This study reports the essential Caulobacter genome at 8 bp resolution determined by saturated transposon mutagenesis and high-throughput sequencing. This strategy is applicable to full genome essentiality studies in a broad class of bacterial species.
The essential Caulobacter genome was determined at 8 bp resolution using hyper-saturated transposon mutagenesis coupled with high-throughput sequencing.Essential protein-coding sequences comprise 90% of the essential genome; the remaining 10% comprising essential non-coding RNA sequences, gene regulatory elements and essential genome replication features.Of the 3876 annotated open reading frames (ORFs), 480 (12.4%) were essential ORFs, 3240 (83.6%) were non-essential ORFs and 156 (4.0%) were ORFs that severely impacted fitness when mutated.The essential elements are preferentially positioned near the origin and terminus of the Caulobacter chromosome.This high-resolution strategy is applicable to high-throughput, full genome essentiality studies and large-scale genetic perturbation experiments in a broad class of bacterial species.
The regulatory events that control polar differentiation and cell-cycle progression in the bacterium Caulobacter crescentus are highly integrated, and they have to occur in the proper order (McAdams and Shapiro, 2011). Components of the core regulatory circuit are largely known. Full discovery of its essential genome, including non-coding, regulatory and coding elements, is a prerequisite for understanding the complete regulatory network of this bacterial cell. We have identified all the essential coding and non-coding elements of the Caulobacter chromosome using a hyper-saturated transposon mutagenesis strategy that is scalable and can be readily extended to obtain rapid and accurate identification of the essential genome elements of any sequenced bacterial species at a resolution of a few base pairs.
We engineered a Tn5 derivative transposon (Tn5Pxyl) that carries at one end an inducible outward pointing Pxyl promoter (Christen et al, 2010). We showed that this transposon construct inserts into the genome randomly where it can activate or disrupt transcription at the site of integration, depending on the insertion orientation. DNA from hundred of thousands of transposon insertion sites reading outward into flanking genomic regions was parallel PCR amplified and sequenced by Illumina paired-end sequencing to locate the insertion site in each mutant strain (Figure 1). A single sequencing run on DNA from a mutagenized cell population yielded 118 million raw sequencing reads. Of these, >90 million (>80%) read outward from the transposon element into adjacent genomic DNA regions and the insertion site could be mapped with single nucleotide resolution. This yielded the location and orientation of 428 735 independent transposon insertions in the 4-Mbp Caulobacter genome.
Within non-coding sequences of the Caulobacter genome, we detected 130 non-disruptable DNA segments between 90 and 393 bp long in addition to all essential promoter elements. Among 27 previously identified and validated sRNAs (Landt et al, 2008), three were contained within non-disruptable DNA segments and another three were partially disruptable, that is, insertions caused a notable growth defect. Two additional small RNAs found to be essential are the transfer-messenger RNA (tmRNA) and the ribozyme RNAseP (Landt et al, 2008). In addition to the 8 non-disruptable sRNAs, 29 out of the 130 intergenic essential non-coding sequences contained non-redundant tRNA genes; duplicated tRNA genes were non-essential. We also identified two non-disruptable DNA segments within the chromosomal origin of replication. Thus, we resolved essential non-coding RNAs, tRNAs and essential replication elements within the origin region of the chromosome. An additional 90 non-disruptable small genome elements of currently unknown function were identified. Eighteen of these are conserved in at least one closely related species. Only 2 could encode a protein of over 50 amino acids.
For each of the 3876 annotated open reading frames (ORFs), we analyzed the distribution, orientation, and genetic context of transposon insertions. There are 480 essential ORFs and 3240 non-essential ORFs. In addition, there were 156 ORFs that severely impacted fitness when mutated. The 8-bp resolution allowed a dissection of the essential and non-essential regions of the coding sequences. Sixty ORFs had transposon insertions within a significant portion of their 3′ region but lacked insertions in the essential 5′ coding region, allowing the identification of non-essential protein segments. For example, transposon insertions in the essential cell-cycle regulatory gene divL, a tyrosine kinase, showed that the last 204 C-terminal amino acids did not impact viability, confirming previous reports that the C-terminal ATPase domain of DivL is dispensable for viability (Reisinger et al, 2007; Iniesta et al, 2010). In addition, we found that 30 out of 480 (6.3%) of the essential ORFs appear to be shorter than the annotated ORF, suggesting that these are probably mis-annotated.
Among the 480 ORFs essential for growth on rich media, there were 10 essential transcriptional regulatory proteins, including 5 previously identified cell-cycle regulators (McAdams and Shapiro, 2003; Holtzendorff et al, 2004; Collier and Shapiro, 2007; Gora et al, 2010; Tan et al, 2010) and 5 uncharacterized predicted transcription factors. In addition, two RNA polymerase sigma factors RpoH and RpoD, as well as the anti-sigma factor ChrR, which mitigates rpoE-dependent stress response under physiological growth conditions (Lourenco and Gomes, 2009), were also found to be essential. Thus, a set of 10 transcription factors, 2 RNA polymerase sigma factors and 1 anti-sigma factor are the core essential transcriptional regulators for growth on rich media. To further characterize the core components of the Caulobacter cell-cycle control network, we identified all essential regulatory sequences and operon transcripts. Altogether, the 480 essential protein-coding and 37 essential RNA-coding Caulobacter genes are organized into operons such that 402 individual promoter regions are sufficient to regulate their expression. Of these 402 essential promoters, the transcription start sites (TSSs) of 105 were previously identified (McGrath et al, 2007).
The essential genome features are non-uniformly distributed on the Caulobacter genome and enriched near the origin and the terminus regions. In contrast, the chromosomal positions of the published E. coli essential coding sequences (Rocha, 2004) are preferentially located at either side of the origin (Figure 4A). This indicates that there are selective pressures on chromosomal positioning of some essential elements (Figure 4A).
The strategy described in this report could be readily extended to quickly determine the essential genome for a large class of bacterial species.
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.