Formation of 'non-culturable' cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation
Formation of 'non-culturable' cells of Mycobacterium smegmatis in stationary phase in response to growth under suboptimal conditions and their Rpf-mediated resuscitation
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DOI：
10.1099/mic.0.26893-0
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发表时间：
2004-06-01
期刊：
影响因子：
2.8
通讯作者：
Kaprelyants, AS
中科院分区：
文献类型：
--
作者：
Shleeva, M;Mukamolova, GV;Kaprelyants, AS
研究了促进分枝杆菌（Myc.）在稳定期内，在丰富培养基中培养后，或在可能被认为对细菌生长最佳的条件下培养后，或在碳、氮或磷饥饿的条件下培养后，细菌未能进入NC状态。然而，当在次优条件下生长时，导致生长速率或最大细胞浓度降低（例如在改良的Hartman ′ s-de Bont培养基中），细菌在稳定期孵育3-4天后采用稳定的NC状态。这些条件不是Myc的purF和devR突变体所特有的。在优化的培养基中生长到稳定期后，但在限氧条件下，smegalovirus也显示出（短暂的）可培养性丧失。相同的突变体在氧气充足但营养不适当的培养基（改良的Hartman 's-de Bont培养基）中的行为与野生型的行为相似（采用稳定的NC状态）。据推测，采用NC状态可能代表细菌的适应性反应，在由于几个因素的同时作用而使其代谢显著受损的条件下生长，所述因素例如使用不适当的营养物或低氧可用性或特定代谢途径的损伤。野生型Myc.当转移到合适的复苏培养基中时，耻垢病恢复生长。值得注意的是，当将重组Rpf蛋白或来源于生长细菌培养物的上清液掺入复苏培养基中时，观察到复苏。此外，与微球菌（Micrococcus（Mcc.）黄体细胞（产生和分泌Rpf）也允许复苏。Myc.携带含有Mcc的质粒的耻垢病。luteus rpf基因在改良Hartman ′ s-de Bont培养基中生长至稳定期后也采取类似的NC状态。然而，与上述行为相反，这些菌株在转移到复苏培养基中时自发复苏，可能是因为它们能够恢复Mcc的内源性合成。鲁特乌斯河在携带缺乏Mcc的质粒的对照菌株中未观察到复苏。黄体反应器与野生型相比，在氧限制条件下获得的purF和devR突变体的NC细胞自发复苏，大概是因为异质群体含有一些残留的活细胞，继续使Rpf样蛋白。
Conditions were investigated that promote the formation of 'non-culturable' (NC) cells of Mycobacterium (Myc.) smegmatis in 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 as purF and devR mutants of Myc. smegmatis also 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-type Myc. smegmatis resume 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 with Micrococcus (Mcc.) luteus cells (producing and secreting Rpf) also permitted resuscitation. Isogenic strains of Myc. smegmatis harbouring plasmids containing the Mcc. luteus rpf gene 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 of Mcc. luteus Rpf. Resuscitation was not observed in the control strain harbouring a plasmid lacking Mcc. luteus rpf. In contrast to wild-type, the NC cells of purF and devR mutants obtained under oxygen-limited conditions resuscitate spontaneously, presumably because the heterogeneous population contains some residual viable cells that continue to make Rpf-like proteins.