To identify the conclusions, the postdoc and I are now independently writing our versions the Abstract. Here's mine: (The target journal is PNAS, so the audience goes beyond microbiologists.)
RR ABSTRACT: Most naturally competent bacteria take up any DNA, but some exhibit a strong preference for fragments containing a specific 'uptake sequence', with a corresponding abundance of the preferred sequence in their genomes. Although these sequences play a major role in the debate about the function of DNA uptake, the actual uptake specificities have not been carefully characterized. Using Illumina sequencing of degenerate DNA fragments recovered from competent Haemophilus influenzae cells, we have produced a very detailed analysis of this species' uptake specificity.
- The uptake consensus sequence matched the known genomic consensus, with the 9 bp core AAGTGCGGT and two flanking AT-rich segments.
- Positions with no genomic signal showed little or no effects on uptake.
- Only four of the core positions (GCGG) made very strong contributions to uptake.
- Other positions of the consensus made relatively weak contributions to uptake.
- Compensatory interactions between bases at some of these positions made substantial contributions to uptake, suggesting that these bases may contact each other during uptake.
And here's his:
POSTDOC ABSTRACT: Many bacterial species are naturally competent, and competent cells are able to take up intact DNA molecules from the surroundings and bring them to the cytosol. Natural competence has a profound effect on genome evolution, due to recombination of taken up fragments with competent cell chromosomes. To bring DNA across cell membranes, the DNA uptake machinery must overcome the physical constraint imposed by stiff highly charged DNA molecules. Haemophilus influenzae competent cells have strong uptake specificity, preferentially taking up DNA fragments through their outer membrane that contain short “uptake signal sequences” (USS); ~2200 sites in the H. influenzae genome conform to a “genomic USS motif”. This genomic USS motif consists of a 9 bp “core” AAGTGCGGT with a strong consensus, flanked by two helically phased AT-tracts with a weaker consensus. We used massively parallel sequencing to dissect the genomic USS motif and find out how the structure of this unusually abundant sequence motif contributes to uptake efficiency. Competent cells were incubated with a complex pool of fragments containing a degenerate version of the consensus genomic USS, and the fragments cells took up were purified from the periplasm. Comparison of sequences from the recovered pool to sequences from the input pool revealed novel aspects of uptake specificity not predicted from genome sequence analysis and subdivides the USS into parts with distinct properties. Four bases in the “inner” core USS (GCGG) are (nearly?) essential for uptake. “Outer” core bases and the AT-tracts make weak individual contributions to uptake, but instead cooperatively contribute to uptake. These results provide a specific mechanistic hypothesis about the interaction of the USS with the DNA uptake machinery, as well as having implications for the evolution of uptake specificity and the accumulation of uptake sequences in genomes by molecular drive.
And here's our consensus:
CONSENSUS ABSTRACT: Most naturally competent bacteria will take up any DNA, but some exhibit a strong preference for fragments containing a specific 'uptake sequence', with a corresponding abundance of the preferred sequence in their genomes. These sequences play a major role in the debate about the function of DNA uptake, but although the genomic motifs are often assumed to directly reflect the uptake specificities, the actual uptake specificity has not been carefully characterized for any species. Using Illumina sequencing of degenerate DNA fragments recovered from competent Haemophilus influenzae cells, we have produced a very detailed analysis of this species' uptake specificity. This work identified an uptake consensus sequence that did indeed match the known genomic consensus, with the 9 bp core AAGTGCGGT and two flanking AT-rich segments. However, positions with moderately strong genomic consensuses had unexpectedly weak effects on uptake, and only four of the core positions (GCGG) made very strong contributions to uptake. Compensatory interactions between bases at some of the minor positions made substantial contributions to uptake, suggesting that these bases may contact each other or the same component of the uptake machinery during uptake. These findings suggest that interaction of the central four bases with a DNA-binding protein may be the main factor in uptake specificity, with minor DNA-DNA and/or DNA-protein interactions also contributing. Cumulative effects of these interactions over evolutionary time may explain the discrepancy between the genomic and uptake motifs. Experimental work on these interactions is likely to clarify how the DNA uptake machinery overcomes the physical constraints imposed by stiff highly charged DNA molecules.