The Impact of Protease during Recovery from Viable but Non-Culturable (VBNC) State in Vibrio cholerae

Vibrio cholerae can survive cold stress by entering into a viable but non-culturable (VBNC) state, and resuscitation can be induced either by temperature upshift only or the addition of an anti-dormancy stimulant such as resuscitation-promoting factors (Rpfs) at suitable temperature. In this study, the role of proteinase K was analyzed as an Rpf in V. cholerae. A VBNC state was induced in V. cholerae AN59 in artificial seawater (ASW) media at 4 °C, and recovery could be achieved in filtered VBNC microcosm, called spent ASW media, merely by a temperature upshift to 37 °C. The resuscitation ability of spent ASW was further enhanced by the addition of proteinase K. The mode of action of proteinase K was investigated by comparing its effect on the growth of the VBNC and culturable state of V. cholerae in ASW and spent ASW media. The presence of proteinase K allowed culturable cells to grow faster in ASW by reducing the generation time. However, this effect of proteinase K was more pronounced in stressed VBNC cells. Moreover, proteinase K-supplemented spent ASW could also accelerate the transition of VBNC into recovered cells followed by rapid growth. Additionally, we found that dead bacterial cells were the substrate on which proteinase K acts to support high growth in spent ASW. So, the conclusion is that the proteinase K could efficiently promote the recovery and growth of dormant VBNC cells at higher temperatures by decreasing the duration of the initial lag phase required for transitioning from the VBNC to recovery state and increasing the growth rate of these recovered cells.

Bacteria differently regulate mRNA abundance to specifically respond to various stresses

Environmental stress is detrimental to cell viability and requires an adequate reprogramming of cellular activities to maximize cell survival. We present a global analysis of the response of Escherichia coli to acute heat and osmotic stress. We combine deep sequencing of total mRNA and ribosome-protected fragments to provide a genome-wide map of the stress response at transcriptional and translational levels. For each type of stress, we observe a unique subset of genes that shape the stress-specific response. Upon temperature upshift, mRNAs with reduced folding stability up- and downstream of the start codon, and thus with more accessible initiation regions, are translationally favoured. Conversely, osmotic upshift causes a global reduction of highly translated transcripts with high copy numbers, allowing reallocation of translation resources to not degraded and newly synthesized mRNAs.

DnaK and GroEL are induced in response to antibiotic and heat shock in Acinetobacter baumannii

We studied the expression of DnaK and GroEL in Acinetobacter baumannii cells (strains ATCC 19606 and RS4) under stress caused by heat shock or antibiotics. A Western blot assay showed that DnaK and GroEL levels increased transiently more than 2-fold after exposure of bacterial cells to heat shock for 20 min at 50 °C. Heat induction of DnaK and GroEL was blocked completely when an inhibitor of transcription, rifampicin, was added 1 min before a temperature upshift to 50 °C, suggesting that the induction of these chaperones depends on transcription. A. baumannii cells pretreated at 45 °C for 30 min were better able to survive at 50 °C for 60 min than cells pretreated at 37 °C, indicating that A. baumannii is able to acquire thermotolerance. DnaK and GroEL were successfully induced in cells pre-incubated with a subinhibitory concentration of streptomycin. Moreover, bacterial cells pretreated for 30 min at 45 °C were better able to survive streptomycin exposure than cells pretreated at physiological temperatures. DnaK expression was upregulated in a multidrug-resistant strain of A. baumannii (RS4) in the presence of different antimicrobials (ampicillin+sulbactam, cefepime, meropenem and sulphamethoxazole+trimethoprim). This study is to the best of our knowledge the first to show that A. baumannii DnaK and GroEL could play an important role in the stress response induced by antibiotics.

DNA Microarray Analysis of the Heat Shock Transcriptome of the Obligate Intracytoplasmic Pathogen Rickettsia prowazekii

ABSTRACT Here we present the first oligonucleotide DNA microarray analysis of global gene expression changes in the obligate intracytoplasmic pathogen Rickettsia prowazekii using temperature upshift as a model stress condition, and we describe a methodology for isolating highly purified rickettsial RNA. In toto, 23 transcripts were significantly increased by temperature upshift (≥2.0-fold; P < 0.05), and no transcripts demonstrated reproducible decreases. Array results for three heat shock-inducible mRNAs were confirmed using quantitative reverse transcription-PCR.

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

ABSTRACT Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4�C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 109 CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.

Hypoxia abolishes transience of the heat-shock response in the methylotrophic yeast Hansenula polymorpha

The heat-shock response is conserved amongst practically all organisms. Almost invariably, the massive heat-shock protein (Hsp) synthesis that it induces is subsequently down-regulated, making this a transient, not a sustained, stress response. This study investigated whether the heat-shock response displays any unusual features in the methylotrophic yeast Hansenula polymorpha, since this organism exhibits the highest growth temperature (49–50 °C) identified to date for any yeast and grows at 47 °C without either thermal death or detriment to final biomass yield. Maximal levels of Hsp induction were observed with a temperature upshift of H. polymorpha from 30 °C to 47–49 °C. This heat shock induces a prolonged growth arrest, heat-shock protein synthesis being down-regulated long before growth resumes at such high temperatures. A 30 °C to 49 °C heat shock also induced thermotolerance, although H. polymorpha cells in balanced growth at 49 °C were intrinsically thermotolerant. Unexpectedly, the normal transience of the H. polymorpha heat-shock response was suppressed completely by imposing the additional stress of hypoxia at the time of the 30 °C to 49 °C temperature upshift. Hypoxia abolishing the transience of the heat-shock response appears to operate at the level of Hsp gene transcription, since the heat-induced Hsp70 mRNA was transiently induced in a heat-shocked normoxic culture but displayed sustained induction in a culture deprived of oxygen at the time of temperature upshift.