Nicholas H. Mann

The Genome of S-PM2, a “Photosynthetic” T4-Type Bacteriophage That Infects Marine Synechococcus Strains

Bacteriophage S-PM2 infects several strains of the abundant and ecologically important marine cyanobacterium Synechococcus. A large lytic phage with an isometric icosahedral head, S-PM2 has a contractile tail and by this criterion is classified as a myovirus (1). The linear, circularly permuted, 196,280-bp double-stranded DNA genome of S-PM2 contains 37.8% G+C residues. It encodes 239 open reading frames (ORFs) and 25 tRNAs. Of these ORFs, 19 appear to encode proteins associated with the cell envelope, including a putative S-layer-associated protein. Twenty additional S-PM2 ORFs have homologues in the genomes of their cyanobacterial hosts. There is a group I self-splicing intron within the gene encoding the D1 protein. A total of 40 ORFs, organized into discrete clusters, encode homologues of T4 proteins involved in virion morphogenesis, nucleotide metabolism, gene regulation, and DNA replication and repair. The S-PM2 genome encodes a few surprisingly large (e.g., 3,779 amino acids) ORFs of unknown function. Our analysis of the S-PM2 genome suggests that many of the unknown S-PM2 functions may be involved in the adaptation of the metabolism of the host cell to the requirements of phage infection. This hypothesis originates from the identification of multiple phage-mediated modifications of the hosts photosynthetic apparatus that appear to be essential for maintaining energy production during the lytic cycle.

THE EFFECT OF PHOSPHATE STATUS ON THE KINETICS OF CYANOPHAGE INFECTION IN THE OCEANIC CYANOBACTERIUM SYNECHOCOCCUS SP. WH78031

Phycoerythrin‐containing Synechococcus species are considered to be major primary producers in nutrient‐limited gyres of subtropical and tropical oceanic provinces, and the cyanophages that infect them are thought to influence marine biogeochemical cycles. This study begins an examination of the effects of nutrient limitation on the dynamics of cyanophage/Synechococcus interactions in oligotrophic environments by analyzing the infection kinetics of cyanophage strain S‐PM2 (Cyanomyoviridae isolated from coastal water off Plymouth, UK) propagated on Synechococcus sp. WH7803 grown in either phosphate‐deplete or phosphate‐replete conditions. When the growth of Synechococcus sp. WH7803 in phosphate‐deplete medium was followed after infection with cyanophage, an 18‐h delay in cell lysis was observed when compared to a phosphate‐replete control. Synechococcus sp. WH7803 cultures grown at two different rates (in the same nutritional conditions) both lysed 24 h postinfection, ruling out growth rate itself as a factor in the delay of cell lysis. One‐step growth kinetics of S‐PM2 propagated on host Synechococcus sp. WH7803, grown in phosphate‐deplete and‐replete media, revealed an apparent 80% decrease in burst size in phosphate‐deplete growth conditions, but phage adsorption kinetics ofS‐PM2 under these conditions showed no differences. These results suggested that the cyanophages established lysogeny in response to phosphate‐deplete growth of host cells. This suggestion was supported by comparison of the proportion of infected cells that lysed under phosphate‐replete and‐deplete conditions, which revealed that only 9.3% of phosphate‐deplete infected cells lysed in contrast to 100% of infected phosphate‐replete cells. Further studies with two independent cyanophage strains also revealed that only approximately 10% of infected phosphate‐deplete host cells released progeny cyanophages. These data strongly support the concept that the phosphate status of the Synechococcus cell will have a profound effect on the eventual outcome of phage‐host interactions and will therefore exert a similarly extensive effect on the dynamics of carbon flow in the marine environment.

Phages of the marine cyanobacterial picophytoplankton

Cyanobacteria of the genera Synechococcus and Prochlorococcus dominate the prokaryotic component of the picophytoplankton in the oceans. It is still less than 10 years since the discovery of phages that infect marine Synechococcus and the beginning of the characterisation of these phages and assessment of their ecological impact. Estimations of the contribution of phages to Synechococcus mortality are highly variable, but there is clear evidence that phages exert a significant selection pressure on Synechococcus community structure. In turn, there are strong selection pressures on the phage community, in terms of both abundance and composition. This review focuses on the factors affecting the diversity of cyanophages in the marine environment, cyanophage interactions with their hosts, and the selective pressures in the marine environment that affect cyanophage evolutionary biology.

A conserved genetic module that encodes the major virion components in both the coliphage T4 and the marine cyanophage S-PM2

Sequence analysis of a 10-kb region of the genome of the marine cyanomyovirus S-PM2 reveals a homology to coliphage T4 that extends as a contiguous block from gene (g)18 to g23. The order of the S-PM2 genes in this region is similar to that of T4, but there are insertions and deletions of small ORFs of unknown function. In T4, g18 codes for the tail sheath, g19, the tail tube, g20, the head portal protein, g21, the prohead core protein, g22, a scaffolding protein, and g23, the major capsid protein. Thus, the entire module that determines the structural components of the phage head and contractile tail is conserved between T4 and this cyanophage. The significant differences in the morphology of these phages must reflect the considerable divergence of the amino acid sequence of their homologous virion proteins, which uniformly exceeds 50%. We suggest that their enormous diversity in the sea could be a result of genetic shuffling between disparate phages mediated by such commonly shared modules. These conserved sequences could facilitate genetic exchange by providing partially homologous substrates for recombination between otherwise divergent phage genomes. Such a mechanism would thus expand the pool of phage genes accessible by recombination to all those phages that share common modules.

Involvement of an FtsH homologue in the assembly of functional photosystem I in the cyanobacterium Synechocystis sp PCC 6803

The Synechocystis sp. PCC 6803 genome encodes four putative homologues of the AAA protease FtsH, two of which (slr0228 and sll1463) have been subjected to insertional mutagenesis in this study. Disruption of sll1463 had no discernible effect but disruption of slr0228 caused a 60% reduction in the abundance of functional photosystem I, without affecting the cellular content of photosystem II or phycobilisomes. Fluorescence and immunoblotting analyses show reductions in PS I polypeptides and possible structural alterations in the residual PS I, indicating an important role for slr0228 in PS I biogenesis.

The response of the picoplanktonic marine cyanobacterium Synechococcus species WH7803 to phosphate starvation involves a protein homologous to the periplasmic phosphate-binding protein of Escherichia coli

During phosphate‐limited growth the marine phycoerythrin‐containing picoplanktonic cyanobacterium Synechococcus sp. WH7803 synthesizes novel polypeptides, including two abundant species of 100 kDa and 32kDa. The 32kDa polypeptide was localized to the cell wall, although in a related strain, Synechococcus sp. WH8103, it could be detected in both the cell wall fraction and the periplasm. The gene (designated pstS) encoding this polypeptide was cloned and shown to be present in a single copy. The deduced amino acid sequence indicated a polypeptide consisting of 326 amino acids with a calculated Mr of 33763. Comparison of this sequence with that obtained by microsequencing the N‐terminus of the 32kDa polypeptide showed that the mature protein was synthesized as a precursor, the first 24 amino acid residues being cleaved between two alanine residues at positions 24 and 25. The amino acid sequence of the mature polypeptide showed 35% identity and 52% similarity to the periplasmic phosphate‐binding protein (PstS) from Escherichia coli, including three regions of much stronger homology which, by comparison with E. coli PstS, are directly involved in phosphate binding. Northern blot analysis revealed a pstS transcript of 1.2 kb in RNA extracted from cells grown in Pi‐replete conditions and one of 1.4 kb in considerably increased abundance under Pi‐depleted conditions. Homologues of the pstS gene were detected in other marine phycoerythrin‐containing Synechococcus strains, but not in freshwater or halotolerant species.

Involvement of Phycobilisome Diffusion in Energy Quenching in Cyanobacteria

Nonphotochemical quenching (NPQ) of excitation energy is a well-established phenomenon in green plants, where it serves to protect the photosynthetic apparatus from photodamage under excess illumination. The induction of NPQ involves a change in the function of the light-harvesting apparatus, with the formation of quenching centers that convert excitation energy into heat. Recently, a comparable phenomenon was demonstrated in cyanobacteria grown under iron-starvation. Under these conditions, an additional integral membrane chlorophyll-protein, IsiA, is synthesized, and it is therefore likely that IsiA is required for NPQ in cyanobacteria. We have previously used fluorescence recovery after photobleaching to show that phycobilisomes diffuse rapidly on the membrane surface, but are immobilized when cells are immersed in high-osmotic strength buffers, apparently because the interaction between phycobilisomes and reaction centers is stabilized. Here, we show that when cells of the cyanobacterium Synechocystis sp. PCC 6803 subjected to prolonged iron-deprivation are immersed in 1 m phosphate buffer, NPQ can still be induced as normal by high light. However, the formation of the quenched state is irreversible under these conditions, suggesting that it involves the coupling of free phycobilisomes to an integral-membrane complex, an interaction that is stabilized by 1 m phosphate. Fluorescence spectra are consistent with this idea. Fluorescence recovery after photobleaching measurements confirm that the induction of NPQ in the presence of 1 m phosphate is accompanied by immobilization of the phycobilisomes. We propose as a working hypothesis that a major component of the fluorescence quenching observed in iron-starved cyanobacteria arises from the coupling of free phycobilisomes to IsiA.

Construction of lacZ promoter probe vectors for use in Synechococcus: application to the identification of CO2-regulated promoters

It was shown that the Escherichia coli lacZ gene could be expressed in the cyanobacterium Synechococcus R2 PCC7942 both as a plasmid-borne form and also integrated into the chromosome. A promoterless form of the lacZ gene was constructed and used as a reporter gene to make transcriptional fusions with cyanobacterial promoters using a shuttle vector system and also via a process of integration by homologous recombination. Synechococcus R2 promoter-lacZ gene fusions were then used to identify CO2-regulated promoters, by quantitatively assessing beta-galactosidase activity under high and low CO2 conditions using a fluorescence assay. Several promoters induced under low CO2 conditions were detected.

Protein phosphorylation in cyanobacteria

The post-translational modification of proteins by phosphorylation may take a variety of forms, but in essence it is always a component of a process of adaptation that enables cells to sense and respond to changes in their external or internal environments. This review presents evidence that not only do the various forms of protein phosphorylation occur in the cyanobacteria, but that they are central to many of the adaptive responses exhibited by these organisms (for review see Tandeau de Marsac & Houmard, 1993) and, indeed, may play a key role in the integration of metabolism. Early studies on protein phosphorylation in prokaryotes were largely confined to phosphate monoester formation with hydroxyl-containing amino acid residues. It was originally thought that protein acyl phosphates and amido phosphates only occurred as intermediates of enzyme catalysis. However, it is now clear that these forms of protein phosphorylation are of enormous significance in the process of sensory transduction via two-component sensory systems involving members of the conserved histidine protein kinase and response regulator families of proteins (for reviews see Stock e t al., 1989; Parkinson & Kofoid, 1992).

The occurrence of rapidly reversible non-photochemical quenching of chlorophyll a fluorescence in cyanobacteria

Cyanobacteria have previously been considered to differ fundamentally from plants and algae in their regulation of light harvesting. We show here that in fact the ecologically important marine prochlorophyte, Prochlorococcus, is capable of forming rapidly reversible non‐photochemical quenching of chlorophyll a fluorescence (NPQf or qE) as are freshwater cyanobacteria when they employ the iron stress induced chlorophyll‐based antenna, IsiA. For Prochlorococcus, the capacity for NPQf is greater in high light‐adapted strains, except during iron starvation which allows for increased quenching in low light‐adapted strains. NPQf formation in freshwater cyanobacteria is accompanied by deep F o quenching which increases with prolonged iron starvation.