The Mycobacteria are quite the unique genus; not quite Gram-positive due to their waxy mycolic acids on their outer surface, but certainly not Gram-negative as they do not have an outer lipid bilayer. (Although, there are some interesting micrographs showing a structural feature that does look a lot like a typical Gram-negative outer membrane on the surface of Mycobacteria.) Much slower growing than the average bacteria studied in the lab and not so easily manipulated genetically (though see here and here for some recent advances), studying this important group of pathogens challenges a researcher's patience for results. (Imagine waiting a month or more for a colony to grow to see if a simple transformation was successful!)
In an effort to study this latent phase, a research group happened upon what appeared to be textbook endospores in very late stationary phase cultures of Mycobacterium marinum, a common model for acute Mycobacteria infection. As they closely examined cell morphology over long periods of time (2 months), they began to see forespore formation, and subsequent endospores. Utilizing TEM, the distinct outer coat and cortex of the spores could be identified in the M. marinum population.
These researchers analyzed rRNA from the sporulating cells and identified it as M. marinum, ruling out contamination issues. Also, they demonstrated heat tolerance, malachite green staining, and the presence of dipicolinic acid an important compound necessary for heat resistance in most spore-forming species. Furthermore, their bioinformatic analysis revealed the presence of homologs of genes utilized in sporulation within the M. marinum genome. Taken all together, the authors hypothesize that M. marinum forms spores, and posit that these spores may be how Mycobacteria stay dormant in a host.
However, here is where the speculation steps in...
A group of rather prominent US labs in the field attempted to replicate M. marinum sporulation in their labs, and were unable to demonstrate sporulation by any of the methods used by the original researchers. Furthermore, in an attempt to see if spores were present during latent M. marinum infection as the original authors postulated, they attempted to isolate heat resistant cells from infected frogs. Although they could recover Bacillus spores from spiked tissue samples, they could never isolate M. marinum.
Additionally, the authors demonstrate that the majority of the genes picked out by the original researchers as being sporulation homologs in Mycobacteria, are actually rather universal Gram-positive genes, with homologs present in a wide variety of species, without being used for sporulation. Some of these genes identified by the original group are also not necessary for sporulation, even in Bacillus sp. Finally, neither group of researchers could identify a group of coat proteins, in M. marinum, that are necessary for spore formation in Bacillus.
The lack of this set of coat proteins could be explained by the hypothesis that M. marinum forms its coat differently. This would be a fine rationale, however the spores the original researchers imaged looked identical to those found in B. subtilis. This is of note, particularly because even within the Bacillus genus (with similar coat proteins), spores between species are highly dissimilar.
With this set of data, the second set of researchers conclude that M. marinum does not form spores, and that the simplest solution is that the original authors were looking at Bacillus sp. spores, and not those of M. marinum.
Looking at these two articles, what do you think? (It's time to stop reading articles that don't pertain to my thesis proposal and get back to studying for quals!)
Ghosh, J., Larsson, P., Singh, B., Pettersson, B., Islam, N., Sarkar, S., Dasgupta, S., & Kirsebom, L. (2009). Sporulation in mycobacteria Proceedings of the National Academy of Sciences, 106 (26), 10781-10786 DOI: 10.1073/pnas.0904104106
Traag, B., Driks, A., Stragier, P., Bitter, W., Broussard, G., Hatfull, G., Chu, F., Adams, K., Ramakrishnan, L., & Losick, R. (2009). Do mycobacteria produce endospores? Proceedings of the National Academy of Sciences, 107 (2), 878-881 DOI: 10.1073/pnas.0911299107