Journal of Molecular Biology
Volume 395, Issue 2, 15 January 2010, Pages 270-281
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Transcription Profile of Thermus thermophilus CRISPR Systems after Phage Infection

https://doi.org/10.1016/j.jmb.2009.10.057Get rights and content

Abstract

The clustered regularly interspaced short palindromic repeat (CRISPR) systems composed of DNA direct repeats designated as CRISPRs and several CRISPR-associated (cas) genes, which are present in many prokaryotic genomes, make up a host defense system against invading foreign replicons such as phages. In order to investigate the altered expression profiles of the systems after phage infection using a model organism, Thermus thermophilus HB8, which has 12 CRISPR loci, genome-wide transcription profiling of the strain infected with lytic phage ΦYS40 was performed by DNA microarray analysis. Significant alteration of overall mRNA expression gradually increased during infection (i.e., from the eclipse period to the period of host cell lysis). Interestingly, the expression of most cAMP receptor protein (CRP)–regulated genes, including two CRISPR-associated (cas) operons, was most markedly up-regulated, especially around the beginning of host cell lysis, although up-regulation of the crp gene was not observed. The expression of the CRP-regulated genes was less up-regulated in a crp-deficient strain than in the wild type. Thus, it is suggested that cAMP is a signaling molecule that transmits information on phage infection to CRP to up-regulate these genes. On the other hand, the expression of several cas genes and that of CRISPRs were up-regulated independent of CRP, suggesting the involvement of unidentified regulatory factor(s) induced by phage infection. On analysis of the expression profile of the entire genome, we could speculate that upon phage infection, the signal was transmitted to the cells, with host response systems including CRISPR defense systems being activated, while the overall efficiencies of transcription, translation, and metabolism in the cells decreased. These findings will facilitate understanding of the host response mechanism following phage infection.

Introduction

Bacteriophages have a major influence on the microbial world.1 Phages regulate host macromolecular synthesis by modifying the transcription and translation machineries to propagate in the cells. In contrast, bacteria have developed various defense systems against phage infection. The clustered regularly interspaced short palindromic repeat (CRISPR) systems that are present in many prokaryotic genomes are a recently discovered host defense system.2, 3, 4, 5 In general, these systems are composed of CRISPR and CRISPR-associated (cas) genes.6, 7 CRISPRs are composed of 24- to 47 -bp direct repeats separated by nonrepetitive unique spacer sequences of similar length. Sequences derived from foreign replicons such as phages and plasmids are found in the spacers of several CRISPRs.8, 9, 10 CRISPR loci are transcribed and processed into small CRISPR RNAs11, 12 that specify acquired immunity against foreign replicons through a mechanism that relies on the strict identity between CRISPR spacers and targets.3, 11 If cells do not have any CRISPR spacers that are identical with the sequences of an invading replicon, a fragment derived from the replicon can be incorporated into a CRISPR locus of the cells as a new spacer after infection by the replicon; this phase is designated as the adaptation phase. The new spacer sequence plays a role in immunity against subsequent infection by the same foreign replicon, this phase being designated as the interference phase. Bioinformatical and experimental studies support that transcribed spacer RNAs directly target DNA or RNA of foreign replicons.2, 4, 13, 14 Several CRISPR system subtypes have been found, and Cas proteins are classified into about 45 families based on their amino acid sequences, most of which remain uncharacterized.7 The Cas protein families are mainly categorized into core proteins, eight subtypes, or the repeat-associated mysterious protein (RAMP) modules.7 Core cas genes cas1 to cas6 are localized in close proximity to CRISPRs, and they are widely distributed in bacterial and archaeal genomes. The amino acid sequences or three-dimensional structures of several Cas proteins are similar to those of RNA- or DNA-binding proteins. In fact, Cas1 is a metal-dependent DNase, and it has been thought to be involved in the acquisition of new CRISPR spacers.15 Cas2 cleaves single-stranded RNAs preferentially within U-rich regions.16 Cas3 and Cas4 resemble helicase and the RecB family of exonucleases, respectively.6 Cas617 and the Ecoli subtype Cas protein complex, also called the cascade complex, which consists of Cse1, Cse2, Cse3, Cse4, and Cas5e,11 cleave a CRISPR RNA precursor in each repeat, with the cleavage products being retained. The X-ray crystal structure of Cse3 is similar to those of many RNA-binding proteins.18 Cse2 and a RAMP module, Cmr5, adopt a novel fold with large continuous basic patches on one side of their surfaces, which possibly bind DNA or RNA.19, 20 Although the molecular mechanism of the CRISPR systems is beginning to be studied, details including those of transcriptional regulation of these systems remain unknown.

Thermus thermophilus HB8 is an extremely thermophilic bacterium that grows in an optimum temperature range of 65–72 °C, and its generation time under optimum conditions is 18–20 min.21 The genome is composed of 1.85-Mbp chromosomal DNA, 0.26-Mbp plasmid pTT27, and 9.32-kbp plasmid pTT8, which encode 1973, 251, and 14 open reading frames (ORFs), respectively (GenBank accession nos. AP008226, AP008227, and AP008228). Structural and functional genomics studies have been carried out on this strain because most proteins from this organism are stable based on crystallization and functional analysis.22 This strain has ten (CRISPR-1–CRISPR-10) and two (CRISPR-11 and CRISPR-12) CRISPR loci on pTT27 and the chromosome, respectively (Fig. 1); that is, four loci have been identified in addition to the eight reported previously.23 On pTT27, several core cas genes (i.e., cas1cas4 and cas6), one set each of the Ecoli and Mtube subtypes, RAMP module genes, and several other cas family genes are encoded (Fig. 1).7 Of them, an operon containing cas1 to cas3 and Ecoli subtype genes and one containing Mtube subtype and cas family genes are positively regulated by cAMP receptor protein (CRP) in a cAMP-dependent manner (Fig. 1).24 ΦYS40, a lytic tailed myophage that infects T. thermophilus HB8,25 is the most characterized at the molecular level among 115 thermophages.26 Its genome sequence has been determined,27 and regulation of gene expression was investigated by means of DNA macroarray analysis, with three temporal classes of phage genes (i.e., early, middle, and late) being identified.28 The sequences of phage DNA are not found in the CRISPR spacer sequences of T. thermophilus HB8.

Here, we show alteration of the transcription profile of the host cells during infection with ΦYS40 by DNA microarray analysis, focusing on the CRISPR systems. This study provides a novel insight into the host response mechanism following phage infection.

Section snippets

Alteration of the overall mRNA expression profile after phage infection

The overall mRNA expression in T. thermophilus HB8 infected by phage ΦYS40 at 25, 50, 75, and 100 min post-infection was analyzed at the ORF level and compared with that in noninfected cells. We confirmed that 99.8% of the phage population had infected the cells at 25 min (data not shown), the phage was still in the eclipse period at 50 min, phage progeny began to be produced at around 75 min, and host cell lysis began at around 100 min (Fig. 2).28 Significant alteration of mRNA expression

Discussion

We found that most CRISPR systems of T. thermophilus HB8 were significantly up-regulated by phage infection. Interestingly, expression of the systems was not simply regulated by some common regulatory factor(s); that is, several cas genes were regulated by CRP, which may be the receiver of a signaling molecule, cAMP, while several other cas genes and CRISPRs were regulated by unidentified factor(s). Notably, CRISPR and the genes in the vicinity of the CRISPRs including cas genes were not always

Strains, cell growth, and phage infection

The crp-deficient T. thermophilus HB8 strain was constructed as described previously.24 The ΦYS40 phage stock solution (0.3 × 1011 to 1.9  × 1011 pfu/ml) was prepared basically as described28 except that the strain was cultured in TT broth33 at 70 °C. T. thermophilus HB8 strains were cultured in 1 liter of TT broth at 70 °C until the A600 value reached ∼0.8 (1.7 × 108 cells/ml), which corresponded to the logarithmic growth phase. For DNA microarray analysis, cells were collected from 50 ml of the

Acknowledgements

We wish to thank Miwa Ohmori and Aimi Osaki for their excellent technical support.

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