399C\_\_965913A G965913ZMO_RS03600WP_011240805.1ATP-dependent **Clp** protease proteolytic subunitS26G\_\_1612573A G1612573\_\_\_\_\_\_\_\_ZMO_RS07065..ZMO_RS07070P_ZM4\*SUBST.P_ER79apLOCUS_TAGPROTEIN_IDPRODUCTP_CHANGEINTERGENIC_REGION78020G C78020ZMO_RS00360WP_012817506.1Bifunctional (p)ppGpp synthetase/guanosine-3\',5\'-bis(diphosphate) 3\'-pyrophosphohydrolase, **SpoT/RelA**R317G\_\_965913A G965913ZMO_RS04250WP_011240805.1ATP-dependent **Clp** protease proteolytic subunitS26G\_\_971444C T971444ZMO_RS09165\_\_IS5/IS1182 family transposase/ pseudogene\_\_\_\_1438735T C1438735ZMO_RS06375WP_011241206.1ATP-dependent chaperone **ClpB**V108A\_\_1612573A G1612573\_\_\_\_\_\_\_\_ZMO_RS07065..ZMO_RS07070

In contrast, ER79ap chromosome possessed three non-synonymous substitutions: the first mutation (S26 G) was located in the gene encoding ClpP and was exactly the same mutation and position as that present in ER79ag. The first substitution occurred in the gene encoding the ClpB chaperone (V108A), a protein involved in suppression and reversal of protein aggregation in conjunction with other heat-shock proteins. The last substitution also fell within the *spoT/relA* gene (R317 G), but in the synthesis/degradation region.

3.6. Effect of the mutant alleles on ethanol tolerance {#sec0085}
------------------------------------------------------

Taking in consideration that strain ER79ap has the best performance when confronting exogenous ethanol, we made an evaluation of the contribution of each one of the mutant alleles to ethanol tolerance. To do this, *clpP*, *clpB* and *spo*T/*rel*A mutant alleles of ER79ap were amplified by PCR, cloned in a replicative vector (pBBR1-MCS3) and introduced to the wild-type strain by conjugation. As a control, the empty vector was also introduced into ZM4. Transconjugant strains were evaluated with inocula at LOD and in culture media with 70 and 90 g/L of exogenous ethanol ([Fig. 4](#fig0020){ref-type="fig"}, [Fig. 5](#fig0025){ref-type="fig"}).Fig. 4Fold change of ER79ap, and comple