7 kDa and the pI is 9.7. Both are predicted to have a short cytoplasmic tail adjacent to a single transmembrane region,
followed by the extracellular part containing the LCP domain, extending from aa 86 to 234 in SA0908 and from aa 90 to 236 in SA2103. The transcriptional start sites (TSS) of sa0908 and sa2103 were identified by primer extension and were 99 and 44 bp upstream of the start codons, Protein Tyrosine Kinase inhibitor respectively, and were preceded by putative promoter elements (Fig. 1a and b). Northern blots revealed that sa0908 and sa0907 were cotranscribed on a single mRNA of ∼2000 nt in wild-type MSSA1112. The deletion of sa0908 in strain RH53 resulted in a shorter, ∼800 bp, sa0907 transcript (Fig. 1c). Two transcripts hybridized to the sa2103 DIG-probe, an ∼1100 bp transcript, which initiated at the TSS, and a larger transcript of ∼2000 bp, representing a bicistronic sa2104–sa2103 transcript, which decreased in size to ∼1000 bp in the Δsa2103 mutant PS47 (Fig. 1d). Promoter–luciferase fusion constructs were used to compare the relative expression
RG7420 manufacturer levels of msrR, sa0908 and sa2103 over growth (Fig. 1D). The expression of all three genes peaked during exponential growth when cells were dividing rapidly, and then decreased as cultures entered the stationary phase. The relative expression levels of msrR were much higher than those of sa0908 and sa2103. To obtain a comprehensive overview of the functions of LCP genes, we created all possible combinations of double mutants: RH72 (Δsa0908/ΔmsrR), PS60 (Δsa2103/ΔmsrR) and PS110 (Δsa2103/Δsa0908), and a triple mutant PS111 (Δsa2103/Δsa0908/ΔmsrR). To further investigate the roles of individual LCP proteins, we complemented the triple mutant with msrR, sa0908 or sa2103 in trans. The deletion of msrR was previously shown to have no effect on
the growth rate (Hubscher et al., 2009). The deletion of sa0908 or sa2103 also had only a small, but complementable effect on growth in RH53 (Δsa0908) and PS47 (Δsa2103). The deletion of a second LCP protein had negligible further effects on the growth characteristics (data not shown). The growth of the triple mutant PS111 was severely Coproporphyrinogen III oxidase retarded, with the growth rate decreasing from 1.39 to 0.95 h−1 at 37 °C (Fig. 2a). This growth defect was further exacerbated at 42 °C (Fig. 2b). The ability of the three proteins to complement this growth defect differed, especially at the elevated temperature of 42 °C: MsrR restored growth almost to the wild-type level, followed by SA0908, which compensated growth to up to ∼70% of the wild type OD600 nm after 7 h, while SA2103 had the lowest effect (Fig. 2b). LCP mutants were analysed by TEM and the cell sizes of a minimum of 100 cells per strain were measured and expressed as the mean±SD. In single mutants, enlarged cells and irregular septa were observed in the msrR mutant (JH100 ∅1.33±0.16 μm) as reported previously (Hubscher et al., 2009). The cells of sa0908 (RH53 ∅1.04±0.07 μm) and sa2103 (PS47 ∅1.