GLOBAL CHANGES IN PROTEIN-COMPOSITION OF N-2-FIXING AZOARCUS SP STRAIN BH72 UPON DIAZOSOME FORMATION

The strictly respiratory, diazotrophic bacterium Azoarcus sp, strain B H72 fixes nitrogen under microaerobic conditions, In empirically optim ized batch cultures at nanomolar O-2 concentrations in the presence of proline, cells can shift into a state of higher activity and respirat ory efficiency of N-2 fixation in which intracytoplasmic membrane stac ks (diazosomes) related to N-2 fixation are formed, Induction of intra cytoplasmic membranes is most pronounced in coculture of Azoarcus sp. strain BH72 with an ascomycete originating from the same host plant, K allar grass, To initiate studies on function of diazosomes and regulat ion of their formation, diazosome-containing bacteria were compared wi th respect to composition of total cellular and membrane proteins with diazosome-free cells fixing nitrogen under standard conditions, In tw o-dimensional protein gels, we detected striking differences in protei n patterns upon diazosome formation: (i) 7.3% of major proteins disapp eared, and only 73% of the total proteins of control cells were detect able, indicating that diazosome-containing cells have a more specializ ed metabolism; (ii) nine new proteins appeared and five proteins incre ased in concentration, designated DP1 to DP15; and (iii) five new majo r membrane proteins (MP1 to MP6) were ere detected, indicating that me mbranes might have specialized functions. N-terminal amino acid sequen ce analysis of DP1 to DP4 allowed us to preliminarily identify DP4 as the glnB gene product P-II, an intracellular signal transmitter known to be involved in the regulation of nitrogen metabolism, According to its electrophoretic mobility, it might be uridylylated in diazosome-fr ee cells but not in diazosome-containing cells, or it may represent a second, not identical P-II protein, Oligonucleotides deduced from N-te rminal sequences of DPI and DP4 specifically hybridized to chromosomal DNA of Azoarcus sp, strain BH72 in Southern hybridizations.