Sinorhizobium meliloti 1021 produces biotin required for growth, but it also responds to external biotin signals from alfalfa plants through the bioS regulatory locus. Mutation of bioS increases biotin uptake, extends stationary phase in the presence of biotin, and impairs competitive growth in the presence of biotin. New data supporting the relevance of this gene to plant-microbe interactions show that a bioS-gusA reporter fusion is expressed by bacteria on plant roots, by bacteria in alfalfa root nodules, and more generally by any stationary-phase bacterial cells in the presence of biotin. Significant transcription of bioS-gusA was detected at 50 pM biotin, and a 16-fold induction was measured in stationary-phase cells with 1 nM biotin. A positive autoregulation of bioS is suggested by the fact that, in the presence of 10 nM biotin, a bioS-gusA fusion in wild-type cells was transcribed at more than twice the rate of the same reporter fusion in a bioS mutant background. Analyses of primer extension products and expression studies with three different bioS-gusA promoter fusions defined major characteristics of the bioS promoter. Expression of bioS in Escherichia coli resulted in the production of a 35-kDa BioS protein with characteristics of an LysR-type regulator. Those traits included a deduced amino acid sequence containing possible helix-turn-helix DNA-binding structures and three T-N(11)-A motifs upstream from the bioS gene. A regulatory role for BioS protein was supported by tests showing that a 321-bp DNA fragment containing the bioS promoter altered the electrophoretic mobility of recombinant BioS and of a corresponding band in protein extracts from S. meliloti 1021. The absence in BioS of any significant homology to known amino acid sequences was consistent with the fact that DNA hybridization tests with seven genera of bacteria detected bioS only in Sinorhizobium.

Most Sinorhizobium meliloti strains lack several key genes involved in microbial biotin biosynthesis, and it is assumed that this may be a special adaptation which allows the microbe to down-regulate metabolic activities in the absence of a host plant. To further explore this hypothesis, we employed two different strategies.

Sinorhizobium meliloti 1021 responds to external biotin signals from alfalfa plants through the bioS regulatory locus. Immunogold labeling and electron microscopy revealed that the BioS protein is located within the S. meliloti cytoplasm. Under biotin-limiting conditions the S. meliloti cell lumen was filled with polyhydroxybutyrate (PHB) granules suggesting that either PHB synthesis or degradation are influenced by biotin. To test this hypothesis a 3-hydroxybutyrate-dehydrogenase-lacZ (bdhA-lacZ) fusion was mobilized into S. meliloti. beta-galactosidase tests revealed an overall 3.6-5.2-fold higher bdhA transcription in the presence of added biotin. Comparison of the bdhA and the bioS promoter regions identified several common motifs.

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