Phage Encoded H-NS: A Potential Achilles Heel in the Bacterial Defence System

Abstract

The relationship between phage and their microbial hosts is difficult to elucidate in complex natural ecosystems. Engineered systems performing enhanced biological phosphorus removal (EBPR), offer stable, lower complexity communities for studying phage-host interactions. Here, metagenomic data from an EBPR reactor dominated by Candidatus Accumulibacter phosphatis (CAP), led to the recovery of three complete and six partial phage genomes. Heat-stable nucleoid structuring (H-NS) protein, a global transcriptional repressor in bacteria, was identified in one of the complete phage genomes (EPV1), and was most similar to a homolog in CAP. We infer that EPV1 is a CAP-specific phage and has the potential to repress up to 6% of host genes based on the presence of putative H-NS binding sites in the CAP genome. These genes include CRISPR associated proteins and a Type III restriction-modification system, which are key host defense mechanisms against phage infection. Further, EPV1 was the only member of the phage community found in an EBPR microbial metagenome collected seven months prior. We propose that EPV1 laterally acquired H-NS from CAP providing it with a means to reduce bacterial defenses, a selective advantage over other phage in the EBPR system. Phage encoded H-NS could constitute a previously unrecognized weapon in the phage-host arms race.

Comparative genomics of two ‘Candidatus Accumulibacter’ clades performing biological phosphorus removal

“…Similar to GI 4, very few of these genes had significant blast hits with sequences in the nr database. Surprisingly, Scaffold00028, which is entirely a GI 2, is nearly identical (498% similar over 92% of its length) to a podovirus (EPV 1) sequenced in a viral metagenome (Skennerton et al, 2011) generated from the same bioreactor 7 months after the R104-IIA sample and roughly 2.5 years earlier when the R107-IA sample was collected. Based on having similar coverage and tetranucleotide frequencies to Clade IA scaffolds, we believe that this is a lysogenic phage in the Clade IA genome.…”

“…Comparison of the spacers in the three loci using blastn determined that there were no spacers shared among them. Interestingly, blast analysis of the spacer sequences from all three loci with the viral metagenome (Skennerton et al, 2011), generated from the same bioreactor 7 months after the R104-IIA sample and roughly 2.5 years before the R107-IA sample, yielded only perfect matches with the Clade IA CRISPR Spacers (Table 5). In total, nine Clade IA CRISPR spacers matched perfectly with the EPV1 phage that was found to be a likely lysogen in the Clade IA genome (Scaffold00028 corresponding to GI 2).…”

“…In contrast, neither of the Clade IIA CRISPR regions matched any viral metagenomic sequences completely. Skennerton et al (2011) predicted that this phage was specific for Accumulibacter and they also noted the presence of a histone-like nucleoidstructuring (H-NS) protein that might make the phage resistant to CRISPR activity. The repeated presence of phage DNA in CRISPR spacers, particularly near the leader region (which represent the most current phage insertion events), suggest that the histone-like nucleoid structuring allows for EPV1 to infect continually Clade IA.…”

“…Although the overall gene content was nearly identical, there were differences in gene counts between the two organisms. The value counts the number of spacer sequences that had complete matches based on blast alignment to the virome sample collected from the bioreactor 7 months after the R104-IIA sample (Skennerton et al, 2011). other clusters share nearly identical structure (i.e. gene order) with rnf gene clusters that have been shown to catalyze the transfer or electron from reduced ferredoxin to NAD þ coupled with Na þ translocation (Muller et al, 2008).…”

Holding a grudge

“…69 The bacterial H-NS transcriptional repressor was also found borne on a phage genome, possibly leading to silencing of CRISPR/Cas in Candidatus Accumulibacter phosphatis. 70 Additional mechanisms can likely serve to shield the prophage from the effects of the CRISPR/Cas system, such as evasive mutations in the proto-spacer adjacent motif. 71,72 Perhaps the most intriguing possibility is that the CRISPR/Cas system may be involved in regulating prophage induction or gene expression.…”