We screened members of a new genus of grass-associated diazotrophs (Azoarcus spp.) for the presence of cellulolytic enzymes. Out of five Azoarcus strains representing different species, only in the endorhizosphere isolate BH72, which is also capable of invading grass roots, was significant endoglucanase activity, in addition to 13-glucosidase and cellobiohydrolase activity, present. Reducing sugars were readily released from medium-viscosity carboxymethylcellulose (CMC), but neither CMC, cellulose filter strips, Avicel, cellobiose, nor D-glucose served as the sole carbon source for growth of Azoarcus spp. Clones from a plasmid library of strain BH72 expressed all three enzymes in Escherichia coli, apparently not from their own promoter. According to restriction endonuclease mapping and subclone analysis, ,-glucosidase and cellobiohydrolase activities were localized on a single 2.6-kb fragment not physically linked to a 1.45-kb fragment from which endoglucanase (egl) was expressed. Two isoenzymes of endoglucanase probably resulting from proteolytic cleavage had pl values of 6.4 and 6.1 and an apparent molecular mass of approximately 36 kDa. Cellobiohydrolase and ,I-glucosidase activity were conferred by one enzyme 41 kDa in size with a pl of 5.4, which we classified as an unspecific exoglycanase (exg) according to substrate utilization and specificity mapping; hydrolysis of various oligomeric substrates differentiated it from endoglucanase, which degraded substituted soluble cellulose derivatives but not microcrystalline cellulose. Both enzymes were not excreted but were associated with the surface of Azoarcus cells. Both activities were only slightly influenced by the presence of CMC or D-glucose in the growth medium but were enhanced by ethanol. egl was located on a large transcript 15 kb in size, which was detectable only in cells grown under microaerobic conditions on N2. Surface-bound exo-and endoglucanases with some unusual regulatory features, detected in this study in a strain which is unable to metabolize cellulose or sugars, might assist Azoarcus sp. strain BH72 in infection of grass roots.

The strictly respiratory, diazotrophic bacterium Azoarcus sp. strain BH72 fixes nitrogen under microaerobic conditions. In empirically optimized batch cultures at nanomolar O 2 concentrations in the presence of proline, cells can shift into a state of higher activity and respiratory efficiency of N 2 fixation in which intracytoplasmic membrane stacks (diazosomes) related to N 2 fixation are formed. Induction of intracytoplasmic membranes is most pronounced in coculture of Azoarcus sp. strain BH72 with an ascomycete originating from the same host plant, Kallar grass. To initiate studies on function of diazosomes and regulation of their formation, diazosomecontaining bacteria were compared with respect to composition of total cellular and membrane proteins with diazosome-free cells fixing nitrogen under standard conditions. In two-dimensional protein gels, we detected striking differences in protein patterns upon diazosome formation: (i) 7.3% of major proteins disappeared, and only 73% of the total proteins of control cells were detectable, indicating that diazosome-containing cells have a more specialized metabolism; (ii) nine new proteins appeared and five proteins increased in concentration, designated DP1 to DP 15; and (iii) five new major membrane proteins (MP1 to MP6) were detected, indicating that membranes might have specialized functions. N-terminal amino acid sequence 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-free cells but not in diazosome-containing cells, or it may represent a second, not identical P II protein. Oligonucleotides deduced from N-terminal sequences of DP1 and DP4 specifically hybridized to chromosomal DNA of Azoarcus sp. strain BH72 in Southern hybridizations.In nitrogen-fixing microorganisms, including aerobic bacteria, nitrogenase activity is sensitive to oxygen. Therefore, aerobic bacteria which have a strictly respiratory metabolism but no efficient mechanism for protection against oxygen fix nitrogen only under microaerobic conditions: the dissolved oxygen concentration (DOC) is crucial to allow respiratory energy generation and the O 2 -sensitive process of nitrogen fixation to occur simultaneously (11). In several grass root-associated bacteria such as Azospirillum spp. and Azoarcus spp., the DOC allowing N 2 fixation is in the range of a few micromolar (13,27). Temporarily, bacteria might face extremely low O 2 concentrations in their natural habitat when soils are flooded; however, how they respond to these changes has not been intensively studied.Azoarcus sp. strain BH72 has some unusual features with respect to nitrogen fixation. When nitrogen-fixing cells shift into extremely low O 2 concentrations in the presence of proline in empirically optimized batch cultures, they reach a hyperinduced state (15) characterized by dramatic physiological and m...

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