Intergeneric conjugal gene transfer from Escherichia coli to the sweet potato pathogen Streptomyces ipomoeae.

AIMS: To develop an effective gene transfer system for Streptomyces ipomoeae, the causative agent of soil rot disease of sweet potatoes. METHODS AND RESULTS: Of the several methods investigated, introduction of genes into S. ipomoeae could only be achieved using intergeneric conjugation from Escherichia coli. However, even results for that method varied greatly and were dependent on using particular media for S. ipomoeae spore preparation and conjugation. Transconjugant to recipient ratios as great as 4.1 x 10(-5) were achieved when International Streptomyces Project Medium 4 was used for both sporulation and conjugation protocols. Both site-specifically integrating and autonomously replicating plasmids could be introduced and maintained in S. ipomoeae, and plasmids could be introduced with approximate equivalent frequencies from either methyl-proficient or methyl- deficient E. coli donors; the latter result indicates a likely absence of relevant methyl-specific restriction in S. ipomoeae. CONCLUSIONS: Efficient transfer of genes into S. ipomoeae was achieved here by using an optimized intergeneric mating procedure. SIGNIFICANCE AND IMPACT OF THE STUDY: The described protocol will facilitate further genetic manipulation of this agriculturally important pathogen.

Expression characteristics of the transfer-related kilB gene product of Streptomyces plasmid pIJ101: implications for the plasmid spread function.

Intermycelial transfer of Streptomyces plasmid pIJ101 occurs prior to cellular differentiation and is mediated by plasmid functions that are also required for production of zones of growth-inhibited recipient cells (i.e., pocks) that develop around individual donors during mating on agar medium. Several other pIJ101 functions, including that of the kilB gene, whose unregulated expression on pIJ101 is lethal, are required for normal pock size and so have been postulated to mediate intramycelial spread of the plasmid throughout recipient cells. Using antibodies raised against a KilB fusion protein expressed in Escherichia coli, native KilB protein was detected throughout development of pIJ101-containing Streptomyces lividans cells, with the concentration of KilB increasing dramatically and reaching a maximum during the final stages (i.e., sporulation and secondary metabolism) of cellular differentiation. Insertion of the kilB gene of pIJ101 into the S. lividans chromosome in cells lacking the pIJ101 KorB protein, which normally represses kilB gene transcription, resulted in elevated but still temporally increasing amounts of KilB. The increased expression or accumulation of the KilB spread protein throughout cellular differentiation of S. lividans, which leads to maximum KilB concentrations during developmental stages that occur far later than when intermycelial transfer of pIJ101 is mediated, supports the existence of a subsequent intramycelial component to the pIJ101 spread function. The results also suggest that intramycelial spread of pIJ101 molecules within the recipient extends beyond intercompartmental movements within the substrate mycelia and includes undetermined steps within the spore-yielding aerial hyphae as well.

Unraveling the essential role in conjugation of the Tra protein of Streptomyces lividans plasmid pIJ101.

Plasmid pIJ101 from Streptomyces lividans encodes a single gene, tra, that is essential for both plasmid transfer and mobilization of chromosomes during mating. The tra gene product (Tra) is a membrane protein, a portion of which shows similarity to transfer proteins of other streptomycete plasmids as well as additional bacterial chromosome partitioning proteins. This paper reviews past and present work that has focused on elucidating the precise role of the Tra protein of pIJ101 in conjugation in Streptomyces.

Minimal and contributing sequence determinants of the cis-acting locus of transfer (clt) of streptomycete plasmid pIJ101 occur within an intrinsically curved plasmid region.

Efficient interbacterial transfer of streptomycete plasmid pIJ101 requires the pIJ101 tra gene, as well as a cis-acting plasmid function known as clt. Here we show that the minimal pIJ101 clt locus consists of a sequence no greater than 54 bp in size that includes essential inverted-repeat and direct-repeat sequences and is located in close proximity to the 3' end of the korB regulatory gene. Evidence that sequences extending beyond the minimal locus and into the korB open reading frame influence clt transfer function and demonstration that clt-korB sequences are intrinsically curved raise the possibility that higher-order structuring of DNA and protein within this plasmid region may be an inherent feature of efficient pIJ101 transfer.

Mutational analysis of the tra locus of the broad-host-range Streptomyces plasmid pIJ101.

The tra gene of Streptomyces lividans plasmid pIJ101 encodes a 621-amino-acid protein that can mediate both plasmid transfer and the interbacterial transfer of chromosomal genes (i.e., chromosome-mobilizing ability [Cma]) during mating. Here we report the results of in-frame insertional mutagenesis studies aimed at defining regions of Tra required for these functions. While hexameric linker insertions throughout the tra gene affected plasmid and chromosomal gene transfer, insertions in a 200-amino-acid region of the Tra protein that contains presumed nucleotide-binding motifs and that is widely conserved among a functionally diverse family of bacterial and plasmid proteins (K. J. Begg, S. J. Dewar, and W. D. Donachie, J. Bacteriol. 177:6211-6222, 1995) had especially prominent effects on both functions. Insertions near the N terminus of Tra reduced Cma for either circular or linear host chromosomes to a much greater extent than pIJ101 plasmid transfer. Our results suggest that Cma involves Tra functions incremental to those needed for plasmid DNA transfer.

Complementation of conjugation functions of Streptomyces lividans plasmid pIJ101 by the related Streptomyces plasmid pSB24.2.

A database search revealed extensive sequence similarity between Streptomyces lividans plasmid pIJ101 and Streptomyces plasmid pSB24. 2, which is a deletion derivative of Streptomyces cyanogenus plasmid pSB24.1. The high degree of relatedness between the two plasmids allowed the construction of a genetic map of pSB24.2, consisting of putative transfer and replication loci. Two pSB24.2 loci, namely, the cis-acting locus for transfer (clt) and the transfer-associated korB gene, were shown to be capable of complementing the pIJ101 clt and korB functions, respectively, a result that is consistent with the notion that pIJ101 and the parental plasmid pSB24.1 encode highly similar, if not identical, conjugation systems.

Diversity Within Streptomyces ipomoeae Based on Inhibitory Interactions, rep-PCR, and Plasmid Profiles.

ABSTRACT Streptomyces soil rot is a destructive disease of sweetpotato (Ipomoea batatas) that causes yield loss resulting from decay of the feeder root system and reduced quality due to the presence of necrotic lesions on the storage roots. It is managed by the use of resistant cultivars, but variability of the pathogen has not been previously assessed. This study compared 36 strains of the pathogen Streptomyces ipomoeae from different locations in the United States and Japan. The strains could be separated into three groups on the basis of their ability to inhibit the growth of one another in in vitro assays. Although some strains contained plasmids of approximately 18, 42, or 270 kb in size, plasmid profiles did not correspond to inhibition grouping. Fingerprinting by repetitive element-based polymerase chain reaction (rep-PCR) using outwardly facing primers for the BOX, enterobacterial repetitive intergenic consensus (ERIC), and repetitive extragenic palindromic (REP) sequences indicated relatively high genomic homogeneity within S. ipomoeae. However, cluster analysis of similarity coefficients among strains using rep-PCR data revealed clusters that correlated with the inhibition grouping. The neotype strain of S. ipomoeae had lower similarity values by rep-PCR than any of the other strains and could not be grouped by inhibitory interactions.

Plasmid transfer and expression of the transfer (tra) gene product of plasmid pIJ101 are temporally regulated during the Streptomyces lividans life cycle.

We previously have shown that a chromosomally integrated copy of the tra gene of plasmid pIJ101 under the control of the KorA repressor protein, which regulates transcription of tra as well as its own synthesis, can promote the conjugal transfer of both chromosomal and plasmid genes in Streptomyces lividans. Using an antibody generated against a fusion protein containing the C-terminal portion of Tra, we show here that this essential conjugation protein is present in membrane fractions of both surface-grown S. lividans, which mate readily, and of cells grown in liquid culture, where mating has not been found. Expression of Tra during the S. lividans life cycle was temporally regulated and was reduced late during vegetative growth so that little or no Tra protein was detected in cells as they began to differentiate morphologically and produce secondary metabolites. Comparison of the membrane concentration of Tra protein with tra mRNA concentration during the S. lividans life cycle indicated that the disappearance of Tra is post-transcriptionally controlled and thus is not mediated by KorA. The results of 'interrupted mating' experiments, together with the time of appearance of Tra in S. lividans membranes, indicate that the intermycelial transfer of pIJ101 in S. lividans is complete by the onset of cellular differentiation.

Transfer of the plJ101 plasmid in Streptomyces lividans requires a cis-acting function dispensable for chromosomal gene transfer.

The tra gene of Streptomyces lividans plasmid plJ101 is required for both plasmid DNA transfer and plJ101-induced mobilization of chromosomal genes during mating. We show that a chromosomally inserted copy of tra mediates transfer of chromosomal DNA at high frequency but promotes efficient transfer of plasmids only when they contain a previously unknown locus, here named clt. Insertional mutation or deletion of clt from plJ101 reduced plasmid transfer mediated by either plasmid-borne or chromosomally located tra by at least three orders of magnitude, abolished the transfer-associated pocking phenomenon, and interfered with the ability of tra+ plasmids to promote transfer of chromosomal DNA. Our results indicate that plasmid transfer in S. lividans involves a cis-acting function dispensable for chromosomal gene transfer and imply that either the S. lividans chromosome encodes its own clt-like function or, alternatively, that transfer of plasmid and chromosomal DNA occurs by different mechanisms.

Promoter and operator determinants for fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system.

The fepA-entD and fes-entF operons in the enterobactin synthesis and transport system are divergently transcribed from overlapping promoters, and both are inhibited by the Fur repressor protein under iron-replete conditions. A plasmid harboring divergent fepA'-phoA and fes-entF'-'lacZ fusions, both under the control of this bidirectional regulatory region, was constructed for the purpose of monitoring changes in expression of the two operons simultaneously. Deletion analysis, site-directed mutagenesis, and primer extension were employed to define both a single promoter governing the expression of fes-entF and two tandemly arranged promoters giving rise to the opposing fepA-entD transcript. A single Fur-binding site that coordinately regulates the expression of all transcripts emanating from this control region was identified by in vitro protection from DNase I nicking. The substitution of one base pair in the Fur recognition sequence relieved Fur repression but did not change the in vitro affinity of Fur for its binding site. Additional mutations in a limited region outside of the promoter determinants for either transcript inhibited expression of both fes and fepA. These observations suggest a mechanism of Fur-mediated regulation in this compact control region that may involve other regulatory components.

Localization and nucleotide sequences of genes mediating site-specific recombination of the SLP1 element in Streptomyces lividans.

SLP1 is a 17.2-kbp genetic element indigenous to the Streptomyces coelicolor chromosome. During conjugation, SLP1 can undergo excision and subsequent site-specific integration into the chromosomes of recipient cells. We report here the localization, nucleotide sequences, and initial characterization of the genes mediating these recombination events. A region of SLP1 adjacent to the previously identified site of integration, attP, was found to be sufficient to promote site-specific integration of an unrelated Streptomyces plasmid. Nucleotide sequence analysis of a 2.2-kb segment of this region reveals two open reading frames that are adjacent to and transcribed toward the attP site. One of these, the 1,365-bp int gene of SLP1, encodes a predicted 50.6-kDa basic protein having substantial amino acid sequence similarity to a family of site-specific recombinases that includes the Escherichia coli bacteriophage lambda integrase. A linker insertion in the 5' end of the cloned int gene prevents integration, indicating that Int is essential for promoting integration. An open reading frame (orf61) lying immediately 5' to int encodes a predicted 7.1-kDa basic peptide showing limited sequence similarity to the excisionase (xis) genes of other site-specific recombination systems.

The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product.

The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a transmembrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA-entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5' to entD, along with RNA hybridization data, suggest that an iron-regulated transcript contains both fepA and entD.

Transcriptional mapping and nucleotide sequence of the Escherichia coli fepA-fes enterobactin region. Identification of a unique iron-regulated bidirectional promoter.

The iron-controlled fepA and fes-entF transcripts from the Escherichia coli enterobactin gene complex are expressed divergently from a limited genetic region, thereby suggesting the existence of a single, possibly overlapping promoter junction for these genes. The nucleotide sequence of a 1,997-base pair HpaI fragment specific for this genetic region allowed for the identification of an 1,122-base pair open reading frame as the previously uncharacterized fes gene. Its product, Fes (approximately Mr 42,573) plays an essential but as yet ambiguous role in the release of ferric iron from the ligand. An additional small open reading frame of 216 nucleotides (encoding a potential product of calculated Mr 8,271) was also identified between fes and entF. A portion of the remaining nucleotide sequence defined a 320-base pair control region for both the fepA and fes-entF messages. Primer extension analyses placed the major in vivo transcription initiation sites to within 18 nucleotides of one another, thereby revealing a novel, extensively overlapping bidirectional promoter as well as long dual leader transcripts. This promoter region contains multiple overlapping nucleotide stretches which show strong homology to the consensus Fur repressor-binding sequence, forms of which are found in all E. coli iron-regulated promoters characterized to date.

Molecular characterization of the Escherichia coli enterobactin cistron entF and coupled expression of entF and the fes gene.

The Escherichia coli entF gene, which encodes the serine-activating enzyme involved in enterobactin synthesis, has been localized to a 4.7-kilobase-pair DNA fragment inserted in the vector pBR328. This recombinant molecule, pITS32, restored the ability of an entF mutant to grow on low-iron medium and to produce enterobactin. Examination of its translation products by minicell and electrophoretic analyses revealed a protein of approximately 160,000 daltons, which we identified as the EntF protein. A small DNA segment from pITS32 containing the translational start site for entF allowed the low constitutive expression of beta-galactosidase when cloned (pITS301) upstream of the lacZ structural gene in the vector pMC1403. In contrast, a clone (pITS312) containing the identical entF-lacZ fusion and a larger region upstream of entF including the entire fes gene and extending into the fepA gene (whose transcription is in the opposite direction relative to entF) expressed beta-galactosidase in high yet inducible amounts in response to fluctuations in the metabolic iron concentration. Transposon insertion mutations in the fes gene but not an insertion near the 5' region of fepA in pITS312 reduced this high inducible expression to the low constitutive level seen for pITS301. These observations are most readily explained by the presence of a regulatory region located upstream of fes which mediates the iron-regulated expression of a transcript that includes the fes and entF genes.

Isolation and characterization of the Vibrio cholerae recA gene.

A 3.6-kilobase PstI fragment was isolated from a Vibrio cholerae chromosomal DNA library and shown to encode RecA-like activity in complementation studies with Escherichia coli recA mutants. Although DNA hybridization experiments failed to detect any homology between the E. coli and V. cholerae recA genes, hyperimmune antiserum produced against purified E. coli RecA protein recognized epitopes shared by the V. cholerae protein. The V. cholerae chromosomal fragments, when cloned and transferred to E. coli, provided the missing recA functions, including resistance to the alkylating agent methyl methanesulfonate, resistance to UV irradiation, and promotion of homologous recombination in Hfr mating experiments.