Defining the DNA uptake specificity of naturally competent Haemophilus influenzae cells

Abstract

Some naturally competent bacteria exhibit both a strong preference for DNA fragments containing specific 'uptake sequences' and dramatic overrepresentation of these sequences in their genomes. Uptake sequences are often assumed to directly reflect the specificity of the DNA uptake machinery, but the actual specificity has not been well characterized for any bacterium. We produced a detailed analysis of Haemophilus influenzae's uptake specificity, using Illumina sequencing of degenerate uptake sequences in fragments recovered from competent cells. This identified an uptake motif with the same consensus as the motif overrepresented in the genome, with a 9 bp core (AAGTGCGGT) and two short flanking T-rich tracts. Only four core bases (GCGG) were critical for uptake, suggesting that these make strong specific contacts with the uptake machinery. Other core bases had weaker roles when considered individually, as did the T-tracts, but interaction effects between these were also determinants of uptake. The properties of genomic uptake sequences are also constrained by mutational biases and selective forces acting on USSs with coding and termination functions. Our findings define constraints on gene transfer by natural transformation and suggest how the DNA uptake machinery overcomes the physical constraints imposed by stiff highly charged DNA molecules.

Figure 1.

DNA uptake and the H. influenzae USS. (A) Mechanism of DNA uptake: uptake initiates at USSs (pink) when type IV pseudopili retract to pull double-stranded DNA through type II secretin pores into the periplasm. Rec2/ComF then translocates a single DNA strand through the inner membrane into the cytoplasm, where DNA is either degraded and reused or recombined with the chromosome. (B) Diagram of the canonical USS consisting of a 9 bp core flanked by two helically phased T-rich tracts.

Figure 2.

Uptake and re-uptake of a degenerate USS pool. (A–C) Experimental overview: a pool of ∼2.6 × 10¹¹ DNA fragments (A) with degenerate USSs was incubated (B) with ∼10⁹ competent cells. DNA that had been taken up (C, ∼1.6 × 10¹⁰ fragments) was purified from the cells’ periplasm. Darker red fragments represent those containing more preferred sequences. (D) Uptake by rec-2 cultures (30 min incubation) of: blue circles, DNA fragments with the consensus USS; red squares, the input degenerate pool; and green diamonds and triangles, reuptake of DNA fragments purified from recovered periplasmic pools from cells incubated with 32 and 256 fragments/cell, respectively. (E) Autoradiogram showing total and periplasmic DNA recovered by organic extractions from wt and rec-2 cultures after 5 min uptake (128 USS fragments/cell). The aqueous fraction of periplasmic extractions retains intact fragments but not labelled chromosomal DNA. (F) The degenerate pool was made by annealing and extending two oligos, followed by PCR; green arrows indicate fixed sequences containing priming sites for single-end Illumina sequencing. (G) The 42-nt reads spanned the 32-nt degenerate USS (24%/position), as well as control flanking bases of 4- and 6-nt.

Figure 3.

Effect of mismatches on uptake efficiency. (A) Histogram of input reads (grey) and recovered reads (green) with different numbers of mismatches from consensus. Inset shows the input in grey and the binomial expectation for 24% degeneracy as a black line. (B) Estimated % recovery of fragments with different numbers of mismatches; red line shows total uptake of 6.2%. (C) and (D) Frequency of reads with mismatches from consensus at each position in the input reads (C) and recovered reads (D). (E) Estimated % recovery of fragments with mismatches at the indicated position; red line shows total uptake.

Figure 4.

USS motifs. (A) Logo diagram of the experimentally derived uptake bias motif, using position-specific normalization of base composition in the recovered reads to that of the input reads. (B) Logo diagram of the genomic USS motif derived from 2206 sites in the genome found by the Gibbs recursive sampler.

Figure 5.

Sources of artefact. In (A) and (B), grey bars show measured information content at each position, as in Figure 4A. (A) Vertical lines show the estimated ranges of information content, given random sequencing error ranging from 0.04–1.5% per position. Horizontal hatches show the medians, and red dots show the capped maximum error rates at highly biased positions. (B) Vertical lines show the ranges of information content, given carry-over contamination ranging from 0.2% (observed uptake of a randomized USS fragments) to 1.3% (the maximum possible contamination level). (C) Histogram of normalized scores of recovered (green) and input (grey) reads using the uptake bias motif model. (D) As in (C), but only for those reads with a normalized score in a non-standard alignment exceeding 84.2. In (C) and (D), the arrow shows the threshold score of 84.2.

Figure 6.

Pairwise interaction effects between USS bases. (A) Effects of three mismatches on information content at other positions: grey bars indicate information content at each position in the total dataset. Yellow, blue and red lines indicate the information content in data subsets where positions 22, 8 and 11 were mismatched. (B) Effects of mismatches at individual focal positions on the information content of other positions. Each row shows the change in information content at non-focal positions (columns) for the subset of recovered and input reads. Purple indicates increased information content, and orange indicates decreased information content. (C) Total residual bits at the remaining positions when the indicated position is mismatched. Red line indicates total bits in the motif derived from the full dataset.

Figure 7.

Analysis of genomic uptake sequences. (A) and (B) Histograms of normalized scores of all 32mer sites in the genome using the uptake (A) or genomic (B) motifs. Shading in zoomed versions (centre panels) indicates the set of 2000 sites used to construct the logos in (C) and (D) (thresholds 80.6 and 71.6, respectively). Grey lines show control histograms of the average of 100 random sequences with H. influenzae genome length and GC-content.

Figure 8.

Information content for USS sites in different genomic partitions. (A) Grey bars show information content in the total set of 2000 sites found by the uptake-bias motif. Blue line: information content in the 1302 sites fully within open reading frames (none were found in non-coding genes). Red line: information content in the 692 intergenic sites. (B) Grey bars: information content in the intergenic sites. Purple lines: information content in the 380 intergenic sites with no terminator function. Orange line: information content in the 312 intergenic terminators sites.