In the present study variant morphotypes of B. mycoides SIN strain were collected in search for mutant gene/s coding for the chiral colony shape. Colonies were found that lost the peculiar wild type shape made of filaments aggregating in bundles with the same final curvature: some acquired a cotton-like appearance; others made round compact colonies; one became less adherent to the agar surface. The new morphotypes were not seen to revert to wild type during several transfers on agar media. All of them lost ability to form aggregates in liquid culture: cells detached easily one from the other and grew in suspension conferring turbidity to the medium.
The observation that mutant morphotypes fall into several different classes strongly suggests that many genes are involved in the process of colony shape formation. We explored the possibility that a genetic determinant could be carried by a plasmid, but strains fully cured of the two cryptic plasmid of the SIN strain maintained wild type morphotype, ruling out this hypothesis.
We compared the sequence of ftsQ, ftsA, ftsZ and murC of wild type SIN with that of the SINett mutant, whose morphotype with round colonies is the most altered, but not a single nucleotide was changed, indicating that mutations affect other genes, probably regulator genes.
It is interesting that mutant morphotypes were able to colonize a reduced territory area compared to wild type: presumably territory conquest must be crucial for a soil inhabitant to reach new nutrients in natural environments. Possibly this trait is under selective pressure and positively selected for. Consistent with the hypothesis of filamentous growth as an adaptation to search for nutrients we noticed an increased colony diameter of SIN strains when grown on media with low nutrient concentration (unpublished observation). Determination of the growth curves in wild type and mutant strains put into evidence that mutants were not negatively affected for the time needed to duplicate, which was found to be similar if not identical during logarithmic growth. On the contrary, mutants were found to persist in the logarithmic period for a longer time during culture in liquid media, resulting in an increased mass compared to wild type strains (unpublished results).
Peculiarities of colony formation were followed by light and scanning electron microscopy. In wild type strains, cells move away from the colony center as single filaments or as bundles. Single filaments can leave a bundle and join other bundles. When filaments coming from different directions meet, they may converge, or alternatively cross one over the other continuing along their route. We do not see twisting of one filament around another as in the case of the B. subtilis
macrofiber-forming mutant [9
Mutant strains also form bundles of filaments, but, instead of leaving the mass, they turn all together back towards the colony center. It is unlikely that a repelling substance, secreted by wild type strains, may be the driving force for cells to spread away from the colony mass since this in contrast with the formation of bundles. We rather hypothesize a greater "independence" of wild type strains from growth substances released from the mass of bacteria. This would permit colonization of empty spaces where no such substances are present. Moreover the strong aggregation that wild type strains show in liquid culture is a consequence of the tight head to head contact of cells after division, probably due to a molecular "glue" or to incomplete separation of the cells after septation. Other attracting forces, like side by side cell stickiness, are present also in mutants, as seen on agar where they form parallel wavy bundles. However this kind of attraction is weak, since mutant cells easily separate in liquid culture mostly as single units.
Very little is known about B. mycoides
at the genetic level. Our soil isolates of DX and SIN share metabolic traits, such as the ability to metabolize substrates, with the exception of sucrose hydrolysis. Genomic organization is not the same, as estimated from ribotyping and plasmid content. At the gene sequence level, as determined for some genes of the division and cell wall cluster, DX and SIN strains appear very close, but not identical. Nucleotide and amino acid sequences are more similar in these two strains than with the ones of the close B. anthracis
and B. cereus
species. The FtsZ sequence of B. pseudomycoides
is the most divergent, confirming a greater phylogenetic distance of this species from the other members of the group [21
As for the molecular mechanism underlying asymmetry of the colony, we obtained mutants that lost this character. This is an essential preliminary step towards the possibility of transforming and complementing the null mutation with wild type long DNA sequences, in search for the gene/s guiding the turn direction morphotype. For this purpose we are now working to increase the very low transformation efficiency of our strains.