11).
Figure 11
Figure 11. DNA content of B. grahamii bacteriophage particles isolated at different growth phases.
Results from microarray hybridizations of DNA extracted from phage particles in (A) exponential phase (B) stationary phase and (C) end of death phase. To exclude possible misinterpretation of repeated probes, probes with more than one exact match in the genome were not plotted unless located in the prophage regions.

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