Formation of ‘non-culturable’ cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation

Abstract

Conditions were investigated that promote the formation of ‘non-culturable’ (NC) cells ofMycobacterium(Myc.)smegmatisin stationary phase. After cultivation in a rich medium, or under conditions that may be considered optimal for bacterial growth, or starvation for carbon, nitrogen or phosphorus, bacteria failed to enter a NC state. However, when grown under suboptimal conditions, resulting in a reduced growth rate or maximal cell concentration (e.g. in modified Hartman's–de Bont medium), bacteria adopted a stable NC state after 3–4 days incubation in stationary phase. Such conditions are not specific aspurFanddevRmutants ofMyc. smegmatisalso showed (transient) loss of culturability following growth to stationary phase in an optimized medium, but under oxygen-limited conditions. The behaviour of the same mutants in oxygen-sufficient but nutrient-inappropriate medium (modified Hartman's–de Bont medium) was similar to that of the wild-type (adoption of a stable NC state). It is hypothesized that adoption of a NC state may represent an adaptive response of the bacteria, grown under conditions when their metabolism is significantly compromised due to the simultaneous action of several factors, such as usage of inappropriate nutrients or low oxygen availability or impairment of a particular metabolic pathway. NC cells of wild-typeMyc. smegmatisresume growth when transferred to a suitable resuscitation medium. Significantly, resuscitation was observed when either recombinant Rpf protein or supernatant derived from a growing bacterial culture was incorporated into the resuscitation medium. Moreover, co-culture withMicrococcus(Mcc.)luteuscells (producing and secreting Rpf) also permitted resuscitation. Isogenic strains ofMyc. smegmatisharbouring plasmids containing theMcc. luteus rpfgene also adopt a similar NC state after growth to stationary phase in modified Hartman's–de Bont medium. However, in contrast to the behaviour noted above, these strains resuscitated spontaneously when transferred to the resuscitation medium, presumably because they are able to resume endogenous synthesis ofMcc. luteusRpf. Resuscitation was not observed in the control strain harbouring a plasmid lackingMcc. luteus rpf. In contrast to wild-type, the NC cells ofpurFanddevRmutants obtained under oxygen-limited conditions resuscitate spontaneously, presumably because the heterogeneous population contains some residual viable cells that continue to make Rpf-like proteins.