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Metabolic flexibility and morphological plasticity in mycobacteria “One wonders how many genetic programs and biochemical/physiological functions remain hidden in the genome, simply because we have not exposed the bacteria to the ‘right’ environmental conditions... ” Mu-Lu Wu1 & Thomas Dick*,1

Mycobacteria are nondifferentiating aerobic bacilli: they do not form specialized resting cells, need oxygen and are rod shaped. That is at least a simplified version of our general text book knowledge of this group of actinobacteria. Although correct if one grows the bacteria in vitro aerobically in standard rich media, it is only part of the story. A type of observational bias is at work here, known as ‘streetlight effect’, where people look for whatever they are searching by looking where it is easiest: under the streetlight [1] . Analyses of mycobacteria grown under various nonstandard in vitro culture conditions and in vivo resulted in a vastly more complex picture of their adaptation and differentiation potential. It turns out that these bacteria possess an amazing metabolic flexibility and morphological plasticity. Here we discuss the recently uncovered complex anaerobic energy metabolism of these ‘obligate’ aerobes as an example for their metabolic flexibility. A multitude of different cell shapes observed under various culture

conditions are discussed to illustrate the morphological plasticity of these bacteria. Finally, we shift the cone of the streetlight a bit and find yet another new type of a mycobacterial resting cell. Implications of this versatility for mycobacterial genomics are discussed.

Keywords 

• adaptation • differentiation • Mycobacterium • streetlight effect

Metabolic flexibility: adaptations to hypoxic environments Despite being obligate aerobes, in other words requiring oxygen for growth, mycobacteria can survive under hypoxic and even anaerobic conditions for extended periods of time in a nonreplicating state without any apparent morphological differentiation  [2] . This has been extensively studied in vitro (in the ‘Wayne model’) for pathogenic tubercle bacilli [3] as well as for the saprophytic Mycobacterium smegmatis  [4] . Mycobacteria have a whole battery of responses to remodel their standard aerobic respiration-based energy metabolism when exposed to oxygen limited or oxygen-free conditions. A 50-gene

1 Department of Microbiology, Yong Loo Lin School of Medicine, National University Health System, National University of Singapore, 5 Science Drive 2, Singapore 117545, Republic of Singapore *Author for correspondence: Tel.: +65 6601 1018; Fax: +65 6776 6872; [email protected]

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Editorial  Wu & Dick regulon controlled by the response regulator dormancy survival regulator (DosR) was found to play a key role in mediating adaptation to oxygen-starved environments [5–7] . The bacteria can increase aerobic respiration efficiency via expression of a high-affinity cytochrome bd oxidase to scavenge oxygen [8] , and they are able to switch to anaerobic respiration using nitrate [9] and fumarate [10] as alternative electron acceptors. Interestingly, the bacilli can also switch to fermentation. Fermentative succinate production involves Sdh2 succinate dehydrogenase and a reverse TCA cycle as well as the glyoxylate shunt [11–13] . Recently, anaerobiosis-induced fermentative production of hydrogen via the Hyd3 hydrogenase was reported [14] . The ability of mycobacteria to switch from low- to highaffinity aerobic respiration, to anaerobic nitrate and fumarate respiration, and to succinate and hydrogen fermentation illustrates the remarkable metabolic flexibility of these ‘obligate’ aerobes.

“Whether mycobacteria

can or cannot develop specific Bacillus-like endospores remains controversial, but appears rather unlikely.”

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Morphological plasticity: all kinds of shapes & sizes Although generally considered nondifferentiating, several nonrod morphotypes have been reported under various conditions in vitro and in vivo. Small, ovoid nonreplicating resting forms have been observed in M. smegmatis after prolonged storage in minimal media [15,16] , and in Mycobacterium tuberculosis after gradual acidification [17] . Small round cells were reported for M. tuberculosis under particular anaerobic culture conditions [18] . Interestingly, cells smaller in size were also reported in a carbon-limited continuous culture model [8] , suggesting that mycobacteria are capable of generating small replicating cells. Unusually small cells were also observed under certain in vivo conditions. For instance, filterable miniforms of M. tuberculosis were isolated from guinea pigs and from patients suffering from extremely drug-resistant (XDR) tuberculosis after 3–6 months of treatment [19] . Small round cells inside rod-shaped cells were detected in XDR strains [20] . Different mechanisms have been proposed for the formation of smaller cells, including folding and budding types of cell division [18] . However, the cellular and genetic mechanisms generating these various small-cell morphotypes remain ill defined. Recently, Ghosh et al.  [21] reported the discovery of endospores in old cultures of Mycobacterium bovis BCG and M. marinum. These endospores showed striking similarities

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to Bacillus subtilis endospores regarding structure (spore coat), properties (spore staining, heat resistance) and chemistry (dipicolinic acid). Traag et al. [22] could not reproduce those results and also pointed out that mycobacteria lack critical orthologous of highly conserved endospore genes. Nevertheless, Lamont et al.  [23] again reported similar endospore formation, this time in M. avium subsp. paratuberculosis. Whether mycobacteria can or cannot develop specific Bacillus-like endospores remains controversial, but appears rather unlikely. It is worth mentioning that not only smaller than usual but also larger morphotypes have been reported. For instance, longer, filamentous forms of M. tuberculosis have been described in IFN-γ-activated macrophages [24] . Taken together, a number of reports describe the observation of nonrod morphotypes under various in vitro and in vivo conditions. In general, these processes are less well defined than the above-mentioned metabolic adaptation processes to oxygen starvation, but nevertheless point to a significant morphological plasticity in mycobacteria. Shifting the light cone a little: a new type of resting cell It is well established that mycobacteria are very hardy and can for instance survive shock starvation in phosphate buffered saline (‘Loebel model’) in a nonreplicating state without any apparent morphological differentiation [25] . We recently decided to have another look at this adaptation but with shifting the cone of the streetlight a bit. Rather than shock starving the bacilli in saline as it had been done since Loebel developed this system almost 100 years ago, we decided to add traces of a carbon source to the saline and observe the response of the bacteria. This idea was stimulated by what we have learnt from the sporulation process in Bacillus subtilis. Endospore formation is triggered under starvation. However, Bacillus is checking on the availability of some carbon sources before committing to the energy consuming and rather lengthy endosporulation process. Interestingly, providing traces of a carbon source to M. smegmatis in saline (i.e., gently as opposed to shock-starve the organism), resulted in the formation of yet another small-cell morphotype: reductive cell division generated very short rodshaped cells with increased long-term viability. Upon addition of rich medium, these small resting cells grew back to larger standard cells before

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Metabolic flexibility & morphological plasticity in mycobacteria  commencement of the regular cell division cycle [Wu ML, Dick T, Unpublished Data] . The fact that a new morphotype can be so easily generated by slight changes in culture conditions supports the notion of a surprising morphological plasticity of mycobacteria. Concluding remarks Mycobacteria live in a large variety of terrestrial, aquatic and host environments. The widely used mycobacterial model organism M. smegmatis, isolated from smegma and living as a saprophyte in soil, is a harmless representative. Other species, for instance Mycobacterium abscessus, living in our municipal water supply systems and being aerosolized by our showerheads, are emerging opportunistic pathogens. With M. tuberculosis we have a pandemic obligate parasite living in the lungs of 2 billion people in a latent state and killing more than a million each year after reactivation. Considering the dynamic nature of any environment it is perhaps not too surprising to find that these ‘nondifferentiating aerobic rods’ in fact can undergo morphological differentiation(s) and complex metabolic adaptations. Many mycobacterial genomes have been sequenced and studied in the past decade. Databases currently contain whole genome sequences of 21 mycobacterial species. Although several studies improved the functional (i.e., biochemical) annotations of large numbers of genes, a considerable fraction of open reading frames is References 1

2

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Wayne LG, Hayes LG. An in vitro model for sequential study of shiftdown of Mycobacterium tuberculosis through two stages of nonreplicating persistence. Infect. Immun. 64(6), 2062–2069 (1996).

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Dick T, Lee BH, Murugasu-Oei B. Oxygen depletion induced dormancy in Mycobacterium smegmatis. FEMS Microbiol. Lett. 163(2), 159–164 (1998).

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still labeled as ‘hypothetical protein’. For many genes we do not know their physiological role, even if we can annotate them with a biochemical function. One wonders how many genetic programs and biochemical/physiological functions remain hidden in the genome, simply because we have not exposed the bacteria to the ‘right’ environmental conditions that would activate those responses. Shifting the cone of the streetlight around should result in many more surprises and will contribute to improve our genome annotations. Acknowledgements The authors thank Martin Gengenbacher, National University of Singapore, for discussion and comments on the manuscript.

Financial & competing interest disclosure T Dick’s research is funded by the Ministry of Health, National Medical Research Council (CBRG/022/2012, CG/013/2013, TCR12dec007/2014), the Singapore MIT Alliance for Research and Technology and the School of Medicine/National University of Singapore. ML Wu receives a research scholarship from the Yong Loo Lin School of Medicine. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript. Mycobacterium tuberculosis DosR regulon assists in metabolic homeostasis and enables rapid recovery from nonrespiring dormancy. J. Bacteriol. 192(6), 1662–1670 (2010).

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Metabolic flexibility and morphological plasticity in mycobacteria.

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