Analysis of the mRNAs in Spores ofBacillus subtilis

ABSTRACTLarge-scale shotgun sequencing (RNA-seq) analysis of mRNAs in dormantBacillus subtilisspores prepared on plates or in liquid generally found the same ∼46 abundant mRNA species, with >250 mRNAs detected at much lower abundances. Knowledge of the amount of phosphate in a singleB. subtilisspore allowed calculation of the amount of mRNA in an individual spore as ∼106 nucleotides (nt). Given the levels of abundant spore mRNAs compared to those of other mRNAs, it was calculated that the great majority of low-abundance mRNAs are present in only small fractions of spores in populations. Almost all of the most abundant spore mRNAs are encoded by genes expressed late in sporulation in the developing spore under the control of the forespore-specific RNA polymerase sigma factor, σG, and most of the encoded proteins are in spores. Levels of the most abundant spore mRNAs were also relatively stable for a week at 4°C after spore harvest. RNA-seq analysis of mRNAs in highly purified and less-well-purified spores made in liquid, as well as from spores that were chemically decoated to remove possible contaminating mRNA, indicated that low-abundance mRNAs in spores were not contaminants in purified spore preparations, and several sources of low-abundance mRNAs in spores are suggested. The function of at least the great majority of spore mRNAs seems most likely to be the generation of ribonucleotides for new RNA synthesis by their degradation early in spore revival.IMPORTANCEPrevious work indicates that dormantBacillus subtilisspores have many hundreds of mRNAs, some of which are suggested to play roles in spores’ “return to life” or revival. The present work finds only ∼46 mRNAs at ≥1 molecule spore, with others in only fractions of spores in populations, often very small fractions. Less-abundant spore mRNAs are not contaminants in spore preparations, but how spores accumulate them is not clear. Almost all abundant spore mRNAs are synthesized in the developing spore late in its development, most encode proteins in spores, and abundant mRNAs in spores are relatively stable at 4°C. These findings will have a major impact on thinking about the roles that spore mRNAs may play in spore revival.

Active depinning of bacterial droplets: The collective surfing ofBacillus subtilis

How systems are endowed with migration capacity is a fascinating question with implications ranging from the design of novel active systems to the control of microbial populations. Bacteria, which can be found in a variety of environments, have developed among the richest set of locomotion mechanisms both at the microscopic and collective levels. Here, we uncover, experimentally, a mode of collective bacterial motility in humid environment through the depinning of bacterial droplets. Although capillary forces are notoriously enormous at the bacterial scale, even capable of pinning water droplets of millimetric size on inclined surfaces, we show that bacteria are able to harness a variety of mechanisms to unpin contact lines, hence inducing a collective slipping of the colony across the surface. Contrary to flagella-dependent migration modes like swarming, we show that this much faster “colony surfing” still occurs in mutant strains ofBacillus subtilislacking flagella. The active unpinning seen in our experiments relies on a variety of microscopic mechanisms, which could each play an important role in the migration of microorganisms in humid environment.

Structure of the RsbX phosphatase involved in the general stress response ofBacillus subtilis

In the general stress response ofBacillus subtilis, which is governed by the sigma factor σB, stress signalling is relayed by a cascade of Rsb proteins that regulate σBactivity. RsbX, a PPM II phosphatase, halts the response by dephosphorylating the stressosome composed of RsbR and RsbS. The crystal structure of RsbX reveals a reorganization of the catalytic centre, with the second Mn2+ion uniquely coordinated by Gly47 O from the β4–α1 loop instead of a water molecule as in PPM I phosphatases. An extra helical turn of α1 tilts the loop towards the metal-binding site, and the β2–β3 loop swings outwards to accommodate this tilting. The residues critical for this defining feature of the PPM II phosphatases are highly conserved. Formation of the catalytic centre is metal-specific, as crystallization with Mg2+ions resulted in a shift of the β4–α1 loop that led to loss of the second ion. RsbX also lacks the flap subdomain characteristic of PPM I phosphatases. On the basis of a stressosome model, the activity of RsbX towards RsbR-P and RsbS-P may be influenced by the different accessibilities of their phosphorylation sites.