Changes in relative transcript amounts caused by ?pbp1a, double ?pbp1a mltG(?488bp), and triple ?pbp1a mltG(?488bp) ?pbp2b mutations in Streptococcus pneumoniae

In ellipsoid-shaped ovococcus bacteria, such as the respiratory pathogen Streptococcus pneumoniae (pneumococcus), side-wall (peripheral) peptidoglycan (PG) synthesis emanates from midcells and is catalyzed by the essential class B penicillin-binding protein PBP2b transpeptidase (TP). We report that mutations that inactivate the pneumococcal YceG-domain protein, Spd_1346 (renamed MltG), remove the requirement for PBP2b. ?mltG mutants in unencapsulated strains accumulate inactivation mutations of class A PBP1a, which possesses TP and transglycosylase (TG) activities. To gain insight into the mechanism of synthetic-viable genetic relationships involving ?pbp1a, we performed RNA-Seq analyses of ?pbp1a, ?pbp1a mltG(?488bp), and ?pbp1a mltG(?488bp) ?pbp2b mutants compared to the isogenic unencapsulated parent strain. Unexpectedly, the ?pbp1a deletion causes an almost exclusive induction in relative transcript amounts of genes that are in the WalRK TCS regulon, which includes established and putative PG hydrolases and division proteins of unknown functions. Overall design: RNA was prepared from cultures of strains IU6741 (?pbp1a), IU8553 (?pbp1a mltG(?488bp)), IU8567 (?pbp1a mltG(?488bp) ?pbp2b) and IU1824 (pbp1a+ mltG+ pbp2b+ parent) grown exponentially in BHI media at 37ºC to OD620 ˜0.15 to 0.2. Three independent biological replicates of RNA were prepared for each strain or condition. cDNA libraries were sequenced and the sequencing results were used as the data base for differential expression analysis.