In this paper, we have shown that it is possible to inactivate the genes encoding the two key ribonucleases RNase J1 and RNase Y in B. subtilis
, contrary to previous data from both the literature and our own experience that suggested that they were essential. The homogenous colony sizes and high transformation efficiencies in four different genetic backgrounds make it relatively unlikely that these deletion strains require second-site suppressor mutations for growth. This observation should serve as a cautionary tale for the choice of method used to declare genes to be essential in bacteria. Without further genetic analysis of the type performed here, there is necessarily some doubt about all 150 genes labeled as being essential based on the failure to obtain viable clones using the plasmid-borne replacement technique in the original paper by Kobayashi et al. (1
The difference in previous attempts to inactivate the rnjA and rny genes seems to be explained in large part by the much higher recombination efficiency of long chromosomal DNA fragments than that of the short homology regions offered on plasmid replacement systems. We managed, with some perseverance, to inactivate both the rnjA and rny genes using a plasmid-borne replacement system in strains containing a second copy of the genes expressed from a xylose-dependent promoter at the amyE locus. Once this construct was established on the chromosome, it was readily transferable into the 168 wild-type background by transformation with chromosomal DNA and grew similarly to the rnjA::spc or rny::spc deletion strain (data not shown). Thus, the major problem is not with the plasmid-borne construct per se, i.e., there are no polar effects on the expression of downstream genes, nor are the truncated peptides synthesized by the Campbell-recombined plasmid a source of toxicity, but rather with the efficiency of recombination. It cannot be solely a question of recombination efficiency, however. A plasmid-borne gene replacement system would be predicted to yield similar numbers of colonies for a gene that is unnecessary for growth on rich medium as for a gene that makes a major contribution to cell growth under these conditions; only colony size should vary. Clearly, the fact that deletion of these genes has a major effect on cell doubling time has an additional negative impact on the ability to recover viable colonies with the plasmid-borne constructs compared to transformation with chromosomal DNA. The reasons for this are unclear.
The RNase J1 and RNase Y deletion strains have major defects in cell growth and morphology. Given their globally important roles, it was surprising to learn that the cell can live without them. This is in contrast, for example, to the major enzyme of E. coli
mRNA degradation, RNase E. While the RNase J1 and RNase Y deletion strains have understandably much longer doubling times than wild-type strains in rich medium, they are not impossible to work with and will be a major asset in future experiments. Although the RNase Y deletion strain has a shorter doubling time than the strain lacking RNase J1 (), it forms smaller colonies (), presumably due to its smaller cell size (). Both deletion stains grow reasonably well in minimal medium and at high temperatures. The RNase J1 mutant is cryosensitive, even at room temperature, presumably explained by its role in rRNA maturation (5
). The increased sensitivity of the RNase J1 and RNase Y mutants to a wide range of antibiotics suggested that they might have cell permeability defects, and we confirmed by electron microscopy that the peptidoglycan layer is disordered. Curiously, however, the RNase Y mutant was more resistant to the topoisomerase/gyrase inhibitor nalidixic acid (as was the PNPase mutant). It will be interesting to explore the basis for this behavior in the future. RNase J1-depleted cells were previously shown by Hunt et al. to be more resistant to the dihydrofolate reductase inhibitor trimethoprim (2
); it is not clear to us why the null mutant is more sensitive to this antibiotic in our hands.
It would not be surprising if the defects in the cell envelope contributed to the sporulation and competence deficiencies of these strains. However, in a previous tiling array experiment (16
), a number of key sporulation genes showed increased expression levels in RNase J1 and RNase Y depletion mutants, including those of the master regulator Spo0A (RNases J1 and Y) and its modulators Spo0B and Spo0E (RNase J1) (see Table S2 in the supplemental material). Phosphorylated SpoOA also plays a key role in competence by first indirectly increasing the expression level of ComK, the master competence regulator, and then directly inhibiting its transcription by binding to its promoter (26
). Interestingly, levels of the comK
mRNA were also significantly increased in RNase J1-depleted strains (16
), while those of other important regulators were decreased in either RNase J1 (ComS and ComQ) or RNase Y (ComN) mutants. It is likely that the perturbation of the optimal balance between these different regulators accounts at least in part for the sporulation and competence deficiencies observed for the ΔrnjA
Under a light microscope, the RNase J1 mutant forms tight spirals interspersed by long chains, while the RNase Y mutant forms thin curvy chains of 2 to 3 cells. Defects in cell morphology were observed previously in cells depleted for RNases J1 and Y (2
), but they appear to be less severe than those observed here with the null mutants. The spiraled phenotype of the RNase J1 deletion mutant is very similar to that of cells lacking the actin-like proteins of the MreB family that are part of the bacterial cytoskeleton (27
). We looked at the mRNA levels of genes involved in cell morphology in strains depleted for RNase J1 or Y in the tiling array data set (16
). The most dramatic effect on the RNase J1 mutant was an 11-fold stabilization of the mreBH
operon mRNA (). Overexpression of MreBH (or MreB) has been shown to lead to a similar spiraled phenotype in B. subtilis
), and this may therefore be a good candidate for a contributor to the morphology phenotype of the RNase J1 mutant. The greatest effect of RNase Y depletion on mRNAs involved in cell morphology was a 12-fold increase in the expression level of the rodA
gene. Depletion of this protein leads to the production of spherical cells in B. subtilis
), but the effect of its overproduction is not known. Mutations in many other genes involved in cell wall biosynthesis also have effects on cell shape, and the expression of a great many of these genes was affected in strains depleted for RNase J1 or RNase Y (see Table S3 in the supplemental material). Indeed, this functional category was overrepresented in cells depleted for RNase Y, with about half of the mRNAs involved in cell wall biosynthesis and cell envelope stress having increased levels in cells deficient for this enzyme (16
). The effects on cell morphology observed in the knockout mutants may thus be far more complicated than just the effects on mreBH
alone. Consistent with this idea, the RNase J1 morphology defect was not corrected by the addition of 25 mM magnesium to cultures (data not shown), which was previously shown to rescue growth and morphology of a B. subtilis mreB
Effect of RNase J1 and RNase Y depletion on mRNAs involved in cell shape determination
We tested whether it was possible to knock out the RNase J1 and RNase Y genes in cells already lacking other components of the degradation machinery. There are currently two known pathways of RNA turnover in B. subtilis
: (i) endonucleolytic cleavage by RNase Y followed by degradation of the downstream fragment by the 5′-to-3′ exoribonuclease activity of RNase J1 and degradation of the upstream fragment by 3′-to-5′ exonucleases, principally PNPase, and (ii) removal of the protecting 5′-triphosphate group of primary transcripts by RppH, followed by degradation from the 5′ end by RNase J1. We were particularly interested in asking whether inhibiting the second pathway, by inactivating RppH, would be synthetic lethal in strains lacking the key endonuclease of the first pathway, RNase Y. Although the rppH rny
double mutant was viable, it did show significantly slower growth than strains lacking RNase Y alone. We suspect that there is a redundant RppH-like activity in B. subtilis
(see reference 32
) that provides sufficient RNA pyrophosphohydrolase activity to maintain the second pathway in the absence of RppH. Strains lacking both RppH and RNase J1 had doubling times similar to those of the single rnjA
mutants. Together, these data are consistent with the idea that RppH acts in the same degradation pathway as RNase J1 but in a different pathway from RNase Y.
We were also able to inactivate RNase J1 in strains already lacking its paralog RNase J2. We have previously shown that these two enzymes form a complex and that RNase J2 has 5′-to-3′ exoribonuclease activity that is about 2 orders of magnitude lower than that of RNase J1, but our ability to make rnjA rnjB
double mutants shows that the viability of RNase J1 deletion strains is not ensured by the residual activity of RNase J2. Indeed, if anything, the rnjA rnjB
double mutant grows slightly faster than the rnjA
mutant alone. We have previously seen that mutation of the RNase J2 catalytic histidine motif (HGHDEN) to match that of RNase J1 (HGHEDH) did not increase exoribonuclease activity as expected but rather abolished it completely (33
), raising questions as to the role of RNase J2. We are currently leaning toward the idea that its principal role is as a scaffold to regulate or stabilize the RNase J1 subunit of the complex. In the absence of RNase J1, the continued presence of RNase J2 may become slightly inhibitory.
We managed to make a pnp rny
double mutant that grew much slower than the strain lacking RNase Y alone. This also suggests that PNPase plays an important role in the cell that is independent of its role in the RNase Y-dependent degradation pathway. Although there is no evidence so far of direct degradation from the 3′ end in B. subtilis
(similar to that performed by the exosome in eukaryotes), this remains a possibility to be considered. Furthermore, PNPase has also been shown to play a role in B. subtilis
DNA damage repair by degrading single-stranded DNA ends (34
We failed to make either pnp rnjA or rnjA rny double mutants in multiple attempts to either transform pnp mutants or simultaneously transform two mutations into wild-type cells. However, given the extremely low competence levels of the PNPase mutant or the low probability of successfully transforming two markers into the same cell, these results are suggestive only, and it is not possible to state definitively that these combinations are synthetic lethal.