ROD-SHAPING PROTEINS · Discovery and characterization of factors coupling the cytoskeleton to cell wall biogenesis in Bacillus subtilis

Periodic Report Summary - ROD-SHAPING PROTEINS (Discovery and characterization of factors coupling the cytoskeleton to cell wall biogenesis in Bacillus subtilis)

Project part A: Discovery and characterisation of factors coupling the cytoskeleton to cell wall biogenesis in Bacillus subtilis. The bacterial cell wall (CW) is a dynamic structure, responsible of the maintenance of the cell shape. It also plays an essential role during cell division. The spatiotemporal coordination between cell wall synthesis and hydrolysis is required to ensure cell proliferation and viability. Among the protein factors known to be involved in determining cell shape are the enzymes forming the CW biosynthetic complex, and the mreB homologues, which constitute the actin cytoskeleton in bacteria. Bacillus subtilis contains three mreB homologues that polymerise into filaments that follow a helical path underneath the cell membrane. Bacterial cytoskeletal proteins seem to coordinate the synthesis of CW, by interacting with many other factors, some of which still remain unknown. In this work, we have identified an uncharacterized putative ABC transporter in B. subtilis homologue of the division protein complex FtsEX in E. coli. We have observed that in the absence of this transporter cells become filamentous and show defects in CW synthesis. Our data indicate that the YknWXYZ transporter localises to the cell septum, interacting mainly with late division proteins. We have discovered that in the absence of this transporter, the onset of sporulation is delayed, and as a consequence the null mutant shows a severe defect in sporulation. Finally, we tested whether the defects in CW synthesis were due to a defect in the synthesis and or secretion of CW associated enzymes. The autolytic enzyme profile shown that a null mutant in this ABC transporter shows a decrease in the level of CW hydrolases associated with the CW. Project part B: Cell shape and division determinants in the absence of cell wall in Bacillus subtilis. The cell wall is a crucial protective layer that surrounds virtually all bacteria. However, some bacteria have developed mechanisms to switch between walled and wall-deficient states. These cell wall-deficient bacteria, known as L-forms, have been isolated from many different species. They represent an interesting model of study considering their potential involvement in antibiotic resistance and persistent infections. But despite decades of study the mechanisms underlying cell division in the absence of the cell wall are poorly understood. To identify genes involved in the transition from the walled to the L-form state, and to unravel the mechanisms underlying L-form proliferation, we have generated a Bacillus subtilis strain that can be induced to generate L-forms in just a few hours. We have implemented new time-lapse microscopy methods that allow us to follow the transition from rods to L-forms, and the localisation of several key cell division proteins during the process. In previous work, we showed that L-form division does not require FtsZ or any residual peptidoglycan synthesis. In fact, many essential proteins for the cell walled form have turned out to be dispensable for L-form survival and proliferation. Therefore, we conducted a genetic screen using random transposon mutagenesis to identify genes exclusively essential for L-form division. Our results have shown the crucial role of membrane lipid composition and the interplay of several stress regulons in L-form biology. The socioeconomic interest in the study of L-forms biology comes from the huge need on the discovery of new antibiotics. On daily bases, we assist to the appearance of new pathogenic bacterial strains with increased levels of antibiotic resistance. Furthermore, our studies in L-form biology would be of clinic relevance if we can prove that they are responsible for chronic infections and persistent diseases. The study of L-form proliferation mechanism will be of great importance to the discovery of new cellular targets and the development of novel agents in the fight against infectious diseases.

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