Transcriptome Analysis of a Bloom-Forming Cyanobacterium Microcystis aeruginosa during Ma-LMM01 Phage Infection

Abstract

Microcystis aeruginosa forms massive blooms in eutrophic freshwaters, where it is constantly exposed to lytic cyanophages. Unlike other marine cyanobacteria, M. aeruginosa possess remarkably abundant and diverse potential antiviral defense genes. Interestingly, T4-like cyanophage Ma-LMM01, which is the sole cultured lytic cyanophage infecting M. aeruginosa, lacks the host-derived genes involved in maintaining host photosynthesis and directing host metabolism that are abundant in other marine cyanophages. Based on genomic comparisons with closely related cyanobacteria and their phages, Ma-LMM01 is predicted to employ a novel infection program that differs from that of other marine cyanophages. Here, we used RNA-seq technology and in silico analysis to examine transcriptional dynamics during Ma-LMM01 infection to reveal host transcriptional responses to phage infection, and to elucidate the infection program used by Ma-LMM01 to avoid the highly abundant host defense systems. Phage-derived reads increased only slightly at 1 h post-infection, but significantly increased from 16% of total cellular reads at 3 h post-infection to 33% of all reads by 6 h post-infection. Strikingly, almost none of the host genes (0.17%) showed a significant change in expression during infection. However, like other lytic dsDNA phages, including marine cyanophages, phage gene dynamics revealed three expression classes: early (host-takeover), middle (replication), and late (virion morphogenesis). The early genes were concentrated in a single ∼5.8-kb window spanning 10 open reading frames (gp054–gp063) on the phage genome. None of the early genes showed homology to the early genes of other T4-like phages, including known marine cyanophages. Bacterial RNA polymerase (σ70) recognition sequences were also found in the upstream region of middle and late genes, whereas phage-specific motifs were not found. Our findings suggest that unlike other known T4-like phages, Ma-LMM01 achieves three sequential gene expression patterns with no change in host promoter activity. This type of infection that does not cause significant change in host transcriptional levels may be advantageous in allowing Ma-LMM01 to escape host defense systems while maintaining host photosynthesis.

Cooccurrence of Broad- and Narrow-Host-Range Viruses Infecting the Bloom-Forming Toxic Cyanobacterium Microcystis aeruginosa

“…1 and Table S3) (12,13,18,33). These two viral contigs contained the middle genes required for DNA replication, recombination/repair, and nucleotide metabolism, in addition to viral structural genes (20). Reconstruction of these Ma-LMM01-related MVGFs from the virome reads corroborated the quality of our sequence assembly and host prediction.…”

“…7). Unlike in the synchronized culture experiment (20), Ma-LMM01 did not clearly show three temporal expression classes (early, middle, and late) in the environment (Fig. 7).…”

“…We next examined the viral gene expression patterns to address whether viral gene expression classes (i.e., early, middle, or late [20]) could be captured in the environment. Of all the mapped reads in the Ma-LMM01 genome, 1.58 to 6.69%, 51.3 to 75.9%, and 17.7 to 32.5% of the metatranscriptome reads were mapped to early, middle, and late Ma-LMM01 genes, respectively, at each time point.…”

“…In addition, the expression of the tail sheath gene (gp091) did not show significant correlations with those of transposase (gp135) and recombinase (gp136) (P values, 0.23 and 0.07, respec- tively, by Pearson's product moment correlation coefficient), although the high correlation between gp135 and gp136 (P value, 0.0015) was observed, as described in a previous study (26). Considering the results of the culture experiment (20), it was possible that at least two distinct transcriptional peaks corresponding to middle and late genes were observed in the environment. Therefore, this result suggested that there is a slightly different infection stage in each infected cell in the environment.…”

“…Ma-LMM01 lacks the virally encoded proteins that are important for switching from host to viral transcription and that are needed to acquire the precursors for replication and virion morphogenesis, as seen in T4-like marine cyanoviruses (16,18,19). We recently reported that Ma-LMM01 achieves three temporal expression classes comprising early (host takeover), middle (DNA replication), and late (virion morphogenesis) periods without affecting the host's physiology in order to escape host defense systems while maintaining host photosynthesis (20). Interestingly, Ma-LMM01-matching spacers are present in very low abundances in natural Microcystis populations (10/995 spacers) (9), suggesting that numerous uncharacterized cyanoviruses exist that could interact with M. aeruginosa in the environment.…”