INTRODUCTION

Top Abstract Introduction Materials and Methods Results and Discussion References

During growth in their natural soil habitat or on agar media, bacteria of the gram-positive genus Streptomyces proceed through a morphologically and physiologically complex developmental program (6). Following germination of spores, cells filament and grow vegetatively as a multinucleate substrate mycelium. Upon nutrient limitation, growth within the substratum ceases, initiating a developmental process that involves the emergence of branching airborne hyphae as well as the production of secondary metabolites. Eventually, aerial hyphal growth slows and individual hyphae develop further into spore chains.

During a portion of their life cycle, Streptomyces bacteria are able to interact conjugally to promote the transfer of DNA. Plasmids, which are prevalent throughout Streptomyces species, play an essential role in the ability of these bacteria to efficiently transfer both chromosomal and plasmid genes (10). On agar plates, transfer of conjugative plasmids between streptomycete cells from individual donors to a surrounding recipient lawn produces circular regions known as pocks, which consist of transconjugant cells showing transiently slowed growth (4). Upon replica plating onto agar selective for the plasmid, transconjugants grow at normal rates in regions that approximate the original pocks (4, 14).

A distinguishing feature of conjugation in Streptomyces is that few plasmid genes are required for this process. For example, the high-copy-number circular plasmid pIJ101, which was originally isolated from Streptomyces lividans but which has since been found to have a broad host range among streptomycetes, carries a single gene (tra) that enables the efficient intermycelial transfer of pIJ101 to up to 100% of potential recipients during mating, while also promoting the movement of chromosomal genes between mating cells such that up to 1% of all cells following mating are recombinants (12, 14). The latter phenotype is commonly referred to as chromosome-mobilizing ability (Cma) (8). An additional cis-acting plasmid element, clt, which is requisite for efficient pIJ101 transfer, was shown to be dispensable for Cma associated with tra, a result that raises the possibility that the Tra-mediated processes of pIJ101 transfer and chromosome mobilization occur by distinct mechanisms (22).

While insertions into either the tra gene or the clt locus eliminate pocking and plasmid transfer, insertions into three other genes, spdA, spdB, and kilB, reduce pock size and thus plasmid "spread" but have little or no effect on either plasmid transfer or Cma (10). Because of their mutant phenotype, spdA, spdB, and kilB have been proposed to be involved in the intramycelial movement of plasmids within recipient hyphae (10, 14).

The tra gene product (Tra), which was predicted from sequence analysis to be a 621-amino-acid protein (11), is present as a 70-kDa component in membrane fractions of S. lividans cells (21). Although its function remains to be determined, Tra, like transfer proteins of other Streptomyces plasmids, contains a Walker type A motif (11), as well as a less-conserved version of the Walker type B motif (5, 15) for presumptive ATP binding. Alteration of amino acid positions within these same domains of the TraB protein of Streptomyces nigrifaciens plasmid pSN22 demonstrated that such sequences are vital for the conjugal function of this protein (15). Energy production via binding and subsequent hydrolysis of ATP is believed to be an integral feature of multiple proteins involved in the processing and translocation of DNA across cell membranes during conjugation in other bacteria (7).

The ATP binding motifs of the pIJ101 Tra protein as well as those of most other essential transfer proteins of Streptomyces plasmids occur within a larger conserved region of amino acids that is shared by proteins from both gram-negative and gram-positive sources; included in this group are, interestingly, proteins that appear to direct the movement of double-stranded DNA molecules across cellular membranes (3, 28). This intriguing conservation of sequences thus raises the possibility that conjugation in Streptomyces may not occur by the interbacterial movement of single-stranded DNA, as is characteristic for conjugation in other bacteria (25), but rather potentially may occur by a double-stranded DNA transfer mechanism.

As a first step towards elucidating the precise role of the pIJ101 Tra protein in intermycelial DNA transfer in Streptomyces, we have used in-frame insertional mutagenesis to define regions of the protein that are important for conjugation. Two insertions in particular, which are located within the conserved portion of Tra but at sites other than the nucleotide-binding folds, greatly reduced or eliminated both plasmid transfer and Cma activities and therefore appear to define additional functional motifs that are common to all proteins sharing these sequences. Other amino acid insertions towards the N terminus of Tra greatly reduced or eliminated Tra's Cma function while leaving the protein still capable of promoting plasmid transfer at significant frequencies; these insertions may thus identify Tra domains that are devoted solely to the task of chromosome mobilization.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results and Discussion References

Bacterial strains, plasmids, and bacteriological methods. S. lividans TK23 (spc-1) and TK64 (str-6 pro-2) have been described previously (9), while S. lividans ZX7 (13) is a better-sporulating derivative of ZX1 (str-6 pro-2 rec-46) (30). S. lividans YSC27-14 is derived from strain ZX7 and contains a circularized chromosome (16). Escherichia coli strain BRL2288 (Life Technologies Inc., Gaithersburg, Md.) was the host for cloning. Plasmids used in this study are described in Table 1. Transformation procedures for S. lividans and E. coli (9) were as described elsewhere. S. lividans was grown in liquid YEME (yeast extract-malt extract) medium (9) or on solid media, including Luria-Bertani agar (23), R5 medium, R2 medium containing 0.1% yeast extract, and minimal medium (9). Plasmid DNA was isolated using the DNA-binding columns and other reagents from Qiagen (Santa Clara, Calif.) either as described by the manufacturer for E. coli or as modified for Streptomyces (D. F. Brolle, T. Henzler, S. Stefani, A. Weissenborn, D. Zell, and W. Wohlleben, Qiagen News 5:9-10, 1997).

Molecular biology and genetic techniques. Cloning using standard molecular techniques was performed as described previously (23). Nucleotide sequencing using relevant primers was performed manually using -³⁵S-dATP and Sequenase, version 2.0 (U. S. Biochemicals, Cleveland, Ohio), or by automated sequencing on a model 310 genetic analyzer (PE Applied Biosystems Inc., Foster City, Calif.).

In-frame linker mutagenesis. Two methods were used to obtain in-frame insertions of the sequence 5'-GAATTC into the pIJ101 tra gene. In the first method, which was based on the protocol of Barany (1, 2), the phosphorylated hexamer 5'-CGAATT was ligated to purified linear pGSP218 DNA that was generated following partial digestion with AhaII and treatment with calf intestinal phosphatase. Sites of insertion for pGSP218 plasmids containing linkers were identified by double digestions using EcoRI and another relevant enzyme. Clones containing linkers in the tra gene were digested to completion with EcoRI, and the purified linear plasmid DNA was religated to create clones that generally contained only a single linker insertion. The number of linkers present was then verified by nucleotide sequencing across the site of insertion. To construct linker-containing pHYG3 plasmids, the 1.8-kb BglII-PvuII fragment of pSP72X:pHYG3 was replaced with the same fragment derived from pGSP218 containing a linker insertion within tra. Digestion of resulting clones with XhoI, followed by ligation and transformation of S. lividans TK23, allowed for isolation of pHYG3 plasmids that contained the various linker insertions.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials and Methods Results and Discussion References

Effects of amino acid insertions on Tra-dependent conjugal activities. To identify regions of the pIJ101 Tra protein that are important for conjugation, we added primarily two codons using in-frame insertion of linkers (see Materials and Methods) at individual positions throughout the tra gene. These derivatives of the conjugative pIJ101-derived plasmid pHYG3 were then tested for conjugal activities dependent on proper Tra protein function. Combined plasmid spread and transfer were tested by a previously described replica plate assay (15) in which donor spores were seeded along defined lines within a lawn of recipient spores and, following growth for one life cycle, the distance that plasmids had been transmitted outward from the original donor line was determined by replica plating of spores to plates selective for transconjugant cells. The effects of linker insertions on plasmid transfer alone and on Cma were assessed following assays of mating between large, approximately equivalent numbers of S. lividans strain TK23 cells containing each of the linker-containing pHYG3 plasmids and a suitable mating partner (i.e., S. lividans TK64 containing the nonconjugative plasmid pGSP196) (22).

View larger version (32K): [in this window] [in a new window]   FIG. 1.   Positions and conjugative effects of amino acid insertions in the pIJ101 Tra protein. Linkers were inserted at individual positions throughout the pIJ101 tra gene (see Materials and Methods), which resulted in the addition of two to four amino acids at the corresponding positions shown (denoted by the linker numbers) in the Tra protein. Hatched area, conserved region of approximately 200 amino acids that shows homology to a family of proteins from both gram-positive and gram-negative sources (see Results and Discussion). Within the conserved region, the positions of sequences that resemble Walker type A and type B nucleotide-binding folds (5, 11, 15) are indicated. The amino acid sequence at each insert junction is shown using one-letter designations, and inserted amino acids are underlined. Insertion of L388 resulted in the loss of the alanine residue at position 389 of Tra (11). Replica plate assays and matings to determine plasmid transfer and Cma capabilities of each of the indicated plasmids were performed and quantified as described in Materials and Methods. Numbers for the replica plate assay are the averages from three independent experiments, while those for plasmid transfer and Cma are averages from at least four independent matings. "Trace" growth in the replica plate test was <0.5 mm and was patchy (i.e., not continuous around the perimeter of the donor line).

2

Cma deficiencies of L13 and L115 Tra derivatives for linear versus circular host chromosomes. How might the L13 and L115 insertions specifically reduce the ability of the Tra protein to promote Cma? The precise role of Tra in promoting Cma is still unknown, as is the overall mechanism of how chromosomes are mobilized during streptomycete conjugation. However, given that the chromosomes of many Streptomyces species including S. lividans have been shown to be linear (17), we wondered whether these insertions may somehow differentially reduce Tra's ability to promote the transfer of linear DNA (e.g., the chromosome of TK23) while leaving its function in the transfer of circular DNA (e.g., pIJ101) relatively intact. To test this notion, we took advantage of recently constructed strains of S. lividans that contain stable, artificially circularized chromosomes (16, 17). pHYG3 plasmids containing relevant linker insertions in tra were introduced into strain YSC27-14, which contains a circularized chromosome (16), and into its parental strain, ZX7, whose chromosome is in the natural linear configuration (17), and the efficiencies of Cma following matings of these strains with a suitable partner (i.e., S. lividans TK23 containing plasmid pGSP196) (22) were determined.