An investigation into the molecular basis of the viable but non-culturable response in bacteria
The viable but non-culturable (VBNC) state is outstanding among bacterial stress responses as being completely uncharacterised at the molecular level. The aim of this investigation was to gain an insight into the molecular basis of the condition by identifying genes whose expression was up-regulated in response to VBNC-inducing stimuli. First, a model experimental system was established where bacteria were induced to enter the state in a routine and predictable manner. Escherichia coli HB101 exhibited a partial viable but non-culturable phenotype when inoculated into microcosms of artificial seawater at 37°C, Pseudomonas fluorescens 10586 became viable but non-culturable in microcosms of drinking water incubated at 37°C, and Vibrio vulnificus MO6-24/T entered a viable, non-culturable state in artificial seawater at 5°C. A transposon mutagenesis strategy utilising a promoter-less bioluminescent reporter cassette, luxAB, was employed in the search for VBNC-associated genes. The mini-Tn5 luxAB transposon was induced to transform into arbitrary positions of the P. fluorescens 10586 chromosome, thus creating a library of P. fluorescens luxAB mutants. This library (consisting of over 1200 transformants) was screened for those which were dark under normal circumstances, but luminesced in response to VBNC stimuli, indicating that the transposon had integrated downstream of a gene up-regulated during the VBNC response. Unfortunately, no mutant examined exhibited such a bioluminescence profile. Differential display of RNA technology was employed subsequently and resulted in the cloning and sequencing of several V. vulnificus transcripts thought to be associated with the VBNC state. Although absolute verification of the involvement of these transcripts was not achieved, hints as to what mechanisms lay at the basis of the VBNC state were gained. Some findings indicated that VBNC cells experience considerable levels of oxidative stress, and it was proposed that this physiological state may lie at the crux of the VBNC phenotype.