## 4 Materials and Methods
### Construction of Mutant Strains  
To obtain a null mutant in the _cdaA_ gene of _B. subtilis_, a 317-bp internal fragment, encompassing from nucleotides +91 to +407, of the gene ORF, was amplified with the specific nucleotide primers 5\({}^{\prime}\)-CGAAAGCTTGTGATACGCGGCACGAAGC-3\({}^{\prime}\) (forward) and \begin{table}
\begin{tabular}{c c c} \hline \hline
**Strain** & **Genotype or Description 1** & **Reference or Source** \\ \hline YB955 & _hisC952 metB5 LeuC427 xin-1 Sp\({}^{\textit{SENS}}\)_ & [18] \\ \hline PERM1647 & YB955 _disA:x.ery_. Em\({}^{\text{R}}\) & This study \\ \hline PERM1665 & YB955 _cdaA:cat disA:x._. [PERSON]\({}^{\text{R}}\) Cm\({}^{\text{R}}\) & PERM1647 \(\rightarrow\) PERM1663 b  \\ \hline PERM1687 & YB955 _cdaA:cat_. Cm\({}^{\text{R}}\) & This study \\ \hline PERM1729 & YB955 _disA:x:ary ampE::Phs-disA_. [PERSON]\({}^{\text{R}}\) Sp\({}^{\text{R}}\) & PERM1647 \(\rightarrow\) PERM1728 \\ \hline PERM1730 & YB955 _cdaA:cat disA:x armyE::Phs-disA_. [PERSON]\({}^{\text{R}}\) Cm\({}^{\text{R}}\) Sp\({}^{\text{R}}\) & PERM1665 \(\rightarrow\) PERM1728 \\ \hline PERM1853 & YB955 _cdaA:cat ampE:Phs-cldaA_. Cm\({}^{\text{R}}\) Sp\({}^{\text{R}}\) & PERM1687 \(\rightarrow\) PERM1845 \\ \hline PERM1854 & YB955 _cdaA:cat disA:x armyE:Phs-cldaA_. Cm\({}^{\text{R}}\) [PERSON]\({}^{\text{R}}\) Sp\({}^{\text{R}}\) & PERM1665 \(\rightarrow\) PERM1845 \\ \hline \hline \end{tabular}
\begin{tabular}{c c} \hline \hline
**Plasmids** & \\ \hline PMUTIN-4 & \
\begin{tabular}{c} Integrative vector for _B. subtilis_. Disrupts the gene after insertion and creates \\ a fusion of the cloned fragment with the _lacZ_ gene. Ery\({}^{\text{R}}\) \\ \end{tabular} & [23] \\ \hline pMUTIN-4-cat &
\begin{tabular}{c} Integrative vector for _B. subtilis_. Disrupts the gene after insertion and creates \\ a fusion of the cloned fragment with the _lacZ_ gene. Cm\({}^{\text{R}}\) \\ \end{tabular} & [41] \\ \hline pDR111 & pHyperspank integrative vector, Amp\({}^{\text{R}}\) Sp\({}^{\text{R}}\) & David Rudner Laboratory \\ \hline PERM732 & pMUTIN-4 plasmid containing an internal fragment of the _B. subtilis disA_ & This study \\ \hline PERM1663 & pMUTIN-4-cat plus an internal fragment of the _B. subtilis daA_ gene. Cm\({}^{\text{R}}\) & This study \\ \hline PERM1845 & pHyperspank-_cdaA_ Integrative vector with _cdaA_ under control of an IPTG & This study \\ \hline PERM1728 & pHyperspank-_cdaA_ Integrative vector with _disA_ under control of an IPTG & This study \\ \hline \hline \end{tabular}
\end{table} 
Table 2: _Bacillus subtilis_ strains and plasmids used in this study. \(5^{\prime}\)-GCGGATCCCGCTCAATGGTCAGGC-\(3^{\prime}\) (reverse) (restriction sites HindIII and BamHI, respectively, underlined) and high-fidelity Vent DNA polymerase (New England BioLabs; Ipswich, MA, USA). This fragment was cloned into the pJET1.2/blunt vector (Thermo Scientific, Waltham, MA, USA). Subsequently, the _cdaA_ fragment was ligated into the HindIII/BamHI sites of the vector pMUTIN-4-cat [41] and the resulting construct pPERM1663 (Table 2) was transformed and replicated in _E. coli_ DH5\(\alpha\) using standard techniques [44]. To obtain a genetic construct for disrupting _disA_, an internal 309-bp internal fragment, encompassing nucleotide positions +274 to +583, of the gene ORF, was amplified with the specific nucleotide primers 5\({}^{\prime}\)-CGGAAATCCCAGCTGATGC-\(3^{\prime}\) (forward) and 5\({}^{\prime}\)-CCGAGACAGACAGACATCACCTG-\(3^{\prime}\) (reverse) (restriction sites EcoRI and BamHI, respectively, underlined) and was cloned in the integrative vector pMUTIN-4 following the strategy for disrupting _cdaA_ above described resulting in construct pPERM732 [14]. To obtain the single _disA_ or _cdaA_ mutants, competent cells of strain _B. subtilis_ YB955, prepared according to the protocol of [PERSON] et al. 1972 [45] were, respectively, transformed with the constructs pPERM1663 and pPERM732 (Table 2). These procedures generated _B.