Microviridae goes temperate: microvirus-related proviruses reside in the genomes of Bacteroidetes

Abstract

The Microviridae comprises icosahedral lytic viruses with circular single-stranded DNA genomes. The family is divided into two distinct groups based on genome characteristics and virion structure. Viruses infecting enterobacteria belong to the genus Microvirus, whereas those infecting obligate parasitic bacteria, such as Chlamydia, Spiroplasma and Bdellovibrio, are classified into a subfamily, the Gokushovirinae. Recent metagenomic studies suggest that members of the Microviridae might also play an important role in marine environments. In this study we present the identification and characterization of Microviridae-related prophages integrated in the genomes of species of the Bacteroidetes, a phylum not previously known to be associated with microviruses. Searches against metagenomic databases revealed the presence of highly similar sequences in the human gut. This is the first report indicating that viruses of the Microviridae lysogenize their hosts. Absence of associated integrase-coding genes and apparent recombination with dif-like sequences suggests that Bacteroidetes-associated microviruses are likely to rely on the cellular chromosome dimer resolution machinery. Phylogenetic analysis of the putative major capsid proteins places the identified proviruses into a group separate from the previously characterized microviruses and gokushoviruses, suggesting that the genetic diversity and host range of bacteriophages in the family Microviridae is wider than currently appreciated.

Figure 1. Bacteroidetes-associated, microvirus-related proviruses BMV1–7.

A. Genomic organization of the putative BMV proviruses residing in the genomes of different species of the phylum Bacteroidetes and two gokushoviruses (family Microviridae), Chlamydia phage 1 (Chp1) and Bdellovibrio-infecting virus φMH2K. Circular genome maps of Chp1 (GenBank accession number: D00624) and φMH2K (GenBank accession number: AF306496) are linearized for convenient alignment. Open reading frames (ORF; arrows) are labeled according to the gokushovirus and microvirus protein nomenclature. ORFs encoding homologous products are coloured similarly. attL and attR, left and right attachment sites, respectively. B. Comparison of the BMV1-containing genomic region of Bacteroides sp. 2_2_4 with the provirus-free genomic region of B. ovatus ATCC 8483 (GenBank accession number: NZ_AAXF02000049; nucleotide coordinates: 301224–301252). The putative attachment sites flanking BMV1 as direct repeats are highlighted in black background.

Figure 2. Comparison of the predicted attachment sites of BMVs with the bacterial dif consensus sequence.

Left arm (XerC binding site), spacer and right arm (XerD binding site) regions of the dif sites are indicated. Bacterial dif consensus sequence is indicated according to the IUPAC code. Nucleotide positions in the att sites of BMVs matching the dif consensus are shown in bold. Identical nucleotide positions in the left (L) and right (R) att sites, flanking the proviruses as imperfect direct repeats (see Figure 1), are shaded gray. BMV att consensus sequence is shown as sequence logo at the bottom of the figure.

Figure 3. BMV-related sequences in environmental databases.

Blastn (nucleotide query against nucleotide database) hits to the human gut metagenome and tblastn (protein query against translated nucleotide database) hits to marine GOS metagenome are depicted below and above the BMV2 genome map, respectively. Hit coverage and respective sequence identities (retrieved contigs are indicated by their GenBank identifiers) are also shown.

Figure 4. Analysis of the putative major capsid protein of BMV1.

A. Alignment of the BMV1 major capsid protein sequence to the corresponding protein sequences of φX174, SpV4 and φMH2K. The proteins are denoted by their GenBank identifier followed by the corresponding (pro)virus name. The alignment is coloured according to sequence conservation (BLOSUM62 matrix). The secondary structure determined from the X-ray structure of φX174 capsid protein F (PDB ID: 1CD3) is shown above the alignment with α helices, β strands, and turns represented by red rectangles, blue arrows, and yellow bulges, respectively. Insertions (>5 aa) relative to the capsid protein F of φX174 are boxed. B. Atomic structure of the major capsid protein F of microvirus φX174 (PDB ID: 1CD3). Magenta spheres highlight the equivalent positions of the putative capsid protein of BMV1, where insertions (larger than 5 aa; numbered 1–7) occur relative to the capsid protein F of φX174 (refer to panel A for the alignment). Size of each insertion is indicated on the right of the figure.

Figure 5. Unrooted phylogenetic tree of the φX174 F-like major capsid proteins.

The evolutionary history was inferred by using the Maximum Likelihood method based on the Whelan and Goldman model . The bootstrap consensus tree was inferred from 1000 replicates. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The scale bar represents the number of substitutions per site. The analysis involved 35 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 356 positions in the final dataset. φX174-like virus group includes φX174 (GI:9626381), WA10 (GI:71843040), S13 (GI:11095662), NC11 (GI:71842956), ID1 (GI:71842872). α3-like virus group includes α3 (GI:9625363), φK (GI:2493329), st-1 (GI:242346750). G4-like virus group includes G4 (GI:9626346), WA6 (GI:71843160), ID12 (GI:71843172). CPAR39-like virus group includes. Chp2-like virus group includes Chp2 (GI:9634949), Chp3 (GI:47566141), Chp4 (GI:77020115), CPAR39 (GI:9791178), φCPG1 (GI:17402851). SARssφ1 (GI:313766927) and SARssφ2 (GI:313766923) are uncultured microviruses the genomes of which were assembled by Tucker et al . GOS sequences are microvirus-like major capsid proteins obtained during the Sorcerer II Global Ocean Sampling (GOS) Expedition : GOS_10590 (GI:142008996), GOS_10391 (GI:142009231), GOS_11182 (GI:142008205), GOS_11146 (GI:142008257), GOS_10803 (GI:142008696).