(i) Construction of gfp reporter plasmids
To create the PmbcS-gfp reporter fusion, a 236-bp
fragment upstream of the translation initiation codon of SAUSA300_2542
(i.e. , mbcS ) was amplified from S. aureus LAC with
Q5 DNA polymerase (NEB) using oAP41 and oAP42 as primers. In parallel,
the promoterless gfp reporter plasmid pMRSI (Waters et al., 2016)
was amplified and linearized using oAP39 and oAP40 as primers. PCR
products were treated with DpnI (NEB), subjected to Gibson Assembly, and
inserted into E. coli NEB 5α (NEB). The resulting plasmid was
named pAP4 (mbcSp+-gfp ). Plasmids pAP10
(mbcS1p-gfp ), and pAP11 (mbcS2p-gfp ), containing the samembcS regulatory region but with point mutations identified during
suppressor analysis, were constructed in a similar way by cloning the
fragments into pMRS1.