Cell-shape regulation in Streptococcus pneumoniae : EloR/KhpA, a new regulatory pathway administering cellelongation

Abstract

The oval shaped Streptococcus pneumoniae utilizes both septal and peripheral peptidoglycan (PG) synthesis in order to maintain its shape. The protein complexes responsible for synthesizing PG are called the elongasome and the divisome, and as the names imply the elongasome synthesizes peripheral PG while the divisome synthesizes the PG responsible for dividing the cell into two [1]. The core of the elongasome and the divisome is the essential class B penicillin binding proteins (PBPs) 2b and 2x, respectively. PBP2b and PBP2x are transpeptidases creating peptide cross-links in PG between glycan strands [1, 2]. They work alongside the glycosyltransferases RodA and FtsW, respectively, to incorporate new PG into the existing PG sacculus [3-5]. The elongasome and divisome activities must be precisely coordinated throughout the cell cycle, but detailed knowledge about the control systems the cells possess to manage these PG machineries are lacking. In the current work, I present a pathway for regulation of elongation that has emerged recently: the EloR/KhpA complex. EloR and KhpA both contain RNA binding domains commonly seen in proteins involved in transcriptional or post-transcriptional regulation [6-8]. We and others have found that in order to survive the loss of pbp2b, S. pneumoniae can create suppressor mutations in the genes encoding EloR, KhpA and MltG rendering the protein products inactive [9, 10]. Our results show that EloR and KhpA work as a complex controlling cell elongation, most likely in a pathway including StkP, a Ser/Thr kinase known to have a regulatory role in cell division [11-15], and the essential lytic transglycosylase MltG.

In paper I we show that the loss of EloR resulted in shorter cells in the laboratory strain R6. We confirmed that EloR is phosphorylated by StkP and conclude that it is likely that phosphorylation of EloR leads to release of bound RNA, stimulating elongation. We speculate that the reason PBP2b and RodA are essential in a wild type background is that these proteins are required in cells where the muralytic activity of MltG has a normal function. The reason pbp2b and rodA can be deleted in an DeloR mutant may be that the MltG activity is reduced without EloR present.

In paper II we show that EloR interacts directly with a small RNA binding protein called KhpA. Using 3D modelling and site directed mutagenesis we identified the interaction surface between the two proteins and two amino acid residues important for this interaction. We could use this information to investigate how cells reacted to the loss of complex formation between the two proteins. A study by Zheng et al., 2017 showed that a khpA deletion mutant phenocopies an eloR deletion mutant [9]. We demonstrate in paper II that EloR and KhpA is one functional unit, and if the direct interaction between EloR and KhpA is broken, the cells behave like a DeloR or DkhpA mutant, i.e. PBP2b/RodA become redundant. We also show that KhpA depends upon EloR interaction to reach its midcell localization.

In addition to two RNA binding domains (KH-II and R3H) at the C-terminal end, EloR has a Jag domain with unknown function at its N-terminus. In the final manuscript, paper III, we set out to unravel the function of the Jag domain. We found that the Jag domain is critical for midcell localization of EloR. Furthermore, by screening for protein-protein interactions between EloR and other elongasome proteins, the Jag domain was found to interact with the cytoplasmic domain of the lytic transglycosylase MltG. We hypothesize that the EloR/KhpA complex is recruited to midcell through the Jag-MltG interaction where it somehow controls the muralytic activity of MltG, either through protein – protein interaction or by RNA binding.

Den ovale bakterien Streptococcus pneumoniae benytter både septal og perifer peptidoglykansyntese for å opprettholde celleformen. Proteinkompleksene som er ansvarlige for å syntetisere peptidoglykan (PG) kalles elongasomet og divisomet, og som navnene tilsier syntetiserer elongasomet perifert PG mens divisomet syntetiserer PG som er ansvarlig for å dele cellen i to [1]. Kjernevirksomheten i elongasomet og divisomet utføres av de essensielle klasse B penicillinbindende proteinene (PBP) 2b og 2x. PBP2b og PBP2x er transpeptidaser som danner peptid-kryssbindinger mellom glykantrådene i PG [1, 2]. De jobber sammen med glykosyltransferasene RodA og FtsW for å inkorporere ny PG i det eksisterende PG nettverket [3-5]. Elongasom- og divisom-aktivitetene må være nøyaktig koordinerte gjennom hele cellesyklusen, men detaljert kunnskap om hvordan cellene kontrollerer disse PG-maskineriene mangler. I dette prosjektet presenterer jeg en vei for regulering av elongering som nylig har blitt oppdaget: EloR/KhpA-komplekset. EloR og KhpA inneholder begge RNA-bindende domener som ofte finnes hos proteiner involvert i transkripsjonell eller post-transkripsjonell regulering [6-8]. Vi og andre har oppdaget at for å overleve tapet av pbp2b kan S. pneumoniae skape suppressormutasjoner i genene som koder for EloR, KhpA og MltG, slik at proteinproduktene blir inaktive [9, 10]. Resultatene våre viser at EloR og KhpA fungerer som et kompleks som kontrollerer celle-elongering sammen med StkP, en Ser/Thr-kinase kjent for å ha en regulerende rolle i celledeling [11-15], og den essensielle lytiske transglykosylasen MltG.