The project studied the molecules that build the septum, either during the processes of sporulation or in vegetative division, and the regulatory mechanisms that control the expression of the genes that code for septal components in Streptomyces, Bacillus, Brevibacterium, and Escherichia coli.
Cell division in Escherichia coli
The phylogenetically ubiquitous ftsZ gene needs to be expressed by its natural regulatory signals to allow the cells to divide within their correct morphological parameters. However, alterations in the contents of FtsZ have no effect on nucleoid segregation. The transcript of ftsZ accumulates in a cell cycle-dependent fashion. The expression of ftsZ is directed by a complex set of promoters, a peculiar feature of the division and cell wall cluster (dcw cluster). Among them there is one gearbox, ftsQ1p, which is dependent on the presence of the specific sigma S factor that participates in the expression of genes which are mainly transcribed at low growth rates. A high complexity in the regulatory pattern of the fts region, resembling the intricate patterns of eukaryotic gene transcription rather than the simplicity of bacterial metabolic operons, is predicted from the analysis of the contribution of each individual promoter to the transcription of ftsZ.
The properties of the essential E. coli protein FtsA match the predictions derived from its structure being akin to the eukaryotic actin. FtsA is able to bind ATP, and it is found in two different forms, one is phosphorylated, able to bind ATP, and appears largely in the cytoplasm. The other form is not phosphorylated, does not bind ATP, and is located in the cytoplasmic membrane, the location where it is likely to exert its activity. Phosphorylation and binding of ATP may have a regulatory role in the proper localization of FtsA in the cell at the time of division.
The cell division genes of Bacillus subtilis
The pbpB and spoVD genes, encoding penicillin binding proteins in the B. subtilis dcw cluster, arose by gene duplication, with spoVD becoming specialised for synthesis of the cortex during sporulation, and pbpB retaining the role in formation of the vegetative division septum. pbpB regulation is complex, with its major promoter also driving transcription of three upstream genes that are conserved in the dcw cluster of E. coli - mraZ, mraW and mraR (ftsL).
The murB gene, encoding UDP-N-acetylenolpyruvoylglucosamine reductase, required for cell wall synthesis, was identified in the dcw cluster of B. subtilis. Unexpectedly, this gene lies outside of the equivalent cluster of E. coli. The divIVA gene, implicated in the regulation of cell division by its isolation in a minicell mutant, was cloned and sequenced. It encodes a protein with striking similarity to myosins - eukaryotic motor proteins. This provides further evidence for the use of cytoskeleton-like elements in bacterial cell division. The spoIIE gene is involved in the initiation of polar division, forming part of the genetic switch from central to polar cell division. This gene has a second function in the regulation of sporulation gene expression. The observed changes in cell cycle parameters during the switch from vegetative (central) to sporulation (polar) division suggest a model to explain the timing and population kinetics of the process.
The cell division genes of Brevibacterium lactofermentum
The ftsZ gene of B. lactofermentum was cloned by PCR using two oligonucleotides designed from two consensus sequences found in the different ftsZ genes cloned from different microorganisms. The cloned gene was located downstream of murC and ftsQ, indicating that the dcw cluster is also present in corynebacteria. In E. coli and B. subtilis ftsZ is immediately preceded by ftsA, however in Streptomyces and B. lactofermentum ftsA, if it exist, is not adjacent to ftsZ.
Several attempts using PCR, oligonucleotide or heterologous probes failed to isolate minCD homologous in B. lactofermentum, suggesting that those genes may be not present in corynebacteria. Interestingly genes homologous to actin, tubulin and myosin were isolated.
Regulation of hyphal division during sporulation in Streptomyces coelicolor
In S. coelicolor sporulation a multigenomic filamentous hypha undergoes synchronous and regular multiple cell division to yield chains of perhaps 100 spores. A seemingly complete collection of `early' sporulation genes comprises six different genes which have been cloned and sequenced (four of them as a result of this project). The whiG gene product is a sigma factor, and one of its target promoters is that of whiH. The whiH gene product is a DNA-binding repressor or transcription factor of the GntR family, many of which bind and respond to intermediary metabolites. The whiB gene product shows evidence of DNA binding. The whiI locus specifies a pair of proteins of the family of two-component response regulators, with one being a presumptive membrane-bound histidine protein kinase, and the other a cytoplasmic DNA-binding transcripton factor containing a consensus phosphorylation target site. The whiA and whiJ gene products do not closely resemble known proteins but both possibly encode transcription factors. The six genes may provide routes for sensory information inputs into the decision to undergo sporulation. Several candidate target genes for these regulators have emerged: ftsQ and Z, glgB2 (spore-associated glycogen synthesis), sigF (late sporulation sigma factor), and two genes of unknown function that appear to be whiG-dependent. The min genes may be absent from actinomycetes, because attempts to isolate the S. coelicolor homologues failed.
MAJOR SCIENTIFIC BREAKTHROUGHS:
-The dcw cluster is conserved in a diversity of bacteria, although individual genes may be absent in some of them.
-The regulation of gene expression in the dcw cluster is very complex and is connected to cell growth processes.
-The mechanisms of bacterial septation include molecules which are similar to the cytoskeletal elements of eukaryotic cells.
-The cellular localization of some of these molecules is regulated by biochemical modifications.