Biochemical and molecular genetic approaches to studying protein export by cyanobacteria

Abstract

We have shown that high molecular weight polypeptides are exported extracellularly by cyanobacteria. This is in addition to the well documented release of amino acids and peptides into the culture medium. Synechococcus R2 secretes two polypeptides of 14,400 molecular weight, and Nostoc sp. MAC a 43,000 molecular weight polypeptide. Their function is unclear, but we have some evidence that the 43,000 polypeptide may be involved in metal-binding. Outer membrane proteins (OMP), i.e. carotenoid-containing cell wall fractions, were isolated from several cyanobacteria and a prochlorophyte, and these showed that the extracellular polypeptides were not just cell wall components released into the culture medium. Functional analysis of Synechococcus R2 OMP showed that these proteins were heat-modifiable, a characteristic of porins, and that the pattern of OMP could be modulated by sucrose, magnesium limitation, phosphate- limitation and iron limitation. The effects of iron limitation on the OMP pattern could be simulated by the addition of EDDA (0.004%) to normal growth medium. Synechocystls sp. PCC6308 also Induced specific OMP under iron limitation. It is possible that Synechococcus R2 OMP are multifunctional. Antibodies raised against a Synechococcus R2 cell wall fraction cross-reacted with several other cyanobacterial OMP, and with an OMP fraction from Prochlorothrix hollandice. Using this antisera several positive plaques were obtained after screening a Synechococcus R2 Agtll library. Antibodies against an iron-regulated inner membrane protein, and a carotenoid-associated thylakoid protein cross-reacted with OMP from Synechococcus R2, suggesting a family of carotenoid- containing membrane proteins may exist. The lacZ gene, encoding the enzyme β-galactosidase, has been expressed in Synechococcus R2 on both a multicopy plasmid and when integrated into the cyanobacterial chromosome. The mechanism of integration relied on homologous recombination events between two pBR-plasmid derivatives. This required the construction of a universal recipient strain of Synechococcus R2. A gene encoding the extracellular enzyme pectate lyase from an Erwinia sp. was introduced into the chromosome of Synechococcus R2, but no detectable enzyme activity was observed. The transposon TnphoA was introduced into a cyanobacterial shuttle vector, but was incapable of transposition. Similarly, transposon Tn5, although stably maintained in the Synechococcus R2 chromosome after its introduction via homologous recombination, was not capable of transposition when introduced on a suicide vector. Even though transposition of Tn5 was observed in another Synechococcus sp. no mutant phenotypes were observed. NTG mutagenesis of Synechococcus R2 and Nostoc sp. MAC produced various pigment mutants, but mutants incapable of growth in chelator-deficient medium could not be obtained. This may reflect the selection procedure. The LacZ promoter probe has been constructed for use in Synechococcus R2 based on a multicopy plasmid capable of replicating in this organism. Differential expression of LacZ by Synechococcus R2 was observed under conditions of magnesium and iron limitation. Similar use of a kanamycin promoter probe suggests this construct will be useful for selecting strong promoter sequences via selection for high antibiotic resistance