Julie Newman

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.

High degree of genetic variation in Prochlorococcus (Prochlorophyta) revealed by RFLP analysis

A genetic characterization of nine strains of Prochlorococcus originating from various depths of the Mediterranean Sea, the Sargasso Sea, the North and the tropical Atlantic Ocean and the Pacific Ocean was performed by restriction fragment length polymorphism (RFLP) mapping using probes for rbcL, rbcS, psbA and woxA. This study revealed extensive genetic variation among strains, which were grouped into two distinct clusters. Unexpectedly, with one exception (TATL1 strain), the strains clustered by isolation depth (i.e. near surface versus deep isolates) rather than by geographic origin. These relationships were confirmed by secondary RFLP analysis of psbA amplification products using Prochlorococcus-specific polymerase chain reaction (PCR) primers.

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.

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

Phosphorylation of β-Phycocyanin in Synechocystis sp. PCC 6803

Regulation of protein function and activity by the monoester phosphorylation of specific hydroxyl-containing amino acid residues in proteins has long been recognized as an important feature of metabolic control in eukaryotes and more recently in prokaryotes. In the specific case of the cyanobacteria there is considerable evidence for the occurrence and metabolic significance of monoester protein phosphorylation [for reviews see 1, 2], but only in the case of the PII protein and its signalling role in nitrogen status has the process been well characterized [see 3, 4]. The harvesting of light is the central process underpinning the photoautotrophic lifestyle of cyanobacteria and there is much evidence for the involvement of protein phosphorylation in this process [for review see 5], though much of the specific details remain to be elucidated. The phycobilisome represents the primary antenna for PSII in cyanobacteria and there is preliminary evidence that a major component of the phycobilisome rod structure, the β-subunit of phycocyanin, is subject to monoester phosphorylation [6, 7]. This work is concerned with firmly establishing whether β-phycocyanin is subject to phosphorylation, and if so, what the physiological significance of the process is. The approach was to identify the phosphorylated amino acid residue in β-phycocyanin and then to carry out site-directed mutagenesis of the β-phycocyanin (cpcB) gene.