Rapid detection of viable Bacillus pumilus SAFR-032 encapsulated spores using novel propidium monoazide-linked fluorescence in situ hybridization

The survival of Bacillus pumilus SAFR-032 spores to standard industrial clean room sterilization practices necessitates the development of rapid molecular diagnostic tool(s) for detection and enumeration of viable bacterial spores in industrial clean room environments. This is of importance to maintaining the sterility of clean room processing products. This paper describes the effect of propidium monoazide (PMA) on fluorescence in situ hybridization (FISH) for detecting and enumerating B. pumilus SAFR-032 viable spores having been artificially encapsulated within poly(methylmethacrylate) (Lucite, Plexiglas) and released via an organic solvent (PolyGone-500). The results of the PMA-FISH experiments discussed herein indicate that PMA was able to permeate only the compromised coat layers of non-viable spores, identifying PMA treatment of bacterial spores prior to FISH analysis as a novel method for selecting out the fraction of the spore population that is non-viable from fluorescence detection. The ability of novel PMA-FISH to selectively distinguish and enumerate only the living spores present in a sample is of potential significance for development of improved strategies to minimize spore-specific microbial burden in a given environment.     

Translation during cold adaptation does not involve mRNA-rRNA base pairing through the downstream box

The downstream box (DB) has been proposed to enhance translation of several mRNAs and to be a key element controlling the expression of cold-shocked mRNAs. However, the proposal that the DB exerts its effects through a base pairing interaction with the complementary anti-downstream box (antiDB) sequence (nt 1469-1483) located in the penultimate stem (helix 44) of 16S rRNA remains controversial. The existence of this interaction during initiation of protein synthesis under cold-shock conditions has been investigated in the present work using an Escherichia coli strain whose ribosomes lack the potential to base pair with mRNA because of a 12 bp inversion of the antiDB sequence in helix 44. Our results show that this strain is capable of cold acclimation, withstands cold shock, and its ribosomes translate mRNAs that contain or lack DB sequences with similar efficiency, comparable to that of the wild type. The structure of helix 44 in 30S ribosomal subunits from cells grown at 37 degrees C and from cells subjected to cold shock was also analyzed by binding a 32P-labeled oligonucleotide complementary to the antiDB region and by chemical probing with DMS and kethoxal. Both approaches clearly indicate that this region is in a double-stranded conformation and therefore not available for base pairing with mRNA.