Prevalence, genetic diversity, and host range of tectiviruses among members of the Bacillus cereus group

Abstract

GIL01, Bam35, GIL16, AP50, and Wip1 are tectiviruses preying on the Bacillus cereus group. Despite the significant contributions of phages in different biological processes, little is known about the dealings taking place between tectiviruses and their Gram-positive bacterial hosts. Therefore, this work focuses on characterizing the interactions between tectiviruses and the B. cereus group by assessing their occurrence and genetic diversity and evaluating their host range. To study the occurrence of tectiviruses in the B. cereus group, 2,000 isolates were evaluated using primers designed to be specific to two variable regions detected in previously described elements. PCR and propagation tests revealed that tectivirus-like elements occurred in less than 3% of the isolates. Regardless of this limited distribution, several novel tectiviruses were found, and partial DNA sequencing indicated that a greater diversity exists within the family Tectiviridae. Analyses of the selected variable regions, along with their host range, showed that tectiviruses in the B. cereus group can be clustered mainly into two different groups: the ones infecting B. anthracis and those isolated from other B. cereus group members. In order to address the host range of some novel tectiviruses, 120 strains were tested for sensitivity. The results showed that all the tested tectiviruses produced lysis in at least one B. cereus sensu lato strain. Moreover, no simple relationship between the infection patterns of the tectiviruses and their diversity was found.

FIG 1

Graphical representation of selected variable regions. (a) Genetic map of phage GIL01. Three gene modules based on functional grouping and similarities to other Tectiviridae in the B. cereus group are displayed. Predicted genes are represented as block arrows, and the color key at the bottom indicates postulated functions. CDS numbers are indicated. The ruler at the top represents base pairs in the GIL01 genome. ITR, inverted terminal repeat; HVR, highly variable region. (b) Whole-genome nucleotide alignments of Gram-positive bacterial tectivirus elements. For visualization, the ClustalW alignment file generated from the Multifasta alignment was summarized using Base-By-Base software (32, 33) after a model given previously (24). The GIL01 genome was used as the base sequence, and changes in other tectiviral genomes are color coded as follows: white, nucleotide identity; blue, single nucleotide polymorphism; green, insertions; and red, deletions. The ruler at the bottom represents base pairs in the whole-genome nucleotide sequence alignments. Selected variable regions (see the text), the RR and the PL regions, which mainly target the genes encoding DNA polymerase B (DNA PolB) and N-acetyl-muramidase (Mur1), respectively, are highlighted by gray dashed rectangles.

FIG 2

Evolutionary relationships displayed by the PL region. (a) Graphical representation of percent pairwise nucleotide sequence identity of the PL region. The color scale bar indicates the percentage of nucleotide sequence identity among the 51 tectiviral isolates. (b) Maximum likelihood tree showing the genetic relationships among the new tectiviral isolates. The general time-reversible plus gamma model of nucleotide substitution was used to build the phylogenetic tree based on the PL region. Bootstrap values (1,000 iterations) above 60% are indicated for each node. Fifty-one nucleotide sequences and 581 cured positions were used for the analysis. The blue box indicates the color key for Bacillus species harboring the respective phages.

FIG 3

Evolutionary relationships displayed by the RR region. (a) Graphical representation of percent pairwise nucleotide sequence identity of the RR region. The color scale bar indicates the percent nucleotide sequence identity among the 28 tectiviral isolates. (b) Maximum likelihood tree showing the genetic relationships among representatives of new tectiviral isolates. The general time-reversible plus gamma model of nucleotide substitution was used to build the phylogenetic tree based on the RR region. Bootstrap values (1,000 iterations) above 60% are indicated for each node. Twenty-eight nucleotide sequences and 1,087 cured positions were used for the analysis. The blue box indicates the color key for Bacillus species harboring the respective phages.

FIG 4

Scatter plot of principal coordinates analysis (PCoA) (axis PC 1 versus axis PC 2) on the basis of the tectivirus spotting host range. Twenty-three tectiviruses and 120 strains were used for host range determination. Squares, tectiviral isolates; gray squares, tectiviruses isolated from B. cereus emetic pathotype strains; white squares, tectiviruses harbored in a B. cereus strain recovered from honey samples; black squares, tectiviruses from different origins that did not cluster. PC, principal coordinates.