A septal chromosome segregator protein evolved into a conjugative DNA-translocator protein

Abstract

Streptomycetes, Gram-positive soil bacteria well known for the production of antibiotics feature a unique conjugative DNA transfer system. In contrast to classical conjugation which is characterized by the secretion of a pilot protein covalently linked to a single-stranded DNA molecule, in Streptomyces a double-stranded DNA molecule is translocated during conjugative transfer. This transfer involves a single plasmid encoded protein, TraB. A detailed biochemical and biophysical characterization of TraB, revealed a close relationship to FtsK, mediating chromosome segregation during bacterial cell division. TraB translocates plasmid DNA by recognizing 8-bp direct repeats located in a specific plasmid region clt. Similar sequences accidentally also occur on chromosomes and have been shown to be bound by TraB. We suggest that TraB mobilizes chromosomal genes by the interaction with these chromosomal clt-like sequences not relying on the integration of the conjugative plasmid into the chromosome.

Elucidation of the molecular function of TraB highlights how nature is able to develop new machineries from other cellular processes. Whereas DNA transfer by classical conjugation is done by specialized type IV protein secretion systems, for the conjugative DNA transfer in Streptomyces a chromosome segregation system was rebuild.

Bacteria evolved distinct machineries to translocate DNA across membranes. Conjugation, the transfer of DNA between two distinct cells, involves a sophisticated protein secretion system that translocates a pilot protein with a covalently attached single-stranded DNA molecule.^1–3 In contrast, segregation of chromosomal DNA during bacterial cell division is characterized by the translocation of doublestranded DNA.⁴

Chromosome Segregation During Cell Division and Sporulation by FtsK

Chromosome segregation is mediated by the septal DNA translocator FtsK, a component of the divisome in nearly all bacteria. FtsK is a multi-domain protein which has several distinct functions during cell division.⁵ It is a HerA-like ATPase translocating double-stranded chromosomal DNA through the closing septum to avoid guillotining of the chromosomes.⁴ By interaction with the XerCD recombinase and topoisomerase IV, FtsK supports decatenation of intertwined chromosomes and the resolution of chromosomal dimers at dif.^6,7 Polarized DNA translocation is achieved by the specific interaction of the C-terminal γ-domain of FtsK with KOPS (FtsK orienting polar sequences).^8,9 KOPS are 8-bp sequence motifs that are unevenly distributed on bacterial chromosomes with a strong bias from the origin to the dif region. It is still a matter of debate, whether KOPS simply represent assembly sites of FtsK or whether FtsK hexamers are able to assemble on any DNA and its DNA translocation activity is modulated by the interaction with KOPS.^10–13

In the gram positive mycelium forming soil bacterium Streptomyces FtsK has a major role during sporulation.¹⁴ In contrast to most other bacteria that divide by binary fission, Streptomyces forms a multiply branching mycelium by apical tip extension which contains only few septal cross walls.¹⁵ The mycelial compartments harbor multiple copies of a linear chromosome. Upon nutrient limitation Streptomyces undergoes morphological differentiation by erecting unbranched aerial hyphae. Subsequently, more than 50 septa are simultaneously formed ending up in a chain of spores. This process involves the Mre-proteins which determine rod-shaped morphology in other bacterial species.¹⁶ Since Streptomyces mycelium contains multiple chromosomal copies, the main role of FtsK in Streptomyces is the segregation of the chromosomes during sporulation to generate spores that contain a single chromosome. Whereas in most other bacteria FtsK segregates a circular chromosome, Streptomyces FtsK has to translocate a linear chromosome.

Conjugative DNA-Translocation by the FtsK-Homolog TraB

Streptomyces was reported to possess unusual conjugative plasmids that require only a single plasmid encoded protein, TraB, for the transfer from the plasmid carrying donor into a plasmid free recipient.^17,18 TraB was shown to interact with the cis acting locus of transfer clt,¹⁹ a small (<200 bp) region required for conjugative plasmid transfer.¹⁷ Deletion of a clt-locus dramatically reduces frequency of plasmid transfer.²⁰ Insertion of a clt locus into a non-transferable plasmid allows its mobilization if the corresponding TraB protein is provided in trans.¹⁷

In adaptation to the mycelial growth, conjugative Streptomyces plasmids encode a second DNA translocation system for the subsequent spreading of the newly transferred plasmid within the recipient mycelium resulting in its rapid colonization. For these secondary DNA-translocations across the septal crosswalls of the recipient mycelium, called plasmid spreading, TraB has to interact with a multi protein complex of several Spd proteins, whose molecular functions are widely unknown.^18,21

The TraB protein is an ATPase which resembles FtsK in many aspects. It has an identical domain architecture, with an N-terminal membrane anchor region.²² This region which might be involved in proper protein localization is connected by a so called linker region to the DNA-translocase domain. The linker region was discussed to be involved in protein interaction but the exact function of this region still awaits its elucidation. The C-terminal wHTH domains of Streptomyces TraB proteins display a previously unrecognized homology to the FtsK γ-domain. A more detailed comparison of the respective domains of the closely related TraB proteins of the plasmids pSG5, pSVH1 and pFP11 revealed highest similarity within the DNA translocase region. Whereas the N-terminal 276 aa of TraB_pSVH1 showed an identity of 75% and 54% to those of TraB_pSG5 and TraB_pFP11, respectively, the DNA-translocase domain (TraB_{psVH1_277–688}) had an identity of 80% and 77%. Lowest similarity was observed for the wHTH domain (TraB_{pSVH1_689–772}) which had an identity of 58% and 49%. The variability of the TraB wHTH domains demonstrates the fast evolution of plasmid TraB proteins as an adaptation to the need of recognizing and transferring a specific plasmid molecule. In contrast, FtsK proteins of different bacteria hardly differ in their γ-domains.

Moreover, the mode of DNA recognition of FtsK and TraB proteins is very similar. Whereas the γ-domain of FtsK recognizes 8-bp KOPS, the wHTH folds of TraB protein interact with 8-bp TRS (TraB Recognition Sequence) repeats, present in the clt region of the corresponding plasmid.²² Generation of chimeric TraB proteins originating from different Streptomyces plasmids located the protein region determining sequence specific DNA recognition to helix α3 of the wHTH fold. Exchange of only 13 aa of TraB_pSVH1 against the 13 aa, comprising helix α3 of TraB_pIJ101 generated a protein with a switched specificity, now recognizing the clt of plasmid pIJ101.²² This is remarkably since recognition of the 8-bp TRS repeats does most probable not involve a monomer but three subunits of the TraB hexamer, as demonstrated for the FtsK γ-domain.¹² During DNA binding of FtsK, each monomer interacts with different bases of the KOPS.

Localization of the sequence specificity determining TraB domain to the wHTH motif and helix α3 explains the high variability of the C-termini of TraB proteins to meet the requirements of recognizing different plasmid clt loci.

Evolution of Plasmid-Encoded TraB Proteins from FtsK-Like Ancestor Proteins

On the one hand, the plasmid encoded TraB proteins are highly similar to FtsK, revealing a common ancestor. On the other hand, they are specifically adapted to the recognition of their cognate clt to direct transfer of a specific plasmid molecule. In contrast to FtsK which mediates DNA translocation between two cellular compartments, Streptomyces TraB proteins are able to translocate DNA between two distinct cells. Rendering a septal chromosome translocator protein into a conjugative plasmid translocator requires three distinct adaptations:

Localization.

Whereas FtsK interacts with other proteins of the divisome at the division septum, Streptomyces conjugation involves the tips, as it is suggested by the localization of a TraB-eGFP fusion protein.¹⁹

Specific DNA recognition.

Whereas FtsK interacts with KOPS distributed all over the chromosome to determine polarity of DNA-translocation, TraB proteins specifically recognize the clt region of a Streptomyces plasmid.²²

Regulation.

The main function of Streptomyces FtsK is the generation of uninuclear spores during septation of the aerial mycelium. Expression of Streptomyces FtsK is therefore highest at the onset of sporulation.²³ In contrast, plasmid transfer takes only place at early time points, when Streptomyces grows as substrate mycelium. Expression of traB is in all Streptomyces plasmids under control of a GntR-type transcriptional repressor which responds to an unknown signal resulting in the expression of TraB during mating.

Transfer of Chromosomal Markers by the Interaction of TraB with clt-like Chromosomal Sequences clc

The plasmid clt-locus containing the 8-bp TRS repeats is only required for plasmid transfer. Surprisingly, the clt-locus is not involved in the transfer of chromosomal marker genes.¹⁷ This is in contrast to the known mechanism of chromosome mobilization in type IV secretion, which relies on the presence of the cis-acting oriT in the chromosome to initiate DNA transfer in a polarized manner.² Identification of multiple clt-like chromosomal sequences (clc) in the S. coelicolor genome provides an explanation for the dispensability of a plasmid clt in the transfer of chromosomal marker genes, though the in vivo functional proof of clcs is still missing.

A PATSCAN analysis of the S. coelicolor genomic sequence for pSVH1 clt-like sequences (four copies of GAC CCG GA, with a spacing of up to 13 bp and allowing one miss match) identified 25 hits (Table 1). Streptomycetes are known to contain multiple plasmids integrated in their genome. S. coelicolor carries SLP1 and several copies of a pSAM2-like plasmid at different positions in the chromosome. Therefore, one could speculate that the clt-like clc sequences are part of or remnants of mobile genetic elements. However, a detailed analysis of the S. coelicolor clt_pSVH1-like sequences in the genome showed that in most cases they were located within genes without disrupting their coding region. E.g., SCO6684 (ramB, probably encoding an ABC transporter) and SCO4600 (encoding the NADH dehydrogenase subunit NuoB2) carry highly repeated sequences of 150 bp (GGC CCG GAG CCG GAG CCG GAG CCG GAG CCG GAG CCG GAG CCG GAG CCG GAG TTG GGG TCG GGG CTG GAG CCA GAG CCA GAG CCA GCG TCA GAG CCA GAG TCG GGG CCG AGC ACC CGA GCG CAT CGG CGG CGG CAT TCG CGG TGC ACA CC) and 123 bp (CCG CCG GAG CCG GAG CCG GAG CCG GAG CCG GAG CCG GAG CCG GAG CCG GCC AGG GGC CGG GCC AGG GGC CGG GCT CGG GTG AGG GCA CGG ACA CGG GCT CGG ATG CGG GGC GAG GCC GAT GA) length, respectively. These insertions contain four TRS repeats (underlined) and encode amino acids GPE PEP EPE PEP EPE PEL GSG LEP EPE PAS EPE SGP STA SAS AAA FAV HT and PPE PEP EPE PEP EPE PGQ GPG QGP GSG EGT DTG SDA GRG R. These insertions are only found in SCO6684 and SCO4600 but not in the corresponding homologs of S. avermitilis [SAV7500 (amfA); SAV4882] or in those of other Streptomyces genomes.

Table 1.

PAT SCAN analysis^* of the S. coelicolor genome for the presence of clt_pSVH1-like chromosomal sequences (clc)^*

Position	Gene Number	Function	TRS
0347832	0343	Hypothetical protein	GAG CCG GA-N9-AAG CCG GA-N4-GAG CCG GT-N4-AAG CCG GA
0512294	0492	Putative peptide synthetase	GAC CGG GA-N8-GAC CGG GA-N7-GAC CGG GA-N7-GAC CGG GG
1355848	1284	Putative aminotransferase	GAC CCG GT-N1-GAC CCG GT-N1-GAC CCG GT-N1-GAC GCG GT
1864064	1744	Putative sensor kinase	GAC CGG GA-N7-GAC CGG GA-N7-GGC CGG GA-N7-GAC CGG GG
2178866	2028	Putative membrane protein	AAC CCG GA-N13-GAC CCG GA-N13-AAC CCG GA-N13-GAC CCG GA
2633080	2450	Put. serine/threonine protein kinase	GAC CGG GA-N4-GAC CGG GA-N4-GAC CGG GA-N5-GGC CGG GA
2817911	2599	Conserved hypothetical protein	GCC CCG GA-N10-GCC CCG GA-N10-GCC CCG GA-N10-GCC CCG GA
2927648	2684	Putative ATP-binding membrane protein	GAC CGG GA-N1-GAC CGG GG-N7-GAC CGC GA-N13-GAC CGG GC
4021120	3642	Putative membrane protein	GAC CCC GA-N4-GAC CCC GA-N4-GAC CCC GA-N4-GAC CCC GA
4013872	3635_3636	intergenic	GAC TCG GA-N1-GAC TCG GG-N1-GAC CCG GA-N1-GAC TCG GG
4418787	4022	Putative glycosyl transferase	GAC CCG CA-N7-GAC CCG CA-N7-GAC CCG CA-N7-GAC CCG CA
4595544	4185_4186	intergenic	GAT CCG GA-N8-GAT CCG GA-N8-GAC CCG GA-N8-GAC CCG GA
4901587	4481	Probable serine/threonine protein kinase	GAG CCG GA-N4-GAG CCA GA-N4-GAG CCG GA-N10-GAG CCG GA
5024597	4600	NADH dehydrogenase subunit NuoB2	GAG CCG GA-N4-GAG CCG GA-N4-GAG CCG GA-N4-GAG CCG GG
6396726	5842	Conserved hypothetical protein	GGC CCG GA-N1-GGA CCG GA-N1-GGG CCG GA-N1-GGA CCG GA
7086737	6417_6418	intergenic	GAC CCG GC-N5-GTC CCG GC-N8-GTC CCG GC-N8-GTC CCG GC
7425531	6684	Putative ABC transporter	GAG CCG GA-N4-GAG CCG GA-N4-GAG CCG GA-N4-GAG CCG GA
7724967	6956	Putative oxidoreductase	GAG CCG GA-N10-GAG CCG GA-N10-GAG CCG GA-N10-GAG CCG GG
7914243	7123	Putative acyltransferase	GAG CCG GA-N4-GAG CCG GA-N4-GAG CCG GA-N10-GAG CCG GC
8189558	7375	Putative acetyltransferase	GAC ACG GA-N1-GAC ATG GA-N1-GGC ACG GA-N1-GAC ACG GA
8189621	7375	Putative acetyltransferase	GAC ACG GA-N10-GGC ACG GA-N1-GAC ACG CA-N1-GAC ACG CA
8236721	7422	Putative sensor histidine-kinase	GAC CGG GA-N13-GAC CGG GA-N4-GAC CAG GA-N10-GAC CGG GA
8236778	7422	Putative sensor histidine-kinase	GAC CAG GA-N4-GAC CAG GA-N4-GAC CAG GA-N4-GAC CGT GA
8475922	7648_7649	intergenic	GAA CCG GA-N4-GAA CCG GA-N4-GAA CCG GG-NN4-GAA CCG GA
8524509	7689	Putative ABC transporter AT P-binding protein	GGC CCG GA-N6-GGC CCG GA-N7-AGC CCG GA-N7-AGC CCG GA

^*p1 = GACCCGGA [1,0,0] 0…13 p1[1,0,0] 0…13 p1 [1,0,0] 0…13 p1 [1,0,0]

Another example is SCO4022 encoding a glycosyl transferase. SCO4022 carries at is 3′end an extension containing five TRS repeats and which encodes PDA PPT THT PDP HLP DPH LPD PHL PDP HLP DPH RTW SDA R*. Again, this C-terminal extension is not present in the homologs of other Streptomyces species (e.g., SAV4196), clearly suggesting that it represents an extra insertion in S. coelicolor genes which does not contribute to the activity of the respective enzyme. Since no remnants of a mobile element are detected in the surrounding regions, these insertion might have been generated by an unknown mechanism without involvement of a plasmid.

Conjugative plasmids of other bacteria transfer chromosomal markers only at low frequencies, since mobilization requires preceding integration at specific chromosomal sites. This indicates a selective pressure for the plasmids to acquire genetic traits, as resistance, degradation of xenobiotics, pathogenicity or biosynthetic capabilities to provide an advantage to the host cell. With the exception of the giant linear plasmids, such traits are normally not found on Streptomyces plasmids. The ability to efficiently mobilize chromosomal markers without plasmid integration might substitute the presence of plasmid encoded genetic traits. Moreover, given the ubiquity of small plasmids in the population, one can speculate that there is an adaptive selection for the presence of clc sequences in Streptomyces genomes to benefit from the presence of a plasmid.

Model of the Streptomyces Conjugative DNA-Translocation System; Open Questions

Streptomyces plasmids do not seem to encode a system for establishing mating pairs. Therefore, conjugative DNA transfer occurs only on solid agar during growth as substrate mycelium. Formation of multiple branching hyphae by apical tip extension provides good probability that mating partners will meet.

During conjugation a plasmid has to pass membranes and peptidoglycan (PG) layers of donor and recipient. Nevertheless only very few Streptomyces plasmids encode a lytic transglycosylase.²⁴ Since Streptomyces hyphae grow by apical tip extension, the machinery for PG remodeling has to be present at the hyphal tip. Therefore, the mycelial tips are the most likely sites, where conjugation takes place. This is supported by the localization of TraB at the hyphal tips.¹⁹ Presence of TraB might direct fusion of the walls of the mating partners, maybe by interaction with a chromosomally encoded lytic transglycosylase.

After fusion of the PG-layer, TraB might also govern fusion of the membranes of donor and recipient. For the FtsK homolog SpoIIIE, involved in the translocation of the chromosome into the fore spore of B. subtilis, a membrane fusing activity has been experimentally demonstrated.^25,26 Membrane insertion of TraB hexamers that assembled at clt sequences provide a pore structure for the DNA translocation. Whereas evidence was reported that FtsK of E. coli might not require pore formation for DNA translocation,²⁷ TraB pores were detected in planar black lipid membranes by single channel recordings.²²

TraB was shown to bind to the clt locus non-covalently without processing the plasmid.¹⁹ This raises the question, how TraB is able to translocate a circular DNA molecule. While during cell division the chromosome is already present at the closing septum, allowing FtsK to assemble at KOPS sequences at each chromosomal arm, TraB has to translocate the DNA across an intact membrane. Since it is highly unlikely that a circular molecule can be transported across the membrane, involvement of a further DNA processing enzyme, e.g., a topoisomerase has to be postulated. Such an activity was described for FtsK which stimulates topoisomerase IV activity to allow decatenation of chromosomes.²⁸ Another possibility would be the fusion of the two TraB hexamers to a single channel structure releasing the circular plasmid end into the recipient. This model was developed to explain release of the transferred chromosome during B. subtilis sporulation.²⁹

A further unsolved problem regards the mobilization of chromosomes. Streptomyces chromosomes are linear and carry covalently linked telomeric proteins (TP) at the ends. Due to the molecular size of the central channel, TraB hexamers should not allow translocation of TP-bound DNA. Therefore one has to postulate a mechanism for removal and reattachment of TP during conjugative transfer of chromosomal DNA. For the FtsK homolog SpoIIIE which segregates chromosomal DNA into the forespore during sporulation of Bacillus subtilis, a so called wire stripping activity removing all DNA bound regulatory proteins during DNA translocation has been demonstrated.³⁰

Elucidation of the TraB-mediated conjugative DNA translocation in Streptomyces demonstrates that nature developed at least two distinct plasmid transfer systems from other cellular processes. On the one side, the classical conjugation system found in nearly all gram negative bacteria and many gram positives, which is derived from a protein secretion system. On the other side, the newly characterized Streptomyces transfer system, which evolved from an intracellular chromosome segregation system.

Figure 1.

Model of conjugative DNA translocation in mycelial streptomycetes. After establishing contact between donor (gray) and recipient mycelium (white, dashed lines indicate neighboring mycelial compartments) and partial fusion of the hyphal tips, TraB hexamers (blue) assemble at clt by specifically recognizing 8-bp TRS repeats. The TraB hexamers form pore structures to the recipient and direct the translocation of a double-stranded plasmid molecule. DNA transfer is energized by ATP hydrolysis. Up to now it is unclear (?) whether the circular plasmid DNA has to be processed into a linear molecule and which enzymes might be involved. For further description see text.