Rapid Identification of Polyhydroxyalkanoate Accumulating Members of Bacillales Using Internal Primers for phaC Gene of Bacillus megaterium

Bacillus megaterium is gaining recognition as an experimental model and biotechnologically important microorganism. Recently, descriptions of new strains of B. megaterium and closely related species isolated from diverse habitats have increased. Therefore, its identification requires several tests in combination which is usually time consuming and difficult to do. We propose using the uniqueness of the polyhydroxyalkanoate synthase C gene of B. megaterium in designing primers that amplify the 0.9 kb region of the phaC for its identification. The PCR method was optimized to amplify 0.9 kb region of phaC gene. After optimization of the PCR reaction, two methods were investigated in detail. Method I gave an amplification of a single band of 0.9 kb only in B. megaterium and was demonstrated by several strains of B. megaterium isolated from different habitats. The use of Method I did not result in the amplification of the phaC gene with other members of Bacillales. The specificity for identification of B. megaterium was confirmed using sequencing of amplicon and RT-PCR. Method II showed multiple banding patterns of nonspecific amplicons among polyhydroxyalkanoate accumulating members of Bacillales unique to the respective species. These methods are rapid and specific for the identification of polyhydroxyalkanoate accumulating B. megaterium and members of Bacillales.

Multiple Roles for the sRNA GcvB in the Regulation of Slp Levels in Escherichia coli

The Escherichia coli gcvB gene encodes a small RNA that regulates many genes involved in the transport of dipeptides, oligopeptides, and amino acids (oppA, dppA, cycA, and sstT). A microarray analysis of RNA isolated from an E. coli wild-type and a ΔgcvB strain grown to midlog phase in Luria-Bertani broth indicated that genes not involved in transport are also regulated by GcvB. One gene identified was slp that encodes an outer membrane lipoprotein of unknown function induced when cells enter stationary phase. The aim of this study was to verify that slp is a new target for GcvB-mediated regulation. In this study we used RT-PCR to show that GcvB regulates slp mRNA levels. GcvB negatively controls slp::lacZ in cells grown in Luria-Bertani broth by preventing an Hfq-mediated activation mechanism for slp::lacZ expression. In contrast, in glucose minimal medium supplemented with glycine, GcvB is required for inhibition of slp::lacZ expression, and Hfq prevents GcvB-mediated repression. Thus, GcvB regulates slp in both LB and in glucose minimal + glycine media and likely by mechanisms different than how it regulates sstT, dppA, cycA, and oppA. Repression of slp by GcvB results in an increase in resistance to chloramphenicol, and overexpression of slp in a ΔgcvB strain results in an increase in sensitivity to chloramphenicol.