Final Report Summary - REGENERATE (Molecular biology of cell wall regeneration in L-form bacteria) Regeneration of walled cells from L-formsThe bacterial cell wall (CW) is a highly conserved structure mainly composed of peptidoglycan (PG). L-forms are cell wall-deficient/defective bacteria (CWDB) that are nevertheless able to proliferate under certain conditions. It has been suggested that most if not all bacterial species can be converted to L-forms, and the most common methods of doing so involve treatments with agents including CW-inhibiting antibiotics, or enzymes that degrade PG. The lack of a wild type CW makes these cells pleomorphic, osmotically sensitive and resistant to antibiotics and enzymes that digest the PG. L-forms are probably important in a number of diseases, particularly those involving chronic or relapsing infections. However, they are also of great interest as a research tool for better understanding the structure, organization and function of bacterial cells. Although the difficulties of reproducibly generating and propagating L-forms has rendered them relatively recalcitrant to experimentation, the Errington lab recently developed genetic methods and mutant strains that enabled L-forms to be rapidly and easily generated from laboratory strains of Bacillus subtilis. These methods have been used to study the generation (escape step) and proliferation processes of L-form cells.Bacterial PG, made of glycan strands cross-linked by short peptides, enables bacteria to resist the intracellular turgor pushing out on the cell membrane, thus preserving cell integrity, and also provides the bacterium with a well-defined shape. The PG sacculus is thought to grow by enlargement of an existing structure, by a hypothetical mechanism known as 'hereditary propagation theory'. Based on this theory the PG polymerization reactions would require a pre-existing primer on the surface of the cell, to which new material would be attached. A long-standing prediction of this hypothesis was that cells propagated for a period of time completely devoid of PG would be unable to regain their original shape. To test this hypothesis we generated an L-form strain in which PG synthesis could be completely shut down by deletion of a key gene needed for synthesis. After propagating the strain for several generations to ensure the complete absence of polymerized PG, we reintroduced the gene and were able to recover progeny walled cells with normal shape. These results show that a 'primer' is not necessary for synthesising a new PG sacculus, which finally disproves the heredity propagation theory. The experimental system we have developed can also now be used for the first time to study the de novo synthesis and polymerization of glycan strands. This promises to provide new insights into a highly important but still poorly understood process.Two other side projects were in progress when the Fellow left the lab for a position in Spain. First, isolation and characterization of mutants impaired specifically in L-form growth. One such mutant was extensively characterised and shown to affect a general area of intermediary metabolism involving methyl group transfer reactions catalysed by a molecule called S-adenosyl-methionine. Unfortunately, this area of metabolism is intricate and highly networked with a range of important cell functions, and despite considerable efforts, the fellow was unable to deduce the molecular basis for this effect. Secondly, the fellow attempted to isolate and characterise mutant L-forms capable of growing in culture medium lacking the osmotic stabilisers that are normally essential for L-form growth. Although interesting mutants were isolated, there was not time to characterise these in any detail., Overall, it is clear that a better understanding of the cell wall regeneration process should facilitate attempts to understand the possible role of L-forms as causative agents of a variety of chronic infections. Furthermore, a better understanding of the mechanisms involved in the transition from the wall-less to the walled state might provide the basis for development of novel antibacterial therapies to deal with persistent infections, which would be of high socio-economic impact.
