DNA sequence heterogeneity of Campylobacter jejuni CJIE4 prophages and expression of prophage genes

Abstract

Campylobacter jejuni carry temperate bacteriophages that can affect the biology or virulence of the host bacterium. Known effects include genomic rearrangements and resistance to DNA transformation. C. jejuni prophage CJIE1 shows sequence variability and variability in the content of morons. Homologs of the CJIE1 prophage enhance both adherence and invasion to cells in culture and increase the expression of a specific subset of bacterial genes. Other C. jejuni temperate phages have so far not been well characterized. In this study we describe investigations into the DNA sequence variability and protein expression in a second prophage, CJIE4. CJIE4 sequences were obtained de novo from DNA sequencing of five C. jejuni isolates, as well as from whole genome sequences submitted to GenBank by other research groups. These CJIE4 DNA sequences were heterogenous, with several different insertions/deletions (indels) in different parts of the prophage genome. Two variants of a 3-4 kb region inserted within CJIE4 had different gene content that distinguished two major conserved CJIE4 prophage families. Additional indels were detected throughout the prophage. Detection of proteins in the five isolates characterized in our laboratory in isobaric Tags for Relative and Absolute Quantitation (iTRAQ) experiments indicated that prophage proteins within each of the two large indel variants were expressed during growth of the bacteria on Mueller Hinton agar plates. These proteins included the extracellular DNase associated with resistance to DNA transformation and prophage repressor proteins. Other proteins associated with known or suspected roles in prophage biology were also expressed from CJIE4, including capsid protein, the phage integrase, and MazF, a type II toxin-antitoxin system protein. Together with the results previously obtained for the CJIE1 prophage these results demonstrate that sequence variability and expression of moron genes are both general properties of temperate bacteriophages in C. jejuni.

Figure 1. Schematic diagram demonstrating differences in Group 1 CJIE4 prophage DNA sequences.

Strains carried the large indel containing genes encoding CJE1439-Cj1442, plus strain 414. The diagram includes prophages investigated in this work (see Materials and Methods) plus those obtained from NCBI databases. Sizes of genes are approximate, and any non-coding sequence between genes has not been shown to conserve space.

Figure 2. Schematic diagram demonstrating differences in Group 2 CJIE4 prophage DNA sequences.

Strains carried the alternate large indel containing ORFs 6-10. The diagram includes prophages investigated in this work (see Materials and Methods) plus those obtained from NCBI databases. The question mark in the 2548 indicates that the fact that no sequence was found for this region may indicate either that there was a problem with the search parameters used to detect contigs encoding proteins homologous to those in other CJIE4 variants or that there was a horizontal gene transfer replacing the region with a different gene repertoire. The colors used to identify putative protein functions were the same as in Fig. 1. Sizes of genes are approximate, and any non-coding sequence between genes has not been shown to conserve space.