Construction and analysis of a library for random insertional mutagenesis in Streptococcus pneumoniae: use for recovery of mutants defective in genetic transformation and for identification of essential genes - PubMed (original) (raw)

Construction and analysis of a library for random insertional mutagenesis in Streptococcus pneumoniae: use for recovery of mutants defective in genetic transformation and for identification of essential genes

M S Lee et al. Appl Environ Microbiol. 1999 May.

Abstract

To explore the use of insertion-duplication mutagenesis (IDM) as a random gene disruption mutagenesis tool for genomic analysis of Streptococcus pneumoniae, a large mutagenic library of chimeric plasmids with 300-bp inserts was constructed. The library was large enough to produce 60,000 independent plasmid clones in Escherichia coli. Sequencing of a random sample of 84 of these clones showed that 85% of the plasmids had inserts which were scattered widely over the genome; 80% of these plasmids had 240- to 360-bp inserts, and 60% of the inserts targeted internal regions of apparent open reading frames. Thus, the library was both complex and highly mutagenic. To evaluate the randomness of mutagenesis during recombination and to test the usefulness of the library for obtaining specific classes of nonessential genes, this library was used to seek competence-related genes by constructing a large pneumococcal transformant library derived from 20,000 mutagenic plasmids. After we screened the mutants exhaustively for transformation defects, 114 competence-related insertion mutations were identified. These competence mutations hit most previously known genes required for transformation as well as a new gene with high similarity to the Bacillus subtilis competence gene comFA. Mapping of the mutation sites at these competence loci showed that the mutagenesis was highly random, with no apparent hot spots. The recovery of a high proportion of competence genes and the absence of hot spots for mutational hits together show that such a transformant library is useful for finding various types of nonessential genes throughout the genome. Since a promoterless lacZ reporter vector was used for the construction of the mutagenic plasmid library, it also serves as a random transcriptional fusion library. Finally, use of a valuable feature of IDM, directed gene targeting, also showed that essential genes, which can be targets for new drug designs, could be identified by simple sequencing and transformation reactions. We estimate that the IDM library used in this study could readily achieve about 90% genome coverage.

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Figures

FIG. 1

FIG. 1

Strategy for construction of a random mutagenic library of insertion plasmids. Elements of pEVP3 indicated are ori, a replication origin active in E. coli but not in S. pneumoniae; cat, the gene for chloramphenicol acetyltransferase, which confers Cmr to both hosts; and ′lacZ, the promoterless gene for β-galactosidase. Small open boxes, 300-bp pneumococcal fragments; horizontal lines, linear pEVP3 molecules; dots, replication origins; vertical line, vector-vector joint within a concatemer of pEVP3 and inserts. conc., concentrations.

FIG. 2

FIG. 2

Dependence of the number of plasmid types (L) represented at least once in a sample on the number of transformants collected (M) after transformation by a pool of 100 plasmids in equimolar concentrations. The probability (PL) of representing at least L plasmid types (Materials and Methods) is shown for several numbers of collected transformants (M).

FIG. 3

FIG. 3

Strategy for recovery of sequences adjacent to an integrated pEVP3 plasmid by LMPCR. In the insertion orientation shown, lacZ is transcribed from the same strand as the target gene. Hatched boxes represent duplications of the target region. In this orientation, the targeted gene is truncated at the right end of the left hatched box. Vertical lines indicate the restriction sites closest to the ends of the plasmid insert for each enzyme. Map positions of primers used for LMPCR are shown by flags. Junction fragments produced by digestion of mutant DNA with one of the enzymes shown were attached to a linker oligonucleotide before PCR amplification with primers complementary to the vector and linker. The combinations of restriction enzymes, linkers, PCR primers, and sequencing primers used are shown in Table 2.

FIG. 4

FIG. 4

Distribution of insertions in competence mutants at six loci. Locations, orientations, and structures of plasmid insertions in 114 transformation-defective mutants. Numbers of mutants were 42 at comAB, 38 at cgl, 20 at cel, 4 at cfl, 9 at ccl, and 1 at dal. Mutations are represented above the maps by a pentagon showing the position of the targeting fragment deduced from a sequenced junction. Pentagons pointing to the right indicate vector insertion in the orientation shown in Fig. 3. Open pentagons, insertion-deletions; filled pentagons, insertion-duplications. Putative genes with high levels of similarity to known genes in other organisms are given the prefix “h” (homologue of); those with no significant homology are given “orf” designations. Index genes for these loci are located in TIGR’s data release of 1997 as follows: cclA (contig 4291, bp 1992 to 1333), celA (contig 4139, bp 5654 to 5004), cflA (contig 4155, bp 6454 to 5156), cglA (contig 4194, bp 17495 to 16554), comA (contig 4105, bp 4693 to 6852), and dalA (contig 4179, bp 2168 to 1308). (celB1 and celB2 are fused in strain Rx [35] to form a full-length homologue of B. subtilis comEC.)

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