The Requirement for Pneumococcal MreC and MreD Is Relieved by Inactivation of the Gene Encoding PBP1a

Abstract

ABSTRACT MreC and MreD, along with the actin homologue MreB, are required to maintain the shape of rod-shaped bacteria. The depletion of MreCD in rod-shaped bacteria leads to the formation of spherical cells and the accumulation of suppressor mutations. Ovococcus bacteria, such as Streptococcus pneumoniae , lack MreB homologues, and the functions of the S. pneumoniae MreCD (MreCD Spn ) proteins are unknown. mreCD are located upstream from the pcsB cell division gene in most Streptococcus species, but we found that mreCD and pcsB are transcribed independently. Similarly to rod-shaped bacteria, we show that mreCD are essential in the virulent serotype 2 D39 strain of S. pneumoniae , and the depletion of MreCD results in cell rounding and lysis. In contrast, laboratory strain R6 contains suppressors that allow the growth of Δ mreCD mutants, and bypass suppressors accumulate in D39 Δ mreCD mutants. One class of suppressors eliminates the function of class A penicillin binding protein 1a (PBP1a). Unencapsulated Δ pbp1a D39 mutants have smaller diameters than their pbp1a + parent or Δ pbp2a and Δ pbp1b mutants, which lack other class A PBPs and do not show the suppression of Δ mreCD mutations. Suppressed Δ mreCD Δ pbp1a double mutants form aberrantly shaped cells, some with misplaced peptidoglycan (PG) biosynthesis compared to that of single Δ pbp1a mutants. Quantitative Western blotting showed that MreC Spn is abundant (≈8,500 dimers per cell), and immunofluorescent microscopy (IFM) located MreCD Spn to the equators and septa of dividing cells, similarly to the PBPs and PG pentapeptides indicative of PG synthesis. These combined results are consistent with a model in which MreCD Spn direct peripheral PG synthesis and control PBP1a localization or activity.