Further studies on AGA production by Pantoea agglomerans strain Eh1087
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Pantoea agglomerans strain Eh1087 produces the phenazine antibiotic Dalanylgriseoluteic acid (AGA). A cluster of 16 genes has previously been shown to be responsible for the production of, and resistance to, AGA. The present study has refined and tested a number of hypotheses arising from the preliminary characterisation of the AGA pathway. The products of the first five genes of the AGA cluster, Group 1, are similar to proteins responsible for phenazine-1-carboxy lie acid by fluorescent pseudomonads. However, Eh1087 appears to be missing a duplication of the ehpA gene, and it was hypothesised that EhpA was responsible for the ability of Eh1087 to produce both phenazine-1- carboxylic acid and phenazine-1,6-dicarboxylic. Comparison of EhpA to related proteins of known structure suggested a catalytic function, and EhpA was found to influence the relative amounts of phenazine-1-carboxylic acid and phenazine-1,6- dicarboxylic acid produced by Group 1. The final step in the AGA pathway is the addition of a D-alanyl residue to griseoluteic acid to form AGA, and is catalysed by the EhpMNO proteins. The previous model for AGA biosynthesis suggested that EhpM was an integral membrane protein and that EhpMNO operated in the periplasm. EhpM and EhpN are similar to components of nonribosomal peptide synthetases, while EhpO is similar to ketosynthases involved in fatty acid and polyketide biosynthesis. Comparison of EhpM with known structures, and preliminary analysis of the subcellular location of EhpMNO suggest that these proteins may be localised to the cytoplasmic side of the inner membrane, rather than the periplasm. An Eh1087 transposon-mutant with an insertion that affected the expression of glnA was isolated. This mutant lacked glutamine synthetase, and was, therefore, not able to incorporate labelled nitrogen from ammonium sulphate, via glutamine, into phenazines. It was concluded that glutamine is the source of the two nitrogen atoms of the phenazine nucleus. In addition, the function of the putative Eh1087 glnA locus was confirmed by complementation of an E.coli glnA mutant and the gene found to encode a type I glutamine synthetase typical of the Enterobacteriaceae.