ClpX and ClpP are essential for the efficient acquisition of genes specifying type IA and IB restriction systems

Abstract

SummaryEfficient acquisition of genes that encode a restriction and modification (R-M) system with specificities different from any already present in the recipient bacterium requires the sequential production of the new modification enzyme followed by the restriction activity in order that the chromosome of the recipient bacterium is protected against attack by the restriction endonuclease. We show that ClpX and ClpP, the components of ClpXP protease, are necessary for the efficient transmission of the genes encoding EcoKI and EcoAI, representatives of two families of type I R-M systems, thus implicating ClpXP in the modulation of restriction activity. Loss of ClpX imposed a bigger barrier than loss of ClpP, consistent with a dual role for ClpX, possibly as a chaperone and as a component of the ClpXP protease. Transmission of genes specifying EcoKI was more dependent on ClpX and ClpP than transmission of the genes for EcoAI. Sensitivity to absence of the protease was also influenced by the mode of gene transfer; conjugative transfer and transformation were more dependent on ClpXP than transduction. In the absence of either ClpX or ClpP transfer of the EcoKI genes by P1-mediated transduction was impaired, transfer of the EcoAI genes was not.

Immigration control of DNA in bacteria: self versus non-self

“…These experiments implicated the protease ClpXP (Makovets et al, 1998), which comprises two components, ClpX and ClpP. In the absence of either ClpX or ClpP, acquisition of hsd genes specifying either EcoKI (type IA) or EcoAI (type IB) led to the death of modification-deficient recipients (Makovets et al, 1998). Together, ClpX and ClpP form a large, but hollow, complex (see Gottesman, 1999) ; ClpX serves to recognize and unfold its substrate so that the polypeptide can be transported to the chamber within the complex where it becomes the target for degradation by ClpP.…”

“…It has been shown that the bacterial chromosome becomes susceptible to restriction as cell growth dilutes the modification enzyme (Handa et al, 2000). However, while the loss of genes that specify some simple R-M systems leads to cell death, the loss of genes that specify other, more complex, R-M systems causes no detectable viability problem (O'Neill et al, 1997 ;Kulik & Bickle, 1996 ;Makovets et al, 1998). In this by EcoKI and thus forms plaques with reduced efficiency (e.o.p.…”

“…criptional regulation would explain why E. coli can cope with the acquisition of type II systems but is sensitive to their loss ; following gene loss, transcriptional control is no longer possible and residual endonuclease will attack unmodified targets within the bacterial chromosome (Handa et al, 2000). The loss of genes encoding type I R-M systems is not associated with any loss of viability (O'Neill et al, 1997 ;Makovets et al, 1998). This may reflect loss of restriction activity by the dissociation of the R subunits of EcoKI to yield a complex (M # S " ) with only modification activity.…”

“…Energy-dependent proteases are known to play important regulatory roles in bacteria (see Gottesman, 1999), therefore mutants deficient in proteases were screened to check whether they might identify the unknown function. These experiments implicated the protease ClpXP (Makovets et al, 1998), which comprises two components, ClpX and ClpP. In the absence of either ClpX or ClpP, acquisition of hsd genes specifying either EcoKI (type IA) or EcoAI (type IB) led to the death of modification-deficient recipients (Makovets et al, 1998).…”

“…No such susceptibility has been detected for strains specifying type I R-M systems (Kulik & Bickle, 1996 ;O'Neill et al, 1997) ; modulation of the restriction activity of type I R-M systems is extraordinarily effective in the protection of the bacterial chromosome. When the genes encoding EcoKI are deleted no viability problem is detected, even in the absence of the ClpXP protease (O'Neill et al, 1997 ;Makovets et al, 1998), and when the genes are replaced with those specifying another system, ClpXP alleviates restriction and permits survival. Furthermore, in contradiction to classical expectations, the presence of a mutation that destroys the modification activity of the EcoKI complex is not lethal : the restriction-proficient cells survive because ClpXP controls the endonuclease activity of the modificationdeficient complex (Makovets et al, 1999 ;Doronina & Murray, 2001).…”