Christian Ibarra

A GENETIC APPROACH TO IDENTIFYING GENES INVOLVED IN BACTERIAL PATTERN FORMATION IN A PAENIBACILLUS ISOLATE

In nature, bacteria exist in multicellular communities that engender advantageous capabilities that they could not achieve as individual cells. These capabilities are dependent upon the bacteria to communicate in a highly cooperative manner. In many Paenibacillus strains, these behaviors manifest themselves as elaborate growth patterns when the bacteria are grown on solid substrates. Many growth patterns or morphotypes often appear as ornate fractal patterns. In our laboratory we are investigating the pattern formation of a Paenibacillus strain (LMB265) which elicits a sunburst morphotype. The overall goal of our work is to use genetic and biochemical methods to study the genes and protein products involved in this particular pattern formation. Our immediate goal is to identify a method for the genetic manipulation of the LMB265 strain. Unlike its closest relative Bacillus subtilis, this strain was found not to be competent for transformation and bears multiple antibiotic resistances. However, it is sensitive to neomycin. Therefore, an integrational plasmid was constructed for transformation of Paenibacillus based on a functional integrative vector for B. subtilis and containing a neomycin resistance cassette. This plasmid, pCI2, also contains the sigD gene for B. subtilis that encodes the alternate sigma factor responsible for the motility regulon in this organism. Preliminary studies indicate that Paenibacillus LMB265 contains a sigD homolog. Thus, successful transformation of LMB265 is predicted to disrupt the sigD homolog and lead to a lack of pattern formation since this phenotype is due to surface-based motility. We have successfully generated neo^R transformants of LMB265 and current studies are aimed at confirming appropriate integration of pCI2 into its genome and a spontaneous non-pattern forming mutant. A gene homologous to gapA sequences of several other bacteria was found to be solely expressed in pattern-forming cells. We are now attempting to disrupt this gene using a derivative of pCI2 to clarify its role in pattern formation. Taken together, these studies provide the first demonstration of genetic transformation in any Paenibacillus species and hold significant promise for a greater understanding of the molecular mechanisms that govern bacterial pattern formation.