We have shown that the yafN
gene of the dinB-yafN-yafO-yafP
) is essential for viability only in the presence of a functional yafO
gene (Fig. ), providing strong evidence that these two genes constitute the antitoxin and toxin genes, respectively, of a type 2 TA pair. Whereas previous in vivo demonstrations of type 2 TA systems have used the method of separate cloning of each gene into differentially inducible plasmids and showing that the toxin induces cell stasis when solely expressed, but not when the antitoxin is also expressed (1
), we used an alternative genetic approach that allows us to rule out possible effects specific to overexpression of either the toxin or the antitoxin. We can therefore conclude that YafO exerts its toxic effect, and YafN can quell that effect, when each gene is expressed at normal levels from its native promoter, in single copy in its normal chromosomal position.
Typically, chromosomal TA systems consist of two genes in an operon, the toxin and the antitoxin. Although there is precedent for a TA system with a third gene element, mazEFG
), we found that yafP
, the third yaf
gene in the operon, is not an integral component of either the toxin or the antitoxin (Fig. ). This conclusion does not preclude the possibility that YafP might play a regulatory role in the YafNO TA system which we have not detected, as mazG
does for mazEF
The possible function of a TA system controlled by the SOS response is an interesting problem. We found that unlike dinB
), the first gene in the operon (43
), the yafNOP
genes do not contribute to stress-induced mutagenesis significantly (Fig. ), a process that requires SOS-induced levels of dinB
, but not SOS-induced levels of any other SOS-controlled component (17
). Other SOS-controlled genes such as recA
) and ruvA
) are required for stress-induced Lac mutagenesis, we now appreciate, at their constitutive levels of expression, not at induced levels (17
). The results presented here rule out a requirement for YafNOP in stress-induced mutagenesis even at their constitutive expression levels. Previously, YafNOP had little effect on spontaneous generation-dependent mutagenesis in nonstressed growing cells (43
We also did not detect effects of yafNOP
on survival of cells following various SOS-inducing treatments, for nonlysogens (Fig. ) and also for cells harboring a wild-type lambda prophage, which is induced leading to cell lysis when the SOS response is activated (Fig. ). Finally, although we could recapitulate the previous results of Pennington and Rosenberg, showing that many spontaneously SOS-induced green fluorescent cells (bearing an SOS-controlled chromosomal gfp
reporter gene) are apparently viable but unable to form colonies when recovered by FACS (47
), we found that yafNOP
is not responsible for their senescence-like state (Fig. ).
What might be the function of an SOS-controlled TA system? For TA systems, the toxic effects ensue when the operon's expression is decreased, so we would expect possible effects of YafO to be manifested as cells recover from an SOS response and return to normal after DNA repair, even though these genes are expressed mid-range in the SOS response (11
). Perhaps YafO induces a transient cell stasis upon recovery. This might function to extend the cell cycle checkpoint caused by SOS-induced expression of the SulA inhibitor of cell division (16
). Although we did not find effects of YafNO on cells after SOS induction, we cannot rule out the possibility of an important role in SOS recovery that our assays might not have detected. Alternatively or in addition, dinB
, and presumably the rest of its operon, is also upregulated slightly by the RpoS general stress response (33
). Perhaps YafO plays a role in promoting cell stasis upon recovery from the RpoS response. Additionally, although the yafNO
genes are transcribed from the upstream SOS-controlled (and RpoS-controlled) dinB
), there is in vitro evidence that an additional promoter may exist immediately upstream of yafN
, possibly creating an SOS (or RpoS)-independent yafNOP
). If so, yafNOP
might act outside the contexts of the SOS (or RpoS) response.
A recent report shows that DinB and also another SOS-inducible DNA polymerase, Pol V, interact directly with the NusA transcription and antitermination factor (10
). The authors suggest that NusA might direct the translesion DNA synthesis activity of DinB to sites of active transcription. Similarly, we can imagine that DinB might affect NusA-dependent transcription termination, which might then provide another level of SOS control of gene expression (negative or positive). Perhaps YafNO or YafP functions in such a process, and perhaps the toxic effect of YafO is related to a transcription-termination-specific effect.
While the manuscript was being prepared, another group reported that the YafO protein is an RNase (58
). Their results support our conclusions and provide a mechanism for the toxic action of the YafO toxin. Important next steps toward understanding the yafNO
TA system include defining when as well as on what targets the toxin acts.