Do mycobacteria produce endospores? Bjorn A. Traag a , Adam Driks b , Patrick Stragier c , Wilbert Bitter d , Gregory Broussard e , Graham Hatfull e , Frances Chu f , Kristin N. Adams f , Lalita Ramakrishnan f , and Richard Losick a,1 a The Biological Laboratories, Harvard University, Cambridge, MA 02138; b Department of Microbiology and Immunology, Loyola University Medical Center, Maywood, IL 60153; c Institut de Biologie Physico-Chimique, Centre National de la Recherche Scientifique-Unité Propre de Recherche R 9073 Université Paris- Diderot, 75005 Paris, France; d Department of Medical Microbiology and Infection Control, VU University Medical Centre, 081 BT Amsterdam, The Netherlands; e Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260; and f Departments of Microbiology, Medicine, and Immunology, University of Washington, Seattle, WA 98195 Edited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved November 6, 2009 (received for review October 1, 2009) The genus Mycobacterium, which is a member of the high G+C group of Gram-positive bacteria, includes important pathogens, such as M. tuberculosis and M. leprae. A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette–Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endo- spores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacte- rium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnos- tic of endospore formation in the genomes of low G+C Gram-positive bacteria. We also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. We conclude that it is un- likely that Mycobacterium is capable of endospore formation. sporulation | tuberculosis T he pathogen Mycobacterium tuberculosis is the leading cause of death worldwide by a single bacterial pathogen (1). An insidious feature of M. tuberculosis is the mysterious phenom- enon of latency in which the pathogen is able to persist in asymptomatic individuals, only to emerge and cause disease many years later (1). Recently, Ghosh et al. (2) reported that the species M. marinum and M. bovis bacillus Calmette–Guérin, a species of the M. tuberculosis complex, produce a type of spore known as an endospore. This discovery, if true, is potentially of great medical significance because it could help explain latency. Endospores are unique among bacterial spores in that they are produced inside of another cell (the mother cell) and, upon maturation, are released as free spores by lysis of the mother cell (3, 4). They are readily recognized under phase-contrast micro- scopy by their phase bright (refractile) appearance. They also exhibit diagnostic features under electron microscopy, such as a protein shell consisting of an inner coat and an electron dense, outer coat (5, 6). Endospores are composed of numerous mol- ecules found, thus far, only in bacterial endospores. These molecules include most of the proteins that encase the spore in a protective shell (called the coat), a family of DNA-protective proteins known as SASP that are bound to the chromosome, and a unique small molecule, dipicolinic acid. All previously known examples of endospore-forming bacteria are members of the low G+C group of Gram-positive bacteria (Firmicutes) belonging either to Bacilli or to Clostridia, and in all cases in which a ge- nome sequence is available, orthologs of genes involved in en- dospore formation are readily seen. The Mycobacterium genus is a member of the high G+C group of Gram-positive bacteria (Actinobacteria) for which there are no prior claims of endo- spore formation. Certain members of the group, such as Strep- tomyces, do produce spores, but spores of a fundamentally different kind that are not produced inside a mother cell (7). Because of the potentially high significance of the discovery of Ghosh et al. (2) for the treatment of tuberculosis, we investigated their claims by carrying out genome sequence analysis and by testing for the production of endospores and for heat-resistant colony forming units by Mycobacterium marinum in vitro and in a frog model. Results and Discussion Mycobacterium and Streptomyces Genomes Lack Orthologs of Highly Conserved Endospore Genes. We carried out genome sequence analysis by using BLAST and Psi-BLAST of the 15 Mycobacte- rium genomes (including those of M. marinum and M. bovis) and the 18 Streptomyces genomes present in the National Center for Biotechnology Information database of microbial genomes. The analysis revealed no orthologs of any of the signature genes for endospore formation (4). Examples are the absence of genes for the above mentioned SASP family, spoIVA, which encodes a highly conserved morphogenetic protein required for coat as- sembly, spoIIR and spoIIGA, which mediate the activation of a mother-cell-specific transcription factor, spoIID, which governs the process by which the forespore is engulfed by the mother cell, spoIIIAE, a critically important membrane protein produced in the mother cell, and the spoVF operon, which encodes a dipi- colinic acid synthetase. [Certain clostridia do, however, lack spoVF and generate dipicolinic acid via an electron transfer flavoprotein that is widely distributed among both endospore- forming and nonendospore-forming bacteria (D. Popham, per- sonal communication).] These genes encode proteins that are almost identical among Bacillus species (E values close to zero), including species that are distantly related to each other, such as B. subtilis and B. anthracis. In contrast, no reliable homologies were detected against predicted proteins from Mycobacterium genomes. Another example is sigG, which encodes the forespore- specific transcription factor σ G . The σ G protein is related to a family of regulatory proteins found in nonendospore forming bacteria but σ G itself has residues that distinguish the sporulation transcription factor from other members of the family. The authors cite examples of M. marinum sporulation genes but they are not in fact diagnostic of endospore formation, as shown on the related pages of the GTOP database (http://spock. genes.nig.ac.jp/~genome/search.html). For example, spo0J (CAB16133.1) encodes a member of the ParB family of proteins involved in DNA segregation (8), spoIIIE (CAB13553.1) enc- odes a member of a family of DNA translocases (9), and spoVE (CAB13394.1) is a homolog of mrdB, encoding a rod-shape de- termining membrane protein (10, 11). The spoVK (CAB13626.1) homolog cited by the authors corresponds to the 3′ half of a gene Author contributions: B.A.T., A.D., P.S., W.B., G.B., G.H., F.C., L.R., and R.L. designed research; B.A.T., A.D., P.S., G.B., F.C., and K.N.A. performed research; P.S. and W.B. contributed new reagents/analytic tools; B.A.T., A.D., P.S., W.B., G.B., G.H., F.C., and L.R. analyzed data; and B.A.T., A.D., P.S., W.B., G.H., F.C., L.R., and R.L. wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. 1 To whom correspondence should be addressed. E-mail: losick@mcb.harvard.edu. 878–881 | PNAS | January 12, 2010 | vol. 107 | no. 2 www.pnas.org/cgi/doi/10.1073/pnas.0911299107