- There are currently 251 and 2 essential protein and RNA-coding genes, respectively, known in B. subtilis.
New resource: A knock-down library to study all essential genes
We are excited to announce the availability of a new collection of Bacillus subtilis 168 mutants designed to explore the functions of 289 essential genes in this organism. The collection is available at the BGSC. For details see at the bottom of the page.
Genes in this functional category
Protein synthesis, secretion and quality control
Protein secretion/ chaperones/ protein quality control
Cell envelope and cell division
Cell wall synthesis
Central energy metabolism
Biosynthesis of amino acids
Biosynthesis of lipids
Biosynthesis of nucleotides
Biosynthesis of cofactors
Metal ion transport
Biosynthesis of iron-sulphur clusters
DNA replication and chromosome maintenance
Chromosome condensation/ segregation
RNA synthesis and degradation
The list of essential genes according to Kobayashi et al. (2003)
The essential gene knock-down collection
The paper describing the construction of this library and its initial characterization will appear in the June 2 edition of Cell. The paper is a collaboration among labs at the University of California, San Francisco, Stanford University, University of California, Berkeley, and McMaster University, Hamilton, Ontario. The co-first authors are Jason M. Peters of UCSF and Alexandre Colavin and Handuo Shi of Stanford.
The library uses a CRISPR interference (CRISPRi) strategy to all a tunable “knockdown” of individual essential genes. Every strain in the library has a Streptococcus pyogenes dcas9 gene integrated into the B. subtilis lacA locus, where it has been placed under control of the xylose-inducible Pxyl promoter. Each strain also has a single-guide RNA (sgRNA) targeting a specific essential gene. The sgRNA coding sequence is integrated into B. subitlis amyE, where it has been placed under the control of the strongly constitutive Pveg promoter. The dCas9 protein lacks nuclease activity. But when dCas9 is present, the sgRNA enables it to bind to the 5’ end of the target gene, where it effectively blocks transcription via steric hindrance. Basal level expression of dcas9 in the absence of xylose knocks down expression of the essential gene about 3-fold. This reduction creates subtle phenotypes, such as increased sensitivity to specific antibiotics and chemical inhibitors, but allows for essentially normal growth under standard laboratory conditions. Full induction of dcas9 with xylose (1%) reduces expression of the essential gene ~150-fold, with drastic consequences for cell morphology and viability. Varying the concentration of xylose between 0.001% and 0.1% allows tunable expression of the essential gene. Peters et al. have not only reported the construction of the library, but have demonstrated its power for analyzing essential genes. They used chemical genomics, for example, to reveal the essential gene network of B. subtilis, revealing interesting connections between seemingly unrelated processes.
These strains provide an invaluable tool for a systematic study of essential genes in a bacterial model system. We thank Jason Peters, Carol Gross, and the entire consortium for donating the library to the BGSC, and we look forward to supplying strains from it to scientists from the B. subtilis research community and beyond. For a complete list of the genes targeted in the library, please see the Peters et al. publication. Summaries of what has been learned previously about most of these genes can be accessed at [SubtiWiki](http://subtiwiki.uni-goettingen.de/wiki/index.php/Essential_genes). It will take a little while for us to update the BGSC online database to include these strains. But their naming convention is simple. The numeric portion of the gene’s locus tag is appended to the prefix “BEC” to produce the strain name. Hence the knockdown strain for the essential gene ligA, which encodes DNA ligase and carries the locus tag BSU06620, is BCE06620. The full genotype of this strain is lacA::Pxyl-dcas9 amyE::Pveg-sgRNA(ligA) trpC2, and it carries resistance markers for erythromycin and chloramphenicol. Users may request these strains by giving us the targeted gene name or locus tag. Standard user fees apply.
Peters et al., A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria, Cell (2016) PubMed
Important original publications