Chapter 7 : Transformation

Model for DNA uptake in transformation of S. pneumoniae. Double-stranded DNA is irreversibly bound to the cell surface and undergoes single-strand cleavage at random sites, possibly by action of a binding protein. A membrane-located nuclease, EndA, initiates entry of the bound strand by endonucleolytic cleavage of the complementary strand to give a double-strand break. Processive action of EndA 5′ to 3′ degrades the complementary strand to oligonucleotides, which remain outside the cell, while donor strands enter from their 3′ end (half-arrowhead). It is not known whether the strand enters without (a) or with (b) a pilot protein. The entering DNA is covered with a single-strand binding protein (c′).

(A) Chromosomal transformation. Heavy line, donor DNA strand segment. Thin line, chromosomal DNA. M and m, marker difference between donor and recipient. For plasmid transformation, substitute resident plasmid for chromosomal DNA. (1) Linear synapsis; (2) integration intermediate; (3) covalent joining. (B) Plasmid establishment. (1) Annealing of complementary strand fragments that entered separately; (2) repair synthesis; (3) completed replicon. (C) Chromosomal facilitation of plasmid establishment. (1) Circular synapsis followed by repair synthesis and ligation to close the plasmid strand; (2) synthesis of the complementary strand from the plasmid origin of replication; (3) release of established plasmid. (D) Ectopic integration of the mal marker in the vicinity of the sul locus. (1) Donor DNA consists of separately cloned mal and sul genes ligated together; (2) circular synapsis of the donor strand fragment at the sul chromosomal locus (a gap is filled by repair synthesis); (3) a single-strand crossover integrates the donor strand into the chromosome; (4) replication of the chromosome converts the integrated single-strand segment to a duplex form, giving a mal segment inserted between duplicated sul segments. (E) Mutagenesis of the ami gene by additive insertion of a nonreplicating plasmid. (1) Donor DNA consists of the ami gene joined to an E. coli plasmid containing an erm gene expressible in S. pneumoniae; (2) circular synapsis of the donor strand at the ami chromosomal locus and repair synthesis; (3) a single-strand crossover integrates the donor strand into the chromosome; (4) replication of the chromosome converts the integrated single-strand segment to a duplex form so that the E. coli plasmid segment is inserted between duplicated ami segments, thereby producing an aminopterin resistance mutation. Letters a to d and a′ to d′ in panels D and E designate parts of the sul and ami loci, respectively.

Model of quorum sensing in the regulation of competence for transformation. Accumulated extracellular CSP signals ComD to phosphorylate ComE, which then enhances synthesis of CSP and ComX. ComX is needed to transcribe genes required for transformation. Relevant genes are shown at the bottom. Open arrows point to gene products. Solid arrows show effects on promoters. Operon control elements: black, SigA promoter; white, weak SigA promoter; horizontal hatch, binding site for ComE enhancer. Other designations: P, protein phosphate; ComC', residual comC product after removal of CSP.

Variation in competence regulatory components of streptococci. (A) CSPs. Species and strain are indicated. Arrows indicate point of cleavage from the precursor. Dots indicate identity to the peptide sequence above. (B) CSP receptor regions of ComD in S. pneumoniae strains. The first 96 amino-terminal residues are compared. (C) Binding sites for ComE in S. pneumoniae. The consensus sequence is from reference 114. Uppercase letters indicate correspondence to relatively invariant bases of the consensus. Numbers in brackets give distance between ComE-binding sequence repeats and between the second repeat and the extended −10 promoter site ( 97 ). (D) Arrangement of the comCDE genes. Boxes depicting genes point in the direction of transcription.

Late competence genes and construction and function of the DNA uptake apparatus in S. pneumoniae. DNA is depicted by heavy lines, with half-arrowheads indicating the 3′ direction. The “translocasome” is a hypothetical structure extruding through the cell wall and formed by CglC-G proteins, which are exported by the CglA-CglB complex and processed by CilC. Other components of the translocasome are CelA, which binds DNA; EndA, which degrades one strand; CelB, which forms a membrane pore for entry of the other strand; and CflA, which may unwind donor DNA. CoiA and CflB also may function in DNA uptake, possibly by nicking and attaching to DNA prior to entry. Calcium and magnesium ions are required for DNA uptake, with the latter needed by EndA. Upon entry, single strands are coated with Ssb. CilB, DpnA, and RecA act subsequent to DNA uptake. Relevant genes are shown at the bottom. Open arrows point to gene products. Solid arrows show effects on promoters. Operon control elements: black, SigA promoter; horizontal hatch, ComE enhancer; crosshatch, ComX promoter. Other designations: P, protein phosphate; m, methyl group on CglC, CglD, and CglF after processing by CilC. Question marks indicate an uncertain role in transformation.

Restriction enzyme systems of S. pneumoniae. (A) Restriction gene cassettes of S. pneumoniae and their products. Symbols: thin bar, S. pneumoniae chromosome; thick bar, Dpn cassette; open boxes, genes in the cassettes or in the adjacent chromosome, showing direction of transcription. (B) Role of DpnA methylase in enabling unmethylated plasmid transfer into cells containing the DpnII restriction system. The degradative processing of DNA entering the cell by the transformation pathway requires the reconstitution of a plasmid from complementary strands that separately enter the cell. In a host lacking the DpnA methyltransferase, unmethylated plasmid DNA, upon reconstitution to a double-stranded form, would be cleaved by the DpnII endonuclease. In a host containing DpnA, single strands are methylated upon entry, so that the reconstituted plasmid is protected from the DpnII endonuclease. (C) Possible survival value of complementary restriction systems. I and II, cells making DpnI and DpnII, respectively. Infection of a mixed population by a single viral particle would destroy only part of the population.