Co-evolution of segregation guide DNA motifs and the FtsK translocase in bacteria: identification of the atypical Lactococcus lactis KOPS motif

Abstract

Bacteria use the global bipolarization of their chromosomes into replichores to control the dynamics and segregation of their genome during the cell cycle. This involves the control of protein activities by recognition of specific short DNA motifs whose orientation along the chromosome is highly skewed. The KOPS motifs act in chromosome segregation by orienting the activity of the FtsK DNA translocase towards the terminal replichore junction. KOPS motifs have been identified in γ-Proteobacteria and in Bacillus subtilis as closely related G-rich octamers. We have identified the KOPS motif of Lactococcus lactis, a model bacteria of the Streptococcaceae family harbouring a compact and low GC% genome. This motif, 5'-GAAGAAG-3, was predicted in silico using the occurrence and skew characteristics of known KOPS motifs. We show that it is specifically recognized by L. lactis FtsK in vitro and controls its activity in vivo. L. lactis KOPS is thus an A-rich heptamer motif. Our results show that KOPS-controlled chromosome segregation is conserved in Streptococcaceae but that KOPS may show important variation in sequence and length between bacterial families. This suggests that FtsK adapts to its host genome by selecting motifs with convenient occurrence frequencies and orientation skews to orient its activity.

Figure 1.

Chimerical protein 3γ_Ec efficiently binds KOPS-containing DNA. (A) Alignment of the winged-helix domains of different FtsKγ. Helices were positioned from the E. coli FtsKγ structure. Coordinates (AA) are those of E. coli FtsK. Invariant residues are highlighted in black and indicated by stars. Double dots indicate conserved and single dots semi-conserved residues. Residues common to E. coli (Ecol), V. Cholerae (Vcho) and P. aeruginosa (Paer), which share the same KOPS motifs, are highlighted in yellow. Residues common to Streptococcaceae are highlighted in grey. Residues of E. coli FtsK whose mutation to alanine results in a KOPS-blind phenotype are highlighted in blue. Residues possibly involved in specific base recognition in the P. aeruginosa FtsKγ/KOPS co-crystal are highlighted in red. Colon and dot indicate conserved and semi-conserved residues, respectively. Abbreviations: Bsub, B. subtilis; Llac, L. lactis subsp. lactis, Lcre, L. lactis subsp. cremoris; Spyo, S. pyogenes; Saga, S. agalactiae; Spne, S. pneumoniae. (B) Schematic representation of a covalent trimer. Three γ subdomains of E. coli FtsK (64AA) were connected by a flexible linker (16AA). The resulting 3γ protein (3γ_Ec) was His-Flag-tagged in the amino-terminal part (see ‘Materials and Methods’). (C) Activation of XerCD/dif recombination by the E. coli 3γ protein. Recombination frequency were measured in Δ(lacI xerC ftsKγ) E. coli strains carrying the dif-lacI-dif recombination cassette in place of the dif site and a plasmid encoding the FtsKγ_EC or 3γ_EC proteins shown in (B), as indicated. Recombination was induced by transformation with plasmid pFC241 (XerC) and measured as previously described (21,29). (D–F) EMSA performed with the indicated DNA substrate containing one KOPS motif (D) or three KOPS motifs (E and F) and the indicated protein: 3γ_EC (D and E) or 3γ_Ll (F). The relevant DNA sequences are shown below the gel. Reactions contained 0.5 and 1 µM of 3γ protein. The presence (+polydIdC) or absence of competitor (−polydIdC) or protein (−) is indicated. The free DNA probes and the shifted DNA are indicated. Asterisk indicates the major complex formed in the absence of competitor DNA.

Figure 2.

Escherichia coli KOPS and B. subtilis SRS motifs are bad candidate motifs for L. lactis KOPS motifs. The graphs show distribution of KOPS or SRS motifs in relevant bacterial genomes. Genomes and motifs are indicated. Coordinates are in bp. Grey arrowheads show the position of the chromosome dimer resolution site. The sequence is red on the top DNA strand; a +1 bar indicates a motif and a −1 bar its complementary sequence. Graphs were generated using an in-house version of the FindOligomers software (5).

Figure 3.

Prediction of the L. lactis KOPS motif. (A) Distribution of octamers with one degenerated position in the L. lactis chromosome. All octamers that have a positive over-representation and skew score and a minimal frequency of 1 every 70 kb on the whole chromosome are represented in grey. Among these, motifs that have a specific distribution in the dif region are shown in red. They represent the 100th most skewed motifs among those with a leading strand skew in the dif region higher than 90%, a minimal frequency of 1 occurrence every 40 kb in this region and a minimal frequency of 1 occurrence every 70 kb in the whole genome. The strength of the red represents the over-representation score in the dif region (the stronger the red, the more over-represented the motif). Four motifs stand out as potential KOPS candidates (highlighted in green) with three additional ones that are slightly less exceptional but share very similar sequence (also in green). (B) Distribution of heptamers in the L. lactis chromosome. Same criteria and color code as in (A). One motif, 5′-GAAGAAG-3′ clearly stands out (shown circled in green).

Figure 4.

The 3γ_Ll protein recognizes the 5′-GAAGAAG-3′ heptamer. (A–C) Same EMSA experiment as in Figure 1D–F, with DNA substrates containing a repetition of 5′-GAA-3′ motifs (A), a single 5′-GAAGAAG-3′ motif (B), or three non-overlapping 5′-GAAGAAG-3′ motifs (C) and the indicated proteins. The relevant DNA sequences are shown below the gels. (D–F) ITC experiments performed by titrating the 3γ_Ll protein with the indicated DNA substrates: (D) containing a single 5′-GAAGAAG-3′ motif, (E) three non-overlapping 5′-GAAGAAG-3′ motifs and (F) a single E. coli KOPS (5′-GGGCAGGG-3′). The best-fitted model curves using a ‘one set of sites’ model are shown (continuous line) with the corresponding stoichiometry values indicated (N).

Figure 5.

The 5′-GAAGAAG-3′ motif controls L. lactis FtsK activity in vivo. (A) Structure of the two FtsK proteins used. Top: wild-type E. coli FtsK with domains and subdomains indicated (grey). Vertical bars represent the transmembrane segments in the N-terminal domain. Coordinates are in AA. Bottom: the C-terminal domain of FtsK has been replaced by its homolog from L. lactis (red) yielding the FtsK_CLl chimeral protein. (B) Measure of the recombination frequencies between dif sites inserted in direct repetition at the dif position on the E. coli chromosome. The relevant structure of the dif-lacI-dif cassette and its derivative after insertion of KOPS motifs is shown. Yellow arrows: Escherichia coli KOPS motifs (5′-GGGCAGGG-3′); red arrows: 5′-GAAGAAG-3′ motifs. Consecutive motifs were separated by 6 bp of random DNA (see Figure 1E and 4C). Bars show means of five independent measures (shown right of the bars) with standard deviations. Frequencies are in percent per cell per generation. Grey bars: strains producing wt FtsK; Red bars: strains producing the FtsK_CLl protein. (C) Distribution of the 5′-GAAGAAG-3′ motif on the L. lactis chromosome. The graphs were obtained as in Figure 2. Coordinates are in base pair. Grey arrowheads show the position of the chromosome dimer resolution site.