The Distribution of a Phage-Related Insertion Sequence Element in the Cyanobacterium, Microcystis aeruginosa

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bViruses play important roles in regulating the abundance, clonal diversity, and composition of their host populations. To assess their impact on the host populations, it is essential to understand the dynamics of virus infections in the natural environment. Cyanophages often carry host-like genes, including photosynthesis genes, which maintain host photosynthesis. This implies a diurnal pattern of cyanophage infection depending on photosynthesis. Here we investigated the infection pattern of Microcystis cyanophage by following the abundances of the Ma-LMM01-type phage tail sheath gene g91 and its transcript in a natural population. The relative g91 mRNA abundance within host cells showed a peak during the daylight hours and was lowest around midnight. The phage g91 DNA copy numbers in host cell fractions, which are predicted to indicate phage replication, increased in the afternoon, followed by an increase in the free-phage fractions. In all fractions, at least 1 of 71 g91 genotypes was observed (in tested host cell, free-phage, and RNA fractions), indicating that the replication cycle of the cyanophage (i.e., injection, transcription, replication, and release of progeny phages) was occurring. Thus, Microcystis cyanophage infection occurs in a diel cycle, which may depend on the light cycle. Additionally, our data show that the abundance of mature cyanophage produced within host cells was 1 to 2 orders of magnitude greater than that of released phages, suggesting that phage production may be higher than previously reported.

Section: Resultsmentioning

confidence: 99%

“…1; see also Table S1 in the supplemental material) (19,20). The reaction conditions for the TAIL-PCR were those described previously (15). The sheathTPF2 and AD2, -4, -5, and -7 primer sets had amplified products (1-to 1.5-kbp fragments) from all of the three fractions in environmental samples from Hirosawanoike Pond.…”

Section: Methodsmentioning

confidence: 99%

Diurnal Infection Patterns and Impact of Microcystis Cyanophages in a Japanese Pond

Kimura

Yoshida

Hosoda

et al. 2012

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“…S1 in the supplemental material. The strains were maintained in CB medium (29) as described previously (31). Late-exponential-phase cultures (2-ml aliquots) were mildly sonicated to remove gas vesicles and then centrifuged at 3,000 ϫ g for 10 min (31).…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, amplification and sequencing were performed using successive thermal asymmetric interlaced PCR (TAIL-PCR) (34) toward the end of the arrays (primers are shown in Table S1 in the supplemental material). Reaction conditions and arbitrary primers for the TAIL-PCR were as described previously (31). A portion of the amplifications were performed using an alternative PCR with MaeCRrGT and outward primers based on CRISPR spacers in the sequenced fragments.…”

Section: Methodsmentioning

confidence: 99%

Intricate Interactions between the Bloom-Forming Cyanobacterium Microcystis aeruginosa and Foreign Genetic Elements, Revealed by Diversified Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Signatures

Kuno

Yoshida

Kaneko

et al. 2012

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bClustered regularly interspaced short palindromic repeats (CRISPR) confer sequence-dependent, adaptive resistance in prokaryotes against viruses and plasmids via incorporation of short sequences, called spacers, derived from foreign genetic elements. CRISPR loci are thus considered to provide records of past infections. To describe the host-parasite (i.e., cyanophages and plasmids) interactions involving the bloom-forming freshwater cyanobacterium Microcystis aeruginosa, we investigated CRISPR in four M. aeruginosa strains and in two previously sequenced genomes. The number of spacers in each locus was larger than the average among prokaryotes. All spacers were strain specific, except for a string of 11 spacers shared in two closely related strains, suggesting diversification of the loci. Using CRISPR repeat-based PCR, 24 CRISPR genotypes were identified in a natural cyanobacterial community. Among 995 unique spacers obtained, only 10 sequences showed similarity to M. aeruginosa phage Ma-LMM01. Of these, six spacers showed only silent or conservative nucleotide mutations compared to Ma-LMM01 sequences, suggesting a strategy by the cyanophage to avert CRISPR immunity dependent on nucleotide identity. These results imply that host-phage interactions can be divided into M. aeruginosa-cyanophage combinations rather than pandemics of population-wide infectious cyanophages. Spacer similarity also showed frequent exposure of M. aeruginosa to small cryptic plasmids that were observed only in a few strains. Thus, the diversification of CRISPR implies that M. aeruginosa has been challenged by diverse communities (almost entirely uncharacterized) of cyanophages and plasmids.

“…Total genomes are invaluable for emerging fields such as ecogenomics, when the plasticity of microorganisms to changing environmental conditions needs to be investigated. So far, it has been speculated that TEs in general are an important source of physiological plasticity in bloom-forming cyanobacteria, such as Microcystis aeruginosa (43,44). The influence of TEs on the restructuring of genomes and the inactivation/rearrangement of genes can be investigated only when completely assembled genome information is available.…”

Section: Discussionmentioning

confidence: 99%

Elucidation of Insertion Elements Carried on Plasmids and In Vitro Construction of Shuttle Vectors from the Toxic Cyanobacterium Planktothrix

Christiansen

Goesmann

Kurmayer

2014

bSeveral gene clusters that are responsible for toxin synthesis in bloom-forming cyanobacteria have been found to be associated with transposable elements (TEs). In particular, insertion sequence (IS) elements were shown to play a role in the inactivation or recombination of the genes responsible for cyanotoxin synthesis. Plasmids have been considered important vectors of IS element distribution to the host. In this study, we aimed to elucidate the IS elements propagated on the plasmids and the chromosome of the toxic cyanobacterium Planktothrix agardhii NIVA-CYA126/8 by means of high-throughput sequencing. In total, five plasmids (pPA5.5, pPA14, pPA50, pPA79, and pPA115, of 5, 6, 50, 79, and 120 kbp, respectively) were elucidated, and two plasmids (pPA5.5, pPA115) were found to propagate full IS element copies. Large stretches of shared DNA information between plasmids were constituted of TEs. Two plasmids (pPA5.5, pPA14) were used as candidates to engineer shuttle vectors (named pPA5.5SV and pPA14SV, respectively) in vitro by PCR amplification and the subsequent transposition of the Tn5 cat transposon containing the R6K␥ origin of replication of Escherichia coli. While pPA5.5SV was found to be fully segregated, pPA14SV consistently co-occurred with its wild-type plasmid even under the highest selective pressure. Interestingly, the Tn5 cat transposon became transferred by homologous recombination into another plasmid, pPA50. The availability of shuttle vectors is considered to be of relevance in investigating genome plasticity as a consequence of homologous recombination events. Combining the potential of high-throughput sequencing and in vitro production of shuttle vectors makes it simple to produce species-specific shuttle vectors for many cultivable prokaryotes.