Article

The essential genome of a bacterium

Department of Developmental Biology, Stanford University, Stanford, CA, USA.
Molecular Systems Biology (Impact Factor: 14.1). 08/2011; 7:528. DOI: 10.1038/msb.2011.58
Source: PubMed

ABSTRACT Caulobacter crescentus is a model organism for the integrated circuitry that runs a bacterial cell cycle. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of a bacterial cell. Using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing, we determined the essential Caulobacter genome at 8 bp resolution, including 1012 essential genome features: 480 ORFs, 402 regulatory sequences and 130 non-coding elements, including 90 intergenic segments of unknown function. The essential transcriptional circuitry for growth on rich media includes 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor. We identified all essential promoter elements for the cell cycle-regulated genes. The essential elements are preferentially positioned near the origin and terminus of the chromosome. The high-resolution strategy used here is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.

Download full-text

Full-text

Available from: Beat Christen, Jul 02, 2015
0 Followers
 · 
150 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Identifying all essential genomic components is critical for the assembly of minimal artificial life. In the genome-reduced bacterium Mycoplasma pneumoniae, we found that small ORFs (smORFs; < 100 residues), accounting for 10% of all ORFs, are the most frequently essential genomic components (53%), followed by conventional ORFs (49%). Essentiality of smORFs may be explained by their function as members of protein and/or DNA/RNA complexes. In larger proteins, essentiality applied to individual domains and not entire proteins, a notion we could confirm by expression of truncated domains. The fraction of essential non-coding RNAs (ncRNAs) non-overlapping with essential genes is 5% higher than of non-transcribed regions (0.9%), pointing to the important functions of the former. We found that the minimal essential genome is comprised of 33% (269,410 bp) of the M. pneumoniae genome. Our data highlight an unexpected hidden layer of smORFs with essential functions, as well as non-coding regions, thus changing the focus when aiming to define the minimal essential genome. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.
    Molecular Systems Biology 01/2015; 11(1):780. DOI:10.15252/msb.20145558 · 14.10 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Quinolone antibiotics are clinically important drugs that target bacterial DNA replication and chromosome segregation. Although the AcrAB-family efflux pumps generally protect bacteria from such drugs, the physiological role of these efflux systems and their interplay with other cellular events are poorly explored. Here, we report an intricate relationship between antibiotic resistance and cell polarity in the model bacterium Caulobacter crescentus. We show that a polarity landmark protein, TipN, identified by virtue of its ability to direct flagellum placement to the new cell pole, protects cells from toxic misregulation of an AcrAB efflux pump through a cis-encoded nalidixic acid-responsive transcriptional repressor. Alongside the importance of polarity in promoting the inheritance and activity of virulence functions including motility, we can now ascribe to it an additional role in drug resistance that is distinct from classical efflux mechanisms.
    Chemistry & biology 04/2014; DOI:10.1016/j.chembiol.2014.02.018 · 6.59 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Understanding how complex networks of genes integrate to produce dividing cells is an important goal that is limited by the difficulty in defining the function of individual genes. Current resources for the systematic identification of gene function such as siRNA libraries and collections of deletion strains are costly and organism specific. We describe here integration profiling, a novel approach to identify the function of eukaryotic genes based upon dense maps of transposon integration. As a proof of concept, we used the transposon Hermes to generate a library of 360,513 insertions in the genome of Schizosaccharomyces pombe. On average we obtained one insertion for every 29 bp of the genome. Hermes integrated more often into nucleosome free sites and 33% of the insertions occurred in ORFs. We found that ORFs with low integration densities successfully identified the genes that are essential for cell division. Importantly, the nonessential ORFs with intermediate levels of insertion correlated with the nonessential genes that have functions required for colonies to reach full size. This finding indicates that integration profiles can measure the contribution of nonessential genes to cell division. While integration profiling succeeded in identifying genes necessary for propagation, it also has the potential to identify genes important for many other functions such as DNA repair, stress response, and meiosis.
    Genetics 07/2013; DOI:10.1534/genetics.113.152744 · 4.87 Impact Factor