The resuscitation promotion escape from prolonged stress by the a variety of chemical and biological factors concept extends to formation of extremely resistant have been shown to promote resuscitation endospores (McKenney et al., 2013). These of VBNC cells (Oliver, 2010). In monocytogenes is a facultative spores can germinate and re-enter into the (high GC-content Gram- pathogenic bacterium that lives in the vegetative life cycle in response to positive ) resuscitation promoting environment as a saprophyte but can turn improved environmental conditions (Fig. factors (Rpfs) were described that have into a harmful pathogen affecting humans 1a). By contrast, L. monocytogenes is unable been shown to induce resuscitation from as well as animals upon ingestion. These to form spores. Thus, L. monocytogenes and starvation-induced dormancy of bacteria have the remarkable capacity to other non-sporulating bacteria must have Mycobacterium tuberculosis and actively invade eukaryotic host cells, to different strategies to survive Micrococcus luteus cells (Mukamolova multiply within them, and to spread to environmental challenges (see Fig. 1a). It et al., 1998; Shleeva et al., 2004). Rpfs are neighbouring cells (Freitag et al., 2009). has been suggested that many Gram- small extracellular proteins with extreme The transition from an environmental positive and Gram-negative bacteria may potency as they are active in very low saprophyte to a pathogen is primarily enter a so-called ‘viable but non- amounts (Mukamolova et al., 1998). The controlled by the transcription factor PrfA. culturable’ (VBNC) state in response to muralytic activity of Rpfs has been proven This regulatory protein directly activates environmental stresses (Fig. 1a; Oliver, to be essential for resuscitation of dormant the expression of the major virulence genes 2005). In this physiological state the cells of Micrococcus luteus (Mukamolova required for the individual steps of the bacteria are alive and metabolically active et al., 1998). infection cycle, i.e. invasion, escape from but the cells are unable to form colonies on Initially, Rpf proteins had not been the phagosome, intracellular motility and conventional culture media (Oliver, 2010). identified in firmicute (low GC-content cell-to-cell spread (de las Heras et al., There is evidence that under certain Gram-positive) bacteria such as L. 2011). Due to its ubiquitous nature, conditions L. monocytogenes cells can also monocytogenes. But later, an in silico contamination of the food processing become VBNC and that these cells escape analysis revealed that many firmicutes chain by L. monocytogenes is a frequent routine detection methods, which are contain a related protein family with event and leads to repeating based on bacterial growth (Cappelier et al., partial similarity to the actinobacterial outbreaks with a high number of severe 2007; Dreux et al., 2007; Lindba¨ck et al., RpfB proteins (Ravagnani et al., 2005). and even fatal cases (Allerberger & 2010). Thus, the VBNC state might be an Two paradigm members of this group of Wagner, 2010). Listeriosis is clearly among important reservoir of harmful bacteria proteins are the YabE and Lmo0186 the deadliest gastrointestinal bacterial and is of major concern for public health proteins from B. subtilis and L. infections as mortality rates of up to 30 % risk and safety assessment. Indeed, harmful monocytogenes, respectively. The Rpf have been reported (Ramaswamy et al., bacteria like Mycobacterium tuberculosis present at the C terminus of the 2007). Consequently, L. monocytogenes has can enter into the VBNC state and return actinobacterial RpfBs is replaced by the Sps become one of the most comprehensively to the infectious state after passaging in or 3D (stationary phase survival/three studied human pathogens (Cossart, 2007, animal hosts (Dhillon et al., 2004). conserved aspartates) domain in the YabE/ 2011). However, we are still far from a The underlying molecular mechanism of Lmo0186 proteins, and these domains complete understanding of virulence of the the reversal of VBNC cells to culturable share partial similarity with the lytic well-characterized and sequenced L. cells is largely unknown. Therefore, the transglycosylase MltA from Escherichia coli monocytogenes strain EGD-e. For example, hypothesis that the VBNC response is a (Ravagnani et al., 2005). The presence of a the function of roughly one-third of all L. programmed response has been faced with domain that is found in proteins with monocytogenes EGD-e genes is still scepticism (Bogosian & Bourneuf, 2001; muralytic activity suggested that Sps unknown according to the KEGG genome Nystro¨m, 2001). However, in recent years, proteins are involved in cell wall database (http://www.genome.jp/dbget- bin/www_bget?genome:T00066). Microbiology Comment provides a forum for discussion of scientific issues arising Like the closely related non-pathogenic directly from papers published in the journal. The authors of papers under discussion will bacterium subtilis, L. be offered an opportunity to respond. monocytogenes is a soil-dwelling bacterium Guidelines on how to submit a Microbiology Comment article can be found in the that frequently encounters harsh Instructions for Authors at http://mic.sgmjournals.org environmental growth conditions, i.e. It should be noted that the Editors of Microbiology do not necessarily agree with the views temperature fluctuations. B. subtilis and expressed in Microbiology Comment. other such as the pathogens Bacillus Agne`s Fouet, Editor-in-Chief anthracis and Clostridium difficile can

1298 G 2013 SGM Printed in Great Britain Microbiology Comment

(a) Starvation In this issue of Microbiology, Pinto et al.

VBNC state stressors Vegetative cycle Sporulation (2013) show for the first time that the Sps proteins Lmo0186 and Lmo2522 in L. monocytogenes are functionally equivalent Lmo0186 to actinobacterial Rpf proteins. Although Lmo2522 Lmo0186 and Lmo2522 are collectively dispensable for growth of L. monocytogenes, cells lacking both proteins had an extended lag phase when cultivated in minimal medium. The growth phenotype of the double knockout could (b) Lmo0186 be restored by the addition of the Listeria eae recombinant Sps proteins Lmo0186 and lac cil ba Lmo2522 to the medium. Moreover, the ni e a observation that the lack of Lmo2522 and P B Lmo2522 all Sps proteins resulted in a small but a c significant increase in cell length during il Lister la ia l e exponential growth and stationary phase, s respectively, suggested a role for the two proteins in cell wall metabolism. Indeed, biochemical analysis revealed that both Sps proteins do have muralytic activity as has

previously been shown for actinobacterial

B

s e

e c l i l a l Rpf proteins (Mukamolova et al., 1998;

Shleeva et al., 2004).

a

i

d

i

r

t

s

o The work by Pinto et al. (2013) provides a l

C well-grounded basis for further studies aimed at understanding the precise biological functions of Sps proteins in a human pathogen. For instance, it would be Fig. 1. (a) Schematic representation of different bacterial life styles. Bacteria like B. subtilis interesting to test whether lack of either can escape from prolonged stress by forming endospores. In response to improved growth Lmo0186 or Lmo2522 individually or in conditions the spores can germinate and re-enter into the vegetative life cycle. In contrast, non-sporulating bacteria such as L. monocytogenes may enter into the viable but non- combination affects pathogenicity of L. culturable (VBNC) state in reponse to prolonged stress. While there is clear evidence that monocytogenes. Moreover, it would be very the resuscitation promoting factor (Rpf) protein from Micrococcus luteus is required for interesting to address the question of resuscitation of dormant cells it is tempting to speculate that the equivalent Sps proteins whether the Sps proteins are needed for Lmo0186 and Lmo2522 of L. monocytogenes serve the same function. (b) Phylogenetic resuscitation of dormant L. monocytogenes distribution of Rpf proteins of the Lmo0186 (SpsB/YabE) and Lmo2522 (SpsA/YocH) VBNC cells (see Fig. 1a). Recently, it has types. Lmo0186 and Lmo02522 were used as query sequences in a BLASTP protein–protein been shown for B. subtilis that the BLAST search (E-value cut-off 1e”30). This yielded 252 homologues of Lmo0186 and 106 of membrane Ser/Thr kinase PrkC detects cell Lmo2522 after removal of truncated protein sequences. The protein sequences were wall-derived muropeptides and stimulates aligned with the CLUSTAL W algorithm and a phylogenetic tree was reconstructed using the synthesis of the Sps protein YocH, which is in-build tree building software of the Geneious Pro 5.5.7 software package. The trees are the homologue of Lmo2522 in L. shown as unrooted cladograms with each end representing one protein sequence monocytogenes (Shah & Dworkin, 2010). homologous to either Lmo0186 or Lmo2522. YocH is secreted into the extracellular space and the hydrolase digests peptidoglycan derived either from the metabolism. Indeed, recently it has been even though members of both Sps domain- same cell or from the surrounding cells. shown that the B. subtilis YocH protein, containing protein families are widespread The released muropeptides may serve as which also contains a Sps domain and is among the firmicutes: YabE/Lmo0186 signalling molecules that trigger regrowth equivalent to the second L. monocytogenes proteins are present in members of the class of cells that were in a resting state (Keep Rpf protein Lmo2522, has muralytic Clostridia, the family Paenibacillaceae and et al., 2006). As a PrkC homologue is also activity and is involved in post-exponential the order as well as the family present in L. monocytogenes (Lmo1820), it growth phase survival (Shah & Dworkin, Listeriaceae, while occurrence of Sps is tempting to speculate that, similar to B. 2010). But as yet, YocH was the only Sps proteins of the YocH/Lmo2522 type is subtilis, Lmo2522 regulates its own protein that had been subjected to a more limited to members of the order Bacillales expression via the PrkC-dependent detailed analysis in a firmicute bacterium and the family Listeriaceae only (Fig. 1b). pathway. Finally, an in-depth expression http://mic.sgmjournals.org 1299 Microbiology Comment analysis of the two Sps protein-encoding proliferation in Listeria. Proc Natl Acad Sci cytokine. Proc Natl Acad Sci U S A 95, 8916– genes might give valuable information USA99, 431–436. 8921. about which growth stage the proteins are Cossart, P. (2007). Listeriology (1926–2007): Nystro¨ m, T. (2001). Not quite dead enough: on synthesized and operate at in the the rise of a model pathogen. Microbes Infect 9, bacterial life, culturability, senescence, and death. Arch Microbiol 176, 159–164. important model pathogen L. 1196–1207. monocytogenes. Cossart, P. (2011). Illuminating the landscape Oliver, J. D. (2005). The viable but of host-pathogen interactions with the nonculturable state in bacteria. J Microbiol 43, bacterium . Proc Natl 93–100. 1 Fabian M. Commichau Acad Sci U S A 108, 19484–19491. Oliver, J. D. (2010). Recent findings on the viable 2 and Sven Halbedel de las Heras, A., Cain, R. J., Bielecka, M. K. & but nonculturable state in pathogenic bacteria. Va´ zquez-Boland, J. A. (2011). FEMS Microbiol Rev 34, 415–425. 1 Regulation of Department of General Microbiology, Listeria virulence: PrfA master and commander. Pinto, D., Sa˜ o-Jose´ , C., Santos, M. A. & Georg-August-University Go¨ttingen, Curr Opin Microbiol 14, 118–127. Chambel, L. (2013). Characterization of two Grisebachstr. 8, 37077 Go¨ttingen, Dhillon, J., Lowrie, D. B. & Mitchison, D. A. resuscitation promoting factors of Listeria Germany (2004). Mycobacterium tuberculosis from chronic monocytogenes. Microbiology 7, 1390–1401. 2Division 11 Enteropathogenic Bacteria murine infections that grows in liquid but not Ramaswamy, V., Cresence, V. M., Rejitha, J. S., Lekshmi, M. U., Dharsana, K. S., Prasad, S. P. & and Legionella, Robert Koch Institute, on solid medium. BMC Infect Dis 4, 51. Vijila, H. M. (2007). Listeria – review of Burgstr. 37, 38855 Wernigerode, Dreux, N., Albagnac, C., Federighi, M., Carlin, F., epidemiology and pathogenesis. J Microbiol Morris, C. E. & Nguyen-the, C. (2007). Viable Germany Immunol Infect 40, 4–13. but non-culturable Listeria monocytogenes on Correspondence: Fabian M. Commichau parsley leaves and absence of recovery to a Ravagnani, A., Finan, C. L. & Young, M. (2005). A novel firmicute protein family related to the ([email protected]) culturable state. J Appl Microbiol 103, 1272– 1281. actinobacterial resuscitation-promoting factors Sven Halbedel by non-orthologous domain displacement. BMC Freitag, N. E., Port, G. C. & Miner, M. D. (2009). 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