Noel G. Carr

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.

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.

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.

The Oceanic Cyanobacterial Picoplankton

The initial interest in the phycoerythrin-containing picoplanktonic cyanobacteria, assigned to the genus Synechococcus, stemmed directly from a recognition of their considerable contribution to marine primary productivity, as well as to their widespread distribution in an environment hitherto characterized by its relative paucity of cyanobacteria. However, recent work has increasingly indicated that these organisms have features of their molecular biology which separate them from the well-characterized freshwater and halotolerant members of the genus and molecular phylogeny may indicate this separation to be deep. Much of the work described relates to molecular biological analyses of the mechanisms by which these organisms harvest light and acquire key nutrients in an environment which is highly variable with regard to the former and acutely oligotrophic with regard to the latter. Where appropriate, comparisons have been made to what is known of the molecular biology of nutrient acquisition by other ecologically significant oceanic cyanobacteria.

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.

Effect of iron and other nutrient limitations on the pattern of outer membrane proteins in the cyanobacterium Synechococcus PCC7942

When cells of Synechococcus PCC7942 were subjected to either iron or magnesium limitation, there was an appearance of specific proteins in the outer membrane (isolated as the cell wall fraction). Under iron limitation outer membrane polypeptides of Mr 92000, 48000–50000 and 35000 appeared. Specific iron-limited outer membrane proteins (IRMPs) of Mr 52000 and 36000 were also induced in iron-limited cultures of Synechocystis PCC6308. Under magnesium limitation polypeptides of Mr 80000, 67000, 62000, 50000, 28000 and 25000 appeared in the outer membrane. phosphate limitation caused minor changes in the outer membrane protein pattern, with polypeptides of Mr 32000 and one of over 100000 being induced, whereas calcium limitation had no apparent affect.

Organization and transcription of the class I phycoerythrin genes of the marine cyanobacterium Synechococcus sp. WH7803.

The nucleotide sequences of the class I phycoerythrin (PE) α-and β-subunit genes (cpeA and cpeB) from the marine cyanobacterium Synechococcus sp. WH7803 are reported. The cpeB gene is located upstream of cpeA with a separation of 56 nucleotides and the two genes are co-transcribed as a transcript of 1.3 kb, with the transcription startpoint being localized to 110–111 bp upstream of cpeB. The sequence of the promoter region bears no similarity to promoters reported for other cyanobacterial PE genes. Pentanucleotide repeats found upstream of some PE operons, particularly in the case of cyanobacterial strains capable of chromatic adaption, are not found in Synechococcus sp. WH7803; instead the sequence 5′-CGGTT-3′ is repeated three times in the promoter region.

Cloning and sequence analysis of the phycocyanin genes of the marine cyanobacterium Synechococcus sp. WH7803.

In 1979 unicellular phycoerythrin-containing cyanobacteria assigned to the genus Synechococcus were discovered to be abundant in the surface waters of temperate and tropical oceans [6, 11], and this group of picoplankters is now recognized to make a large contribution to the primary productivity of the oceans [see 12]. Two distinct populations of marine Synechococcus strains may be distinguished on the basis of the predominant chromophore associated with phycoerythrin [9]; the phycourobilin-rich strains are characteristic of the open ocean whereas those with a lower phycourobilin content are found in shelf waters. In addition to phycoerythrin, the major lightharvesting phycobiliprotein, marine Synechococcus species contain phycocyanin, allophycocyanin and occasionally phycoerythrocyanin [ 10]. In this report we describe the cloning and sequencing of the genes encoding the ~ and fl subunits of phycocyanin of Synechococcus sp. WH7803, a low-urobilin strain characteristic of shelf isolates [9]. Chromosomal DNA, partially digested with Sau3a, from Synechococcus sp. WH7803 was used to construct a library in lambda charon 35. One clone from this library was isolated which hybridized strongly with plasmid pAQPR1 [4]: pAQPR1 carries the 0c(cpcA) and fl-phycocyanin (cpcB) genes of the freshwater cyanobacterium Synechococcus sp. PCC7002. A 1.6 kb Bam HI fragment from this clone was sub-cloned into pBR322 to yield plasmid pJN12.1. We determined the nucleotide sequence (Fig. 1) of this 1.6 kb Bam HI fragment by the dideoxy chaintermination method following a combination of random and directed subcloning into M 13 mp 18 and mpl0. Analysis of the nucleotide sequence reveals two open reading frames, at positions 257-775 and 820-1308, which on the basis of homologies detected with previously sequenced phycocyanin genes were identified as the cpcB (fl-subunit) and cpcA (~-subunit) genes. The cpcB and cpcA genes are, respectively, 84.8~o and 83.1 ~o homologous to the equivalent genes in the freshwater species Anacystis nidulans R2 (Synechococcus sp. PCC7942) [7, 8] and are organized in the same fashion as that seen in the other cyanobacteria with the cpcB upstream from the cpcA gene [see 3]. The two genes are separated by an intergenic region of 44 bp and there is a possible

Freiburg to Vienna—Looking at Cyanobacteria Over The Twenty-Five Years

In following an invitation to present an overview of the last twenty-five years of cyanobacteriological research it will come as no surprise that my first response will be some kind of general disclaimer with respect to synoptic balance or indeed total accuracy of what follows. The best that one can hope for is a personal, hopefully dispassionate, version of some of the developments and constraints that have happened in our own field during what has surely been a second golden age of biology, coming more or less a century after the first. I will advance an argument that, separate from the revolution of molecular biology that has pervaded virtually the whole of biology, there have been two themes of particular importance which have changed the way in which we look at cyanobacteria and indeed many other bacteria. Firstly there ha been a tighter integration between the understanding of biochemistry and ecology. The role of the organism in its natural environment and the ways in which it interacts with other microbes increasingly forms the background in which laboratory experiments are designed. This has consequences with regard to the use and value of “type species” and this will be alluded to further. More importantly, there has been the emergence of the need to understand the evolutionary significance of our knowledge of bacteria. This has arisen directly from the triumph of molecular phylogeny, which has provided for the first time a coherent measure of relatedness. Dobzhansky’s well known dictum about understanding biology must now be applied to microorganisms and is perhaps particularly appropriate to cyanobacteria.

Microbial Cultures and Natural Populations

For some years there have been descriptions, particularly from oligotrophy environments, of microorganisms that evade culture. Initially these were based upon the very low proportion of colonies obtained relative to the number of microscopically observed bacteria in a sample. Recently there has been several papers that, using molecular biological methods, describe a much greater phylogenetic diversity of bacteria in the open ocean than could have been forecast from traditional means of bacterial culture (see Giovannoni, 1995). Without doubt this is partly a result of our less than perfect skills in isolation but the very low recovery rate reported, often 1 in 103 or 104 leads one to think that other factors may be involved. When bacteria are brought into culture and maintained in the laboratory their growth rate and maximum culture density often increase over periods of months or even years. They ‘adapt’ to laboratory culture. The roots of the idea that the organisms that microbiologists study in the laboratory are often but not always only representative, rather than a cross section, of those from their natural environment is not new and goes back to what has been termed the golden age of bacteriology around the end of the nineteenth century.