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The essential genome of a bacterium. Mol Syst Biol 7:528

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.

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Available from: Beat Christen, Aug 30, 2015
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    • "This illustrates the limitation of transposon essentiality studies using deep sequencing for fitness genes. Notably, we found that the insertions were not evenly distributed along the entire ORFs as previously observed in Caulobacter crescentus (Christen et al, 2011). In this respect, it is important to note that our mini-transposon has an internal promoter that could allow expression of downstream genes or domains if there is a start codon for translation. "
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    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
    • "Transposon insertion sequencing, a technique first reported in 2009 that allows genome-wide analysis of insertions impairing bacterial fitness, was however suitable for identifying sRNA and other non-coding regulatory sequences (reviewed in Van Opijnen and Camilli, 2013). High-density transposon libraries identified non-coding RNAs important for growth of Caulobacter crescentus (Christen et al., 2011) and the pathogens Mycobacterium tuberculosis and Streptococcus pneumoniae (Mann et al., 2012; Zhang et al., 2012). Analysis of S. pneumoniae libraries in in vivo infection models also revealed sRNAs required for virulence (Mann et al., 2012). "
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    ABSTRACT: Intracellular bacterial pathogens have evolved distinct lifestyles inside eukaryotic cells. Some pathogens coexist with the infected cell in an obligate intracellular state, whereas others transit between the extracellular and intracellular environment. Adaptation to these intracellular lifestyles is regulated in both space and time. Non-coding small RNAs (sRNAs) are post-transcriptional regulatory molecules that fine-tune important processes in bacterial physiology including cell envelope architecture, intermediate metabolism, bacterial communication, biofilm formation, and virulence. Recent studies have shown production of defined sRNA species by intracellular bacteria located inside eukaryotic cells. The molecules targeted by these sRNAs and their expression dynamics along the intracellular infection cycle remain, however, poorly characterized. Technical difficulties linked to the isolation of "intact" intracellular bacteria from infected host cells might explain why sRNA regulation in these specialized pathogens is still a largely unexplored field. Transition from the extracellular to the intracellular lifestyle provides an ideal scenario in which regulatory sRNAs are intended to participate; so much work must be done in this direction. This review focuses on sRNAs expressed by intracellular bacterial pathogens during the infection of eukaryotic cells, strategies used with these pathogens to identify sRNAs required for virulence, and the experimental technical challenges associated to this type of studies. We also discuss varied techniques for their potential application to study RNA regulation in intracellular bacterial infections.
    Frontiers in Cellular and Infection Microbiology 11/2014; 4:162. DOI:10.3389/fcimb.2014.00162 · 2.62 Impact Factor
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    • "Similarly , B. subvibrioides has a MipZ homologue and no apparent homologues to Min proteins, Noc or SlmA. MipZ was characterized as essential in C. crescentus along with the ParA and ParB proteins necessary for MipZ gradient formation (Thanbichler and Shapiro, 2006; Christen et al., 2011). It was later shown that mipZ can be deleted from C. crescentus, but deletion results in severely filamentous and slow-growing cells (Radhakrishnan et al., 2010). "
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    ABSTRACT: The cell cycle of Caulobacter crescentus is controlled by a complex signaling network that coordinates events. Genome sequencing has revealed many C. crescentus cell cycle genes are conserved in other Alphaproteobacteria, but it is not clear to what extent their function is conserved. As many cell cycle regulatory genes are essential in C. crescentus, the essential genes of two Alphaproteobacteria, Agrobacterium tumefaciens (Rhizobiales) and Brevundimonas subvibrioides (Caulobacterales), were elucidated to identify changes in cell cycle protein function over different phylogenetic distances as demonstrated by changes in essentiality. The results show the majority of conserved essential genes are involved in critical cell cycle processes. Changes in component essentiality reflect major changes in lifestyle, such as divisome components in A. tumefaciens resulting from that organism's different growth pattern. Larger variability of essentiality was observed in cell cycle regulators, suggesting regulatory mechanisms are more customizable than the processes they regulate. Examples include variability in the essentiality of divJ and divK spatial cell cycle regulators, and non-essentiality of the highly conserved and usually essential DNA methyltransferase CcrM. These results show that while essential cell functions are conserved across varying genetic distance, much of a given organism's essential gene pool is specific to that organism.
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