RESUMO
To address the need for new antibacterials, a number of bacterial genomes have been systematically disrupted to identify essential genes. Such programs have focused on the disruption of single genes and may have missed functions encoded by gene pairs or multiple genes. In this work, we hypothesized that we could predict the identity of pairs of proteins within one organism that have the same function. We identified 135 putative protein pairs in Bacillus subtilis and attempted to disrupt the genes forming these, singly and then in pairs. The single gene disruptions revealed new genes that could not be disrupted individually and other genes required for growth in minimal medium or for sporulation. The pairwise disruptions revealed seven pairs of proteins that are likely to have the same function, as the presence of one protein can compensate for the absence of the other. Six of these pairs are essential for bacterial viability and in four cases show a pattern of species conservation appropriate for potential antibacterial development. This work highlights the importance of combinatorial studies in understanding gene duplication and identifying functional redundancy.
Assuntos
Genes Bacterianos/genética , Genes Duplicados , Genes Essenciais/genética , Sequência de Aminoácidos , Bacillus subtilis/genética , Bacillus subtilis/crescimento & desenvolvimento , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Biologia Computacional , Deleção de Genes , Duplicação Gênica , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/fisiologia , Genes Essenciais/fisiologia , Genoma Bacteriano , Dados de Sequência Molecular , Mutação , Alinhamento de SequênciaRESUMO
Systematic inactivation of Bacillus subtilis genes has previously revealed that 271 are indispensable for growth. In the present study, 11 of these (yacA, ydiB, ydiC, ykqC, ylaN, yloQ, ymdA, yneS, yqeI, yqjK and ywlC) were identified as genes encoding proteins of unknown function. By analysing the effects of protein depletion, and examining the subcellular localization of these proteins, a start has been made in elucidating their functions. It was found that four of these genes (ydiB, yloQ, yqeI and ywlC) were not required for B. subtilis viability. Analysis of the localization of YkqC suggests that it co-localizes with ribosomes, and it is proposed that it is involved in processing either rRNA or specific mRNAs when they are associated with the ribosome. The results suggest that other novel essential proteins may be involved in lipid synthesis and control of cell wall synthesis.
Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Genes Bacterianos , Genes Essenciais , Antibacterianos/farmacologia , Bacillus subtilis/química , Bacillus subtilis/citologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/fisiologia , Biomassa , Parede Celular/química , Parede Celular/genética , Citoplasma/química , Densitometria , Deleção de Genes , Lipídeos/genética , Microscopia de Fluorescência , Mutagênese Insercional , Fenótipo , Proteínas Recombinantes de Fusão/metabolismo , Ribossomos/química , Trimetoprima/farmacologiaRESUMO
The continuous emergence of antibiotic resistance demands that novel classes of antibiotics continue to be developed. The division machinery of bacteria is an attractive target because it comprises seven or more essential proteins that are conserved almost throughout the bacteria but are absent from humans. We describe the development of a cell-based assay for inhibitors of cell division and its use to isolate a new inhibitor of FtsZ protein, a key player in the division machinery. Biochemical, cytological, and genetic data are presented that demonstrate that FtsZ is the specific target for the compound. We also describe the effects of more potent analogues of the original hit compound that act on important pathogens, again at the level of cell division. The assay and the compounds have the potential to provide novel antibiotics with no pool of pre-existing resistance. They have provided new insight into cytokinesis in bacteria and offer important reagents for further studies of the cell division machinery.
