k, ,* e I ELSEVIER Biochimica et Biophysica Acta 1290(1996) 210-214 Biochi~ic~a et Biophysica A~ta Short sequence-paper Cloning and functional analysis of the pmmA gene encoding phosphomannomutase from the photosynthetic prokaryote Prochlorothrix hollandica Kshitij Dwivedi ~, Anton F. Post a,b,2, S. Bullerjahn a,* ~ Departmentof Biological Sciences. Bowling Green State Unilersity, Bowling Green, OH 43403-0212, USA b Departmentof Microbial and Molecular Ecology, Hebrew Unit.ersityof Jerusalem, GloatRam 91904, Israel Received 10 April 1996:revised 31 May 1996:accepted 3 June 1996 Abstract The pmmA gene encoding a bifunctional phosphomannomutase/phosphoglucomutase (PMM/PGM) from the photosynthetic prokary- ote, Prochlorothrix hollandica has been cloned and sequenced. The gene encodes a 51 827 Da polypeptide 48% identical to the PMM of Azospirillum brasilense, 37% identical to the PGMs of pathogenic Neisseria sp. and 37% identical to the bifunctionai AigC PGM/PMM of Pseudomonas aeruginosa. The pmmA gene encodes an enzyme having both PGM and PMM activities as judged by both enzyme assays and complementation analysis in which the cloned gene partially corrected the pgm-1 mutation of Escherichia coli. Keywords: Phosphomannomutase;Phosphoglucomutase; rfbK; algC: Polysaccharide: (Prochlorothrix) Prochlorothrix hollandica is a filamentous, oxygenic photosynthetic prokaryote that has been shown to be a divergent member of the cyanobacteria [1,2] review [3]. Growth patterns of the organism in the lab and in natural environments indicate that it can exhibit both planktonic and benthic lifestyles. Analyses of the cell envelope struc- tures have revealed that the cell wall is similar in many respects to other cyanobacteria, having multiple layers of peptidoglycan and covalently attached polysaccharides containing abundant N-acetyl muramic acid and N-acetyl mannosamine [4]. Such polysaccharide likely is involved in the tight attachment of the organism to smooth surfaces during stationary phase, as this material accumulates in cultures only after long term storage of cultures (Post and Bullerjahn, unpublished). We are interested in the regula- tion of growth phase-inducible genes in cyanobacteria, and possible candidates for such genes could be those involved in cell surface polysaccharide synthesis. Owing to the high * Corresponding author. Fax: + 1 (419) 3722024: e-mail: bullerjahn @ opie.bgsu.edu. IThe nucleotide sequence data reported in this paper have been submitted to the GenBank database under the accession number U23551. " Present address: Interuniversity Institute, H. Steinitz Marine BioLogy Laboratory, Eilat 88103, Israel. mannosamine content of the polysaccharide [4], and the observation that the growth-phase regulated synthesis of Pseudomonas aeruginosa alginate is in part dependent on the regulation of the algC gene (encoding phosphomanno- mutase) by a pathway involving an alternative sigma factor [5,6], we have sequenced the pmmA gene encoding phos- phomannomutase (PMM) from P. hollandica. PMM is an enzyme that catalyzes the conversion of mannose 6-phos- phate to mannose l-phosphate early in the pathway of polysaccharide synthesis, and thus is likely involved in the synthesis of the mannosamine-containing cell surface poly- saccharide. Further characterization of the gene suggests that the gene product can also use glucose 6-phosphate as a substrate, indicating the PmmA protein also has a phos- phoglucomutase (PGM) activity. The pmmA gene was identified as an open reading frame observed while sequencing through a lambda EMBL3 clone, termed FAP9, that was originally retrieved by probing the EMBL3 library for genes associated with protein kinase activity, pmmA was adjacent to the region hybridizing to the kinase gene probe (data not shown). Confirmation that the genetic region including pmmA encoded a PMM activity was provided by experiments in which the gene was introduced into the Escherichia coli K12 PGMI strain lacking PGM activity [7]; additionally, 0304-4165/96/$15.00 Copyright © 1996 Elsevier Science B.V. All rights reserved. PII S0304-4 165(96)0006 I-X