The Bacteriophage Carrier State of Campylobacter jejuni Features Changes in Host Non-coding RNAs and the Acquisition of New Host-derived CRISPR Spacer Sequences

Abstract

Incorporation of self-derived CRISPR DNA protospacers in Campylobacter jejuni PT14 occurs in the presence of bacteriophages encoding a CRISPR-like Cas4 protein. This phenomenon was evident in carrier state infections where both bacteriophages and host are maintained for seemingly indefinite periods as stable populations following serial passage. Carrier state cultures of C. jejuni PT14 have greater aerotolerance in nutrient limited conditions, and may have arisen as an evolutionary response to selective pressures imposed during periods in the extra-intestinal environment. A consequence of this is that bacteriophage and host remain associated and able to survive transition periods where the chances of replicative success are greatly diminished. The majority of the bacteriophage population do not commit to lytic infection, and conversely the bacterial population tolerates low-level bacteriophage replication. We recently examined the effects of Campylobacter bacteriophage/C. jejuni PT14 CRISPR spacer acquisition using deep sequencing strategies of DNA and RNA-Seq to analyze carrier state cultures. This approach identified de novo spacer acquisition in C. jejuni PT14 associated with Class III Campylobacter phages CP8/CP30A but spacer acquisition was oriented toward the capture of host DNA. In the absence of bacteriophage predation the CRISPR spacers in uninfected C. jejuni PT14 cultures remain unchanged. A distinct preference was observed for incorporation of self-derived protospacers into the third spacer position of the C. jejuni PT14 CRISPR array, with the first and second spacers remaining fixed. RNA-Seq also revealed the variation in the synthesis of non-coding RNAs with the potential to bind bacteriophage genes and/or transcript sequences.

Keywords: CRISPR; RNA-Seq; bacteriophage; campylobacter; carrier state lifecycle; ncRNA.

Figure 1

Organization of the genomic loci of representative Type II CRISPR-Cas systems. The type II-C arrangement represents the Campylobacter jejuni carrier state with the addition of bacteriophage encoded cas4 in trans. The red and white sections represent, respectively, direct repeats and spacer sequences. The analogous genes in the Type II systems are labeled and color coded, where Type II-B is Legionella pneumophila and Type II-A Streptococcus thermophilus.

Figure 2

Phylogenetic analysis of the Cas4 protein family related to the proteins encoded in class II and III Campylobacter bacteriophages using a neighbor-joining tree methodology (ClustalW2 alignment and FigTree v1.4.2.).