Kevin P. Francis

Bacillus weihenstephanensis sp. nov. is a new psychrotolerant species of the Bacillus cereus group.

The Bacillus cereus group comprises the four valid species Bacillus cereus, Bacillus mycoides, Bacillus thuringiensis and bacillus anthracis. Some isolates of B. cereus are known to be psychrotolerant (growth at 7 degrees C or below). Here, specific sequence differences are described between the 16S rDNA, the 23S rDNA, the 16S-23S rDNA spacer region and the genes of the major cold-shock protein homologue cspA in a variety of psychrotolerant and mesophilic B. cereus and B. mycoides strains. Randomly amplified polymorphic DNA analysis using three different primers clearly separated psychrotolerant strains of both species from the rest of the B. cereus group, as did inverse PCR patterns of the rDNA operons. These data strongly support a hitherto unrecognized fifth sub-group within the B. cereus species group comprising psychrotolerant, but not mesophilic, B. cereus strains. Despite the latter finding, the DNA sequences investigated exhibited a high degree of sequence similarity indicating a close relationship between the species of the B. cereus group. Considering the unusual importance of B. cereus in both food poisoning and food spoilage and to avoid merging all species of the group, a new species, Bacillus weihenstephanensis sp. nov., comprising psychrotolerant cereus strains, is proposed. Isolates of the new species grow at 4-7 degrees C but not at 43 degrees C and can be identified rapidly using rDNA or cspA targeted PCR. The type strain is B. weihenstephanensis WSBC 10204T (= DSM 11821T).

Rapid discrimination of psychrotolerant and mesophilic strains of the Bacillus cereus group by PCR targeting of 16S rDNA

The paper describes a novel PCR assay for discriminating psychrotolerant and mesophilic strains of the Bacillus cereus group by targeting of 16S rDNA signatures. Application of the assay circumvents long-term growth tests at low temperature currently used to detect psychrotolerant strains. PCR was performed with pure cultures. A 100% correlation of PCR and growth data at 7°C was obtained for the 194 B. cereus group strains tested. Potential applications of the assay for the dairy industry and agriculture are suggested.

Restart of Exponential Growth of Cold-Shocked Yersinia enterocolitica Occurs after Down-Regulation of cspA1/A2 mRNA

The cellular content of major cold shock protein (MCSP) mRNA transcribed from the tandem gene duplication cspA1/A2 and growth of Yersinia enterocolitica were compared when exponentially growing cultures of this bacterium were cold shocked from 30 to 20, 15, 10, 5, or 0 degrees C, respectively. A clear correlation between the time point when exponential growth resumes after cold shock and the degradation of cspA1/A2 mRNA was found. A polynucleotide phosphorylase-deficient mutant was unable to degrade cspA1/A2 mRNA properly and showed a delay, as well as a lower rate, of growth after cold shock. For this mutant, a correlation between decreasing cspA1/A2 mRNA and restart of growth after cold shock was also observed. For both wild-type and mutant cells, no correlation of restart of growth with the cellular content of MCSPs was found. We suggest that, after synthesis of cold shock proteins and cold adaptation of the cells, MCSP mRNAs must be degraded; otherwise, they trap ribosomes, prevent translation of bulk mRNA, and thus inhibit growth of this bacterium at low temperatures.

Sensitive In Situ Monitoring of a Recombinant Bioluminescent Yersinia enterocolitica Reporter Mutant in Real Time on Camembert Cheese

ABSTRACT Bioluminescent mutants of Yersinia enterocolitica were generated by transposon mutagenesis using a promoterless, complete lux operon (luxCDABE) derived from Photorhabdus luminescens, and their production of light in the cheese environment was monitored. Mutant B94, which had the lux cassette inserted into an open reading frame of unknown function was used for direct monitoring of Y. enterocolitica cells on cheeses stored at 10°C by quantifying bioluminescence using a photon-counting, intensified charge-coupled device camera. The detection limit on cheese was 200 CFU/cm2. Bioluminescence of the reporter mutant was significantly regulated by its environment (NaCl, temperature, and cheese), as well as by growth phase, via the promoter the lux operon had acquired upon transposition. At low temperatures, mutant B94 did not exhibit the often-reported decrease of photon emission in older cells. It was not necessary to include either antibiotics or aldehyde in the food matrix in order to gain quantitative, reproducible bioluminescence data. As far as we know, this is the first time a pathogen has been monitored in situ, in real time, in a “real-product” status, and at a low temperature.

The AGUAAA motif in cspA1/A2 mRNA is important for adaptation of Yersinia enterocolitica to grow at low temperature.

Acclimatization of the psychrotolerant Yersinia enterocolitica after a cold shock from 30°C to 10°C causes transcription of the major cold shock protein (CSP) bicistronic gene cspA1/A2 to increase by up to 300‐fold. Northern blot analysis of cspA1/A2 using four probes that hybridize specifically to different regions of CSP mRNA revealed the appearance of a number of cspA1/A2 transcripts that are smaller than the original transcript and transiently visible at the end of the acclimation period. Primer extension and RNA protection experiments demonstrated that these smaller mRNAs have 5′ ends located in the same core sequence (5′‐AGUAAA‐3′) at five different places within the mRNA, indicating preferential cleavage of the CSP mRNA transcripts. A similar result was obtained for cspB of Escherichia coli, containing two such core sequences. Furthermore, this motif is present in the major CSP genes of a variety of Gram‐negative and Gram‐positive bacteria. We have therefore termed this sequence cold shock cut box (CSC‐box). After inserting a CSC‐box into a plasmid‐bound lacZ gene in Y. enterocolitica, the mRNA of this construct was cleaved within the CSC‐box, and a change in this CSC‐box from AGUAAA to AGUCCC dramatically reduced cleavage of the mutated lacZ gene. Mutating all CSC‐boxes in Y. enterocolitica of a plasmid bound cspA1/A2 dramatically increases the lag time after a cold shock before re‐growth occurs. Based on these results, we suggest that the role of the CSC‐box is related to downregulation of cspA mRNA after acclimation to low temperature.