The proposal deals with the genetic basis of complex pattern formation defined as morphotypes referring to the distinctive geometrical shapes of individual bacterial patterns occurring in nutritionally stressed Bacilli. The five characterized types found under nutrient-poor conditions -hard agar are: 1. compact growth pattern with rough edges, (B); 2. branched tip-splitting pattern, (T); 3. branched with the same handedness or chiral pattern, (C); 4. the vortex pattern, (V) with branching led by a droplet of bacteria that spin around a common center and 5. spiral vortex (SV). These forms are stably inherited and distinct from Bacillus subtilis. They exhibit new properties such as multiple antibiotic resistance, excretion of """"""""wetting fluid"""""""" and the induction of a beta-galactosidase-like activity (blue colonies on Xgal). Souther hybridizations and 16S ribosomal DNA sequences of these morphotypes show that they are not B. subtilis, a group II Bacillus, but instead they belong to the group I Bacilli; the T and C have identical 16S sequences and differ from V/SV and we named them Bacillus tipchairales and Bacillus vortex respectively. The project has four major goals: 1) To design """"""""molecular beacon"""""""" probes that fluoresce only when they bind to their targets-16S rRNA regions of T/C/V at 25 degrees Celsius in fixed B cells or by liquid hybridization to total RNA from B cells or by PCR assays. 2) To visualize a field of whole and sectioned B spores by transmission, electron microscopy for the presence of aberrant morphologies like fusion structures or typical T, C and V spores. 3) To determine if single B. subtilis spores (B) obtained by micromanipulation give rise to T/C/V morphotypes in a traditional fluctuation analyses. 4) To isolate mutants in strains T/C/V that are defective in pattern formation, in motility in the production of """"""""wetting fluid"""""""", lost their multiple drug resistance phenotypes and mutants unable to accumulate (p)ppGpp by chemical and insertional mutagenesis with transposons from Gram-positive bacteria. The results will show if a symbiotic career state exists in which presumptive pure cultures and spore suspensions are actually mixtures of two or three Bacilli species, or in a novel genetic state exists in which B. subtilis cells can carry both expressed and silent genomes where the latter is activated during certain hostile conditions.
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