Low Efficiency of Homology-Facilitated Illegitimate Recombination during Conjugation in Escherichia coli

Homology-facilitated illegitimate recombination has been described in three naturally competent bacterial species. It permits integration of small linear DNA molecules into the chromosome by homologous recombination at one end of the linear DNA substrate, and illegitimate recombination at the other end. We report that homology-facilitated illegitimate recombination also occurs in Escherichia coli during conjugation with small non-replicative plasmids, but at a low frequency of 3×10−10 per recipient cell. The fate of linear DNA in E. coli is either RecBCD-dependent degradation, or circularisation by ligation, and integration into the chromosome by single crossing-over. We also report that the observed single crossing-overs are recA-dependent, but essentially recBCD, and recFOR independent. This suggests that other, still unknown, proteins may act as mediator for the loading of RecA on DNA during single crossing-over recombination in E. coli

Inferred chromosomal recombination products obtained with plasmid pJA2.

<p><b>Left part</b>: Single crossing-over (SCO) between circular pJA2 and the chromosome, as produced if pJA2 remains uncut (or if pJA2 is cut and resealed). The resulting exconjugants are Lac⁺ Cm^R. <b>Central part</b>: HFIR between linear pJA2 and the chromosome. Exconjugants are Lac⁻ Cm^R, and therefore distinguishable from the previous SCO events. <b>Right part</b>: If pJA2 is cut, degraded up into the <i>lacZ</i> sequence, and recircularized, a SCO between a circular, deleted (wavy bold line) pJA2 molecule and the chromosome will give rise to Lac⁻ Cm^R exconjugants. White arrow, 3′ part of the <i>lacZ</i> gene. Grey box, remaining part of the <i>lacZ</i> gene. Wavy lines, other sequences of the <i>E. coli</i> chromosome. Straight line, pJA2 plasmid sequences. Black box, “cut cassette”. Primers j18, j19, j17, j5, j16 complementary to the plasmid pJA2 and the chromosome, are shown above each chromosomal recombination product.</p

Sequence of the oligonucleotides used in this study.

<p>Sequence of the oligonucleotides used in this study.</p

Properties of the HFIR clones obtained.

a<p>The joint is composed of the concatenation of the two bold sequences from each parent.</p

Recombination frequencies with linear pJA2 in <i>E. coli</i> mutant derivatives.

(1)<p>*Significant below 5%, ** Significant below1% with a Student test.</p>(2)<p>Frequencies calculated with the first term of a Poisson law.</p

Efficiency of post-conjugative recombination with plasmids pJA3, pJA2 and pJA1.

<p>The Lac+ (blue bars) and Lac− (white bars, when present) Cm^R ex-conjugants reported to the viable recipient cells, are scored separately.</p

Surface proteome analysis of a natural isolate of Lactococcus lactis reveals the presence of pili able to bind human intestinal epithelial cells.

Surface proteins of Gram-positive bacteria play crucial roles in bacterial adhesion to host tissues. Regarding commensal or probiotic bacteria, adhesion to intestinal mucosa may promote their persistence in the gastro-intestinal tract and their beneficial effects to the host. In this study, seven Lactococcus lactis strains exhibiting variable surface physico-chemical properties were compared for their adhesion to Caco-2 intestinal epithelial cells. In this test, only one vegetal isolate TIL448 expressed a high-adhesion phenotype. A nonadhesive derivative was obtained by plasmid curing from TIL448, indicating that the adhesion determinants were plasmid-encoded. Surface-exposed proteins in TIL448 were analyzed by a proteomic approach consisting in shaving of the bacterial surface with trypsin and analysis of the released peptides by LC-MS/MS. As the TIL448 complete genome sequence was not available, the tryptic peptides were identified by a mass matching approach against a database including all Lactococcus protein sequences and the sequences deduced from partial DNA sequences of the TIL448 plasmids. Two surface proteins, encoded by plasmids in TIL448, were identified as candidate adhesins, the first one displaying pilin characteristics and the second one containing two mucus-binding domains. Inactivation of the pilin gene abolished adhesion to Caco-2 cells whereas inactivation of the mucus-binding protein gene had no effect on adhesion. The pilin gene is located inside a cluster of four genes encoding two other pilin-like proteins and one class-C sortase. Synthesis of pili was confirmed by immunoblotting detection of high molecular weight forms of pilins associated to the cell wall as well as by electron and atomic force microscopy observations. As a conclusion, surface proteome analysis allowed us to detect pilins at the surface of L. lactis TIL448. Moreover we showed that pili appendages are formed and involved in adhesion to Caco-2 intestinal epithelial cells