Generalized transduction in Streptomyces coelicolor

Autor: Julie A. Burke, Janet Westpheling, David A. Schneider
Rok vydání: 2001
Předmět:
Zdroj: Proceedings of the National Academy of Sciences. 98:6289-6294
ISSN: 1091-6490
0027-8424
DOI: 10.1073/pnas.101589398
Popis: It would be difficult to overestimate the contribution generalized transduction has made to the study of prokaryote biology since the discovery of phage P22 in Salmonella typhimurium and phage P1 in Escherichia coli in the early 1950s (1, 2). Generalized transduction remains an important genetic tool for fine-structure mapping, site-directed mutagenesis, and transposon-related genetic manipulation even in highly developed model systems such as E. coli, S. typhimurium, and Bacillus subtilis. In organisms such as Streptomyces coelicolor the need for such genetic tools is even greater. S. coelicolor is the most genetically well characterized actinomycete, an extremely diverse group of filamentous prokaryotic organisms, which includes the producers of the majority of therapeutically important natural product antibiotics (3). The mycelial growth mode and sporulation cycle of these unusual bacteria also offers one of the most dramatic examples of prokaryotic morphological differentiation. They grow as multicellular, multinucleoid, branching hyphae that penetrate and solubilize organic material in the soil, forming a mycelial mass. In response to extracellular signals (4), they initiate a cycle of differentiation that begins with the production of aerial hyphae that septate into uninucleoid compartments that give rise to spores. S. coelicolor, therefore, has attracted wide interest as a model system for understanding how bacteria regulate changes in gene expression during differentiation and coordinate these changes temporally and spatially with complex changes in cellular morphology (5, 6). Despite their interesting biology and commercial importance, relatively little is known about the gene expression pathways that regulate morphological development or antibiotic biosynthesis. A major limitation in the study of morphogenesis in Streptomyces has been the inability to clone genes identified by morphological mutations. For example, despite intensive efforts to clone and study the bld loci, sites of mutations that cause pleiotropic defects in morphological development, antibiotic production, and extracellular signaling, only five bld genes have been characterized at the molecular level since the first description of these mutations in 1976 (7–11). The reasons for such slow progress are that the obvious genetic approaches for recovering genes identified by chemically induced mutations have been difficult to implement in Streptomyces. Relatively few genetic markers exist, making fine structure mapping impossible. Cloning by complementation is slow and tedious. Transformation of plasmid libraries constructed in either E. coli or Streptomyces is inefficient, and the libraries are often incomplete. Transposons indigenous to Streptomyces (12, 13) or derived from other bacteria (14, 15) have been identified, but they have not proven effective for insertional mutagenesis in S. coelicolor, in part due to the nature of transposon delivery systems currently available that typically depend on temperature-sensitive plasmid vectors. Curing is not effective, and exposure to high temperatures is mutagenic, which results in a high background of mutations not caused by transposition. Mutations resulting from transposition are not easily distinguished from this background because the genetic tools have not been available to establish causal relationships between transposon insertions and mutant phenotypes. Gehring et al. (16) recently have developed a method for efficient in vitro transposition of Tn5 in S. coelicolor that takes advantage of chromosomal transformation (17) to show linkage between phenotypes of interest and transposon insertions. Cotransformation, however, may not be efficient enough to allow genome scale screening of transposon-generated mutations. It was widely recognized that an efficient system for generalized transduction was needed in Streptomyces, but attempts to identify such phages for S. coelicolor were unsuccessful, as were attempts to transduce markers by the most extensively studied Streptomyces phages PhiC31, VP5, and R4 (18). A generalized transducing phage was reported for Streptomyces venezuelae (19), but it was thought to be an anomaly and somehow specific to S. venezuelae because the approaches used to identify transducing phages for S. venezuelae did not work for S. coelicolor. It was even suggested that most streptomycetes lacked a host factor necessary for the propagation of generalized transducing phages and were thus incapable of supporting generalized transduction (20). Here we report the isolation and partial characterization of four phages that are capable of efficient generalized transduction in S. coelicolor. These phages exhibit a broad host range and will very likely be useful for genetic manipulation of other Streptomyces spp. At least three of the phages transduce plasmids between strains of S. coelicolor and other Streptomyces spp. The ability to transduce plasmids from S. coelicolor to S. verticillus, for example, a species that has been refractory to genetic manipulation will allow genetic analysis of the bleomycin biosynthetic pathway. We also describe methods for minimizing superinfection killing during the selection of transductants that may allow the isolation and characterization of generalized transducing phages for any Streptomyces spp.
Databáze: OpenAIRE
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