The essential genome of a bacterium

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.

Figure 1

Genomic high-resolution transposon scanning strategy. Insertion mutants are pooled to generate a hyper-saturated Tn5 mutant library. Subsequent parallel amplification of individual transposon junctions by a nested arbitrary PCR yields DNA fragments reading out of transposon elements into adjacent genomic DNA sequences. DNA fragments carry terminal adapters (orange, blue) compatible to the Illumina flow-cell and are sequenced in parallel by standard paired-end sequencing.

Figure 2

Identification of essential genome features. (A) 28 735 unique Tn5 insertions sites (red) were mapped onto the 4-Mbp Caulobacter genome that encodes 3876 annotated ORFs shown in the inner (minus strand) and middle (plus strand) tracks by black lines. (B) An 192-bp essential genome segment (no Tn5 insertions) contains a stationary phase expressed non-coding sRNA (Landt et al, 2008). The rectangular heat map above shows the micro-array probe cross-correlation pattern of the sRNA (Landt et al, 2008). The locations of transposon insertions (red marks) are shown above the genome track. P-values for essentiality for the different gap sizes observed are below. (C) A small non-disruptable segment containing an essential tRNA. (D) Two non-disruptable genome regions include two regulatory sequences required for chromosome replication. (E) Locations of mapped transposon insertion sites (red marks) on a segment of the Caulobacter genome. Non-essential ORFs (blue) have dense Tn5 transposon insertions, while large non-disruptable genome regions contain essential ORFs (light red). For every non-disruptable genome region, a P-value for gene essentiality is calculated assuming uniform distributed Tn5 insertion frequency and neutral fitness costs. (F) For each of the 3876 Caulobacter ORFs, the number of Tn5 insertions is plotted against the corresponding ORF length. Non-essential ORFs (blue), fitness relevant ORFs (Supplementary information) (green) and essential ORFs (red) have different transposon insertion frequencies. (G) The essential cell-cycle gene divL had multiple transposon insertions within the 3′ tail. This dispensable region encodes parts of the histidine kinase domain as well as an ATPase domain that is non-essential. (H) One of the ORFs with mis-annotated start site. The essential cell-cycle gene chpT tolerates disruptive Tn5 insertions in the 5′ region of the mis-annotated ORF. The native promoter element and TSS are located downstream of the mis-annotated start codon as confirmed by lacZ promoter activity assays.

Figure 3

Genome-wide identification of essential gene regulatory sequences. (A) Transposon insertions within the promoter region of an essential gene are only viable if the transposon-specific promoter points toward the gene (sense insertion); insertions in the opposite orientation (anti-sense insertions) are lethal. The distance between the annotated start codon and the first detected occurrence of an anti-sense insertion within the upstream region of an essential gene defines its essential promoter region. (B) Anti-sense insertions (red marks below genome track) are absent throughout the entire ATP synthase operon while sense insertions (red marks above genome track) are only tolerated within the non-essential lead gene and within non-coding regions of co-transcribed downstream genes. (C) Lengths of promoter regions that extend upstream of predicted transcriptional start sites. (D) Size distribution of essential promoter regions. The cell-cycle master regulators ctrA, dnaA and gcrA, which are subjected to complex cell cycle-dependent regulation, ranked among the longest essential promoter regions identified. (E) Essential cell cycle-regulated genes are clustered according to their temporal expression profile. Essential genes with long essential promoter regions indicate cell-cycle hub nodes subjected to complex transcriptional regulation. (F) Only insertions in the sense strand (red lines above the genome track) are tolerated within the 171 bp long essential promoter region of the cell-cycle master regulator ctrA. Insertions in the anti-sense strand (red lines below the genome track) are absent from the essential ctrA promoter. Both transcription start sites (arrows P1, P2), two DNA binding sites for CtrA (gray boxes) and one of two reported binding site for the SciP transcription factor (yellow boxes) are contained within the identified essential promoter region.

Figure 4

Chromosomal distribution of essential transcripts and phylogenetic conservation of essential Caulobacter ORFs. (A) Chromosome distribution of essential transcripts and ORFs found within a 500-kb window. Top panel: Essential Caulobacter transcripts are non-uniformly distributed and are enriched near the replication origin (ori) and terminus (ter) regions of the chromosome. The P-values for enrichment of essential Caulobacter ORFs (middle panel) and E. coli ORFs (bottom panel) are graphed as a function of chromosomal position. Inserts show a schematic representation of the corresponding circular chromosomes indicating regions of enrichment in red. (B) A heat map showing the sequence conservation of essential Caulobacter proteins across the α, β, γ, δ and ε classes of proteobacteria. Highly conserved essential proteins are in red while poorly conserved proteins are in black. (C) Venn diagram of overlap between Caulobacter and E. coli ORFs (outer circles) as well as their subsets of essential ORFs (inner circles). Less than 38% of essential Caulobacter ORFs are conserved and essential in E. coli. Only essential Caulobacter ORFs present in the STING database were considered, leading to a small disparity in the total number of essential Caulobacter ORFs.