Mura, Andrea
Characterization of ftsA conditional lethal mutants shows that FtsA is required at early and late stages of cell division in Streptococcus pneumoniae [Tesi di dottorato]

FtsA is an essential cell division protein fairly conserved among Eubacteria. It is an actin-like protein that structurally differs from actin because it lacks one of the four subdomains that is replaced by an additional one located elsewhere in the structure. FtsA localizes early at the cell division site, together or immediately after FtsZ, where it is needed both to tether FtsZ to the membrane and also to recruit to midcell the other cell division proteins. In agreement with this, FtsA interacts, at least, with itself, FtsZ, ZapA and the septal PBP, FtsI. Despite the advances in understanding cell division in Streptococcus pneumoniae, little is known about the early stages of the process. Here I present the characterization of S. pneumoniae ftsA thermosensitive (Ts) mutants, obtained by error-prone random PCR, allelic replacement and subsequent screening for ability to grow at 28°C (permissive temperature) but not at 40°C (non permissive temperature). Out of 8000 transformants screened, three, named A19TS, A20TS and A21TS, were identified and shown to carry mutated ftsA alleles and express the corresponding mutated protein. The mutations mapped in different domains of the FtsA structure, thought to be involved in FtsA polymerization, interactions with FtsZ and/or with other cell division proteins. Temperature shifting experiments, carried out in rich medium, showed that in contrast to the Rx1 wild type, that grew well at both 28°C and 40°C, the FtsA Ts mutants stopped growing and started lysing about 60 to 90 minutes after shifting to the non permissive temperature, in a mutation specific manner. The lysis was greatly reduced when the shift to 40°C was performed in a chemically-defined medium or in a lytA- background. Detection of FtsAwt and FtsATS, in crude extracts, derived from cultures after shifting at 28°C and 40°C, revealed that all the FtsA Ts proteins are stable at both temperatures, indicating that protein degradation is not the cause of the Ts phenotype. Protein-protein interaction assays showed that all three FtsATS proteins lost their ability to self-interact but retained their ability to interact with the FtsAWT protein and FtsZ. Consistently, GFP-FtsAWT, expressed under the control of the PZn inducible promoter at the ectopic bga locus of the S. pneumoniae chromosome, fully complemented the Ts phenotype of two, A20TS and A21TS, mutants. Interestingly, the Ts phenotype of the A19TS mutant was only partially complemented by GFP-FtsAWT, which localized as multiple rings in the partially complemented elongated A19TS+PZn-GFP-FtsAWT cells, suggesting the FtsA19TS may be dominant over FtsAWT. Localization studies, using fluorescence microscopy, showed that CFP-FtsZ, DivIVA-GFP, GFP-StkP, GFP-GpsB and GFP-PBP2x exhibited proper localization in the Rx1WT at 28°C and 40°C, while in the FtsA Ts mutants these cell division markers were localized properly at 28°C but retained or lost their localization upon shifting to 40°C depending on the specific mutant. Notably, in all FtsA Ts mutants, FtsZ lost its septal localization after shifting to 40°C. The results are consistent with what was observed in a zinc dependent ftsA conditional lethal S. pneumoniae mutant, where the only source of FtsA was GFP-FtsA expressed under the control of the PZn inducible promoter, indicating that S. pneumoniae FtsA is required for efficient midcell localization of FtsZ and supporting the notion that in Gram-positives, which lack ZipA, FtsA may be absolutely necessary for anchoring FtsZ to the membrane. Together these results confirm that FtsA is essential in S. pneumoniae and further validate it as a target to develop novel inhibitors active on septation. Moreover, the data suggest that an early block in cell division in S. pneumoniae impairs both growth and division, supporting a model in which a single machinery, containing both complexes, one for peripheral elongation and one for septum formation, directed by FtsZ, and likely by FtsA, is present at the midcell of oval-shaped cocci.

In relazione con
BIO/19 - Microbiologia generale

Tesi di dottorato. | Lingua: | Paese: | BID: TD18027175