Outer membrane protein changes during bacteroid development are independent of nitrogen fixation and differ between indeterminate and determinate nodulating host plants of Rhizobium leguminosarum

The outer membrane of bacteroids contains largely decreased levels of protein antigen groups II and III in comparison with that of free-living rhizobia (R. A. de Maagd, R. de Rijk, I. H. M. Mulders, and B. J, J. Lugtenberg, J.Bacteriol, 171:1136-1142, 1989). Since we intend to study the molecular basis of the development of bacterium to bacteroid, we wanted to know whether these outer membrane protein differences are conserved in various plant-Rhizobium combinations, For this purpose we developed a faster assay in which cell lysates instead of isolated cell envelopes were used to analyze these outer membrane changes, With this method the previously described low levels of antigen groups II and III in isolated bacteroid cell envelopes were confirmed, Moreover the described decrease in antigen groups II and III was also found in bacteroids of Rhizobium leguminosarum by. viciae with a mutated nifA or nifK gene as well as in the non-fixing pea mutant FN1 inoculated with the wild-type strain 248, This indicates that the decrease in the antigen levels is not restricted to effective nodules, The results also showed that the decrease in antigen group II not only occurs in bacteroids from pea, but also in bacteroids from vetch, broadbean, white clover, and common bean, Antigen group III, however, remained present in bacteroids from common bean, It is concluded that the changes in antigen group II are not restricted to a specific cross-inoculation group but represent a general phenomenon in the rhizobial bacteroid differentiation process, Of the tested plants, the decrease in antigen group III was not found in bacteroids from common bean and appeared to be restricted to bacteroids from indeterminate nodules. Therefore one should expect that at least two molecular mechanisms are responsible for these outer membrane protein changes and that elucidation of these mechanisms will contribute to our understanding of bacteroid development

Down-regulation of expression of the Rhizobium leguminosarum outer membrane protein gene ropA occurs abruptly in interzone II-III of pea nodules and can be uncoupled from nif gene activation.

Microbial Biotechnolog

Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition

The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4'-phosphopantetheine prosthetic group by holo-ACP synthase. The K(m) value for NodF is 61 μM, as compared to 2 μM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for β-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed comprising part of the E. coli acpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1-33 of AcpP fused to amino acids 43-93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes)