Do mycobacteria produce endospores?

Abstract

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 endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic 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 unlikely that Mycobacterium is capable of endospore formation.

Fig. 1.

Shown is a reproduction of figure 2B from Ghosh et al. (2) with an inset of a published electron micrograph of a B. subtilis endospore inside of sporangium [Reproduced with permission from ref. (Copyright 2001, American Society for Microbiology).].

Fig. 2.

Shown is a gallery of thin-section electron micrographs of endospores from the indicated Bacillus species. Samples were sporulated and prepared for electron microscopy as described (28). The image of B. subtilis (Top Left) was published previously [Reproduced with permission from ref. (Copyright 2004, American Society for Microbiology).].

Fig. 3.

Light microscopy images of a M. marinum culture upon prolonged incubation. (A) Strain Mm T CCUG 20998 (ATCC 927) (a kind gift from L. Kirsebom, Uppsala University, Uppsala, Sweden) was streaked for single colonies on 7H10 agar plates supplemented with 0.5% glycerol, 10% acid-albumin-dextrose complex, and cycloheximide [as in Ghosh et al. (2)]. After 1 week, four individual single colonies were streaked on fresh agar plates and followed by phase-contrast light microscopy every week for 12 weeks. Samples were fixed with 70% EtOH (2) or 4% paraformaldehyde in PBS with essentially the same results. (B) A field of sporulating Bacillus subtilis cells, including sporangia with endospores and free spores.

Fig. 4.

Phase-contrast light microscopy images of M. marinum cultures. (A) Laboratory stock cultures that had been grown in liquid for ≈8.5 months at 33°C but with intervals at 4 °C. Arrows indicate phase-gray structures. (B) M. marinum cultures that had been grown in liquid culture for either 4 weeks (Left) or 7.5 months (Right) and spiked with the addition 1% or 5% vol/vol of B. subtilis endospores, respectively. Arrowheads indicate B. subtilis phase-bright endospores. Cultures were spiked by a different individual (K.N.A.) than the individual (F.C.) who did the microscopy, who did not know in advance which cultures had been spiked and which not.

Fig. 5.

Approximately 1,000 colony forming units of M. marinum cells from a 2-week-old culture were plated with (Left) or without (Right) 15 min of heat treatment at 65 °C.