Current address: School of Natural Sciences, University of California, Merced, California, United States of America
Current address: CNRS UMR8030, Génomique Métabolique, and CEA - Institut de Génomique - Genoscope, Laboratoire de Génomique Comparative, Evry, France
Current address: Department of Microbiology, The Swedish Agricultural University, Uppsala, Sweden
Conceived and designed the experiments: SGEA. Performed the experiments: ECB OVP FG ASE KN. Analyzed the data: ECB ACF AC SGEA. Contributed reagents/materials/analysis tools: MH HL. Wrote the paper: ECB ACF SGEA.
The authors have declared that no competing interests exist.
The genus
Emerging infectious diseases represent an increasing human health problem with many examples of disease outbreaks caused by transmissions from animals to humans, such as, most recently, the bird flu virus. Genes involved in virulence and antibiotic resistance are often carried by mobile elements like plasmids and viruses, which mediate transfer between cells at an amazing speed. Rodents represent a major carrier of infectious agents, and it is therefore particularly important to study the gene transfer processes in bacteria that use rodents as their natural host reservoir. We have studied the genome of a bacterium that is naturally adapted to mice and identified many more putative host-interaction genes than were observed in previously recognized human pathogens. Furthermore, most of these genes are located in a segment of about 25% of the genome, which was massively amplified and packaged into viral particles. This is the first demonstration of targeted packaging of a portion of the bacterial chromosome into viral particles, and we propose that this is a novel strategy for increased exchange of genes involved in the infectious process.
Horizontal gene transfer contributes to phenomena such as pathogen emergence and antibiotics resistance, with major implications for human health. However, little is known about the mechanisms and ecological factors that influence the intensity of horizontal gene transfer in bacteria adapted to wild host populations. With about 170 cases of emerging infectious diseases during the last two decades, more than half of which are caused by bacterial infections
A recent genomic survey identified many putative host-adaptability genes essential for bloodstream infection of
Several of the genes for secretion systems are located in a dynamic region of the genome, up to several hundred kb in size, which is thought to have originated by the integration of an auxiliary replicon
Bacteriophage particles have been identified in several species of
To learn more about the mechanisms and selective forces driving run-off replication, we determined the genome sequence of
We demonstrate that the highly dynamic region of the chromosome, that contains many gene clusters for secretion systems, is extensively amplified and packaged into bacteriophage particles. This is the first report that associates run-off replication with bacteriophage particles. We propose that the combination of these two systems promotes diversification and rapid spread of selectively favored host-adaptability genes within and among
A schematic illustration of the phylogenetic relationship of the five sequenced
Genome size chromosome (bp) | 2341328 |
pBGR3 size (bp) | 28192 |
Protein-coding genes (chromosome) | 1737 |
Protein-coding genes (pBGR3) | 31 |
Pseudogenes (chromosome) | 236 |
tRNA | 42 |
rRNA | 6 |
tmRNA | 1 |
Orthologs |
1245 |
Orthologs |
1237 |
Orthologs |
1135 |
Orthologs |
1042 |
Orthologs |
955 |
The number of
The number of
The
Name | First gene |
Last gene |
Size | Genes |
Content |
BgGI 1 | Bgr_01430 | Bgr_01610 | 61 kb | 10 | |
BgGI 2 | Bgr_03020 | Bgr_03830 | 60 kb | 73 | |
BgGI 3 | Bgr_03980 | Bgr_04180 | 23 kb | 14 | Membrane proteins |
BgGI 4 | Bgr_07490 | Bgr_09310 | 166 kb | 163 | |
BgGI 5 | Bgr_09700 | Bgr_10280 | 53 kb | 55 | Phage related |
BgGI 6 | Bgr_12450 | Bgr_12590 | 20 kb | 7 | Membrane proteins |
BgGI 7 | Bgr_12750 | Bgr_12950 | 24 kb | 13 | |
BgGI 8 | Bgr_13490 | Bgr_13590 | 11 kb | 5 | Unknown |
BgGI 9 | Bgr_14480 | Bgr_14750 | 40 kb | 20 | Autotransporters, phage |
BgGI 10 | Bgr_15090 | Bgr_15450 | 43 kb | 25 | |
BgGI 11 | Bgr_15690 | Bgr_15890 | 34 kb | 16 | |
BgGI 12 | Bgr_16030 | Bgr_16480 | 70 kb | 36 | Autotransporters, phage |
BgGI 13 | Bgr_16650 | Bgr_16880 | 32 kb | 16 | |
BgGI 14 | Bgr_16900 | Bgr_17120 | 37 kb | 16 | |
BgGI 15 | Bgr_17370 | Bgr_17750 | 41 kb | 36 | |
BgGI 16 | Bgr_17950 | Bgr_18150 | 18 kb | 17 | Vbh T4SS |
Prophage Ia | Bgr_03240 | Bgr_03830 | 44 kb | 56 | Prophage |
Prophage Ib | Bgr_08620 | Bgr_09310 | 47 kb | 67 | Prophage |
The locus_tag of the first and the last gene in each genomic island.
The number of predicted protein-coding genes in each genomic island.
A prediction of the total amount of horizontally transferred genes by phylogenetic analysis (excluding plasmid genes and hypothetical genes solely identified in
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
A functional dissection of the 457 imported genes in
Function | Number of genes |
Phage-related | 165 |
T4SS | 58 |
T5SS | 35 |
Membrane proteins | 30 |
Transport proteins | 41 |
Hypothetical proteins | 62 |
Other function | 66 |
Total | 457 |
Our analysis suggests dramatic changes in the mobile gene pool in the rodent- versus the human-associated
The innermost circle represents the genome of
A closer inspection of the high plasticity zone revealed two conserved segments, here called
Comparative gene map of the chromosomal high plasticity zone in the five sequenced
Located between or near the conserved segments
Secretion system | Gene family | Bg | Bt | Bh | Bq | Bb |
T4SS | 18 | 18 | 18 | 15 | 0 | |
22 | 10 | 0 | 0 | 0 | ||
29 | 30 | 23 | 23 | 0 | ||
Total | 69 | 58 | 41 | 38 | 0 | |
T5SS | autotransporters | 17 | 17 | 10 | 6 | 5 |
5 | 1 | 2 | 3 | 3 | ||
13 | 8 | 7 | 0 | 0 | ||
Total | 35 | 26 | 19 | 9 | 8 | |
All | 104 | 84 | 60 | 47 | 8 |
Only full-length genes are included.
The autotransporters are ubiquitously present in all five
Phylogenetic trees of (A) autotransporters and (B) filamentous hemagglutinin.
The
Of the genes for secretion systems, the
Since phage related genes contribute much of the variability in
(A) Circular overview of the
Several shorter clusters of phage genes may represent remnants of other integrated phages. Two such clusters, here called
We observed bacteriophage particles consisting of a round to icosahedral head of 50–70 nm in diameter and a sheathed tail of approximately 100 nm by transmission electron microscopy of bacteriophage preparations from
Transmission electron microscopy of bacteriophage particles isolated from
We examined the protein profiles of the phage particles from all three strains using SDS-PAGE and mass spectrometry (
Additionally, hemin binding protein A (HbpA), also known as Pap31, was present in bacteriophage preparations from
We extracted bacteriophage DNA from all three strains, and agarose gel electrophoresis revealed two bands of circa 14 and 45 kb in
Agarose gel electrophoresis of
To examine the gene content of the bacteriophage DNA, we constructed a microarray from 4,438 oligomers of 60 bp in size, which covers 1703 protein-coding genes (96% of the predicted protein-coding genes in
Results from microarray hybridizations of phage DNA versus cellular DNA of the same strain. The x-axis represents the genome of
To specifically assess the content of the 14 kb and the 45 kb bands in strain af165up, we extracted DNA from each band, and hybridized against cellular DNA. The results showed that the hybridization signal from the 45 kb band was concentrated to
To examine whether induction of the prophage and run-off replication occur predominantly in dying cells, or possibly is the cause of bacterial death, we collected phage particles from three different time points from a liquid culture of
Growth curve of
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.
In this study, we have resolved the long-standing debate about the content of the
The identification of a GTA encoded by
Despite the absence of sequence similarity between these two GTAs in the alpha-proteobacteria, their overall organization is similar, including genes coding for terminase, portal protein, and putative capsid and tail proteins. We identified gene clusters homologous to the
Could run-off replication be initiated from a plasmid replication initiation site inherited from a once self-replicating megaplasmid? Such a phenomenon is believed to have occurred in the archaea, where multiple replication initiation sites have been demonstrated
It is more likely that replication starts from an origin derived from an inactivated prophage, as does escape replication in
An important question is whether run-off replication and GTA-production are two independent phenomena, resulting from an error in the phage replication machinery and a degrading prophage, or whether the systems driving these processes co-evolve under selection and control of the bacterial genome. Several lines of evidence argue in favor of selection, particularly the conservation of
What could be the selective advantage of linking run-off replication with a gene transfer agent? One advantage that we can think of is gene diversification by recombination and rapid spread of new gene variants. Because many genes for secretion systems are located in the amplified region, host adaptation and host switches could select for linkage of the two systems. Indeed, rapid diversification and horizontal transfer of host-adaptability genes within
If run-off replication and production of GTAs are driven by selection, what are the cellular mechanisms and conditions responsible for regulating these processes? Our observation that the
An exciting avenue for future work is to investigate how expression of the
Starting from single colonies,
A total of 31,166 shotgun sequences were obtained from the M13 library of
Bacteria were grown on hematin agar plates for 10 days. Prior to DNA extraction, the cells were suspended in TNE buffer (10 mM TRIS pH 8.0, 150 mM NaCl and 1 mM EDTA) and centrifuged; washes were repeated twice. DNA was isolated in agarose plugs, made using 2% SeaPlaque GTG agarose (Cambrex Bio Science, ME, USA) in 0.5× TBE buffer as described in
Curation and annotation of the genome were done with the annotation platform GenDB
Orthologs between
Genes were defined as orphans if there was no convincing Blast hit in the GenBank nr database. Genes were defined as
Genomic islands were manually located in
Protein sequences for FHA and autotransporters were aligned with Kalign
Bacterial cells were either harvested after 5 to 15 days of culture on hematin agar plates, or at selected time points during growth in liquid culture. While samples from liquid culture were collected directly, plate-grown bacteria were either overlaid with 5 ml Brain-Heart Infusion Broth (Difco) the day prior to collection or re-suspended in SM buffer. After centrifugation at ca. 4000×g for 15 min at 4°C, supernatants were collected, filtered through 0.2 µm filters and enriched using polyethylene glycol as described in
Extraction of DNA from the bacterial pellets was performed using the AquaPure Genomic DNA Kit (Bio-Rad) according to the manufacturer's instructions. Phage DNA from plate cultures was extracted as described in
Size selection of phage DNA fragments from non-amplified preparations was carried out by separation in 0.7% (w/v) low melting point agarose (SeaKem Plaque, Cambrex) gels containing ethidium bromide, in 1× TAE buffer at 4°C at 6 v/cm overnight. The fragments were visualized under near-UV light (λ = 360 nm) and excised with a sterile scalpel blade. Two volumes of ß-agarase I buffer (New England Biolabs) amended with 100 mM NaCl, 30 µM spermine and 70 µM spermidine
The protein components of the bacteriophage particles were separated by SDS-PAGE (12%) after being heat-denaturized at 95°C for 5 minutes. Mass spectrometry was performed for each band. Proteins were identified based on computational searches in the NCBI non-redundant (nr) database and a local database consisting of the
We designed 4438 60-mer oligonucleotides, representing 1703 protein-coding genes, 110 pseudogenes and 663 spacers with OligoArray2.1
Scanning and image analysis were performed with a GenePix 4100A scanner and the GenePix 5.1 software (Axon Instruments, Molecular Devices) as described previously
The genome sequence of
Comparative gene map of the genomic region containing BgGI 1.
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Electron micrographs of bacteriophage particles isolated from cultivations with
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PFGE migration of undigested DNA from bacteriophage particles isolated from
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Proteins identified in mass spectrometry of bacteriophage preparations from
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Rhizobiales species with homologs to the genes in
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We thank Kirsten Ellegaard at the Department of Molecular Evolution, Uppsala University, for aid with whole-genome amplification of the extracted phage DNA; Leif Ljung at the Department of Medical Cell Biology, Uppsala University, for assistance with electron microscopy; and Åke Engström at the Department of Medical Biochemistry and Microbiology, Uppsala University, for assistance with mass spectrometry. We thank Annelie Waldén and Max Käller at the Royal Institute of Technology, Stockholm, Sweden, for microarray printing.