Identification of genetic determinants for the hemolytic activity of Streptococcus agalactiae by ISS1 transposition

Abstract

Streptococcus agalactiae is a poorly transformable bacterium and studies of molecular mechanisms are difficult due to the limitations of genetic tools. Employing the novel pGh9:ISS1 transposition vector we generated plasmid-based mutant libraries of S. agalactiae strains O90R and AC475 by random chromosomal integration. A screen for mutants with a nonhemolytic phenotype on sheep blood agar led to the identification of a genetic locus harboring several genes that are essential for the hemolytic function and pigment production of S. agalactiae. Nucleotide sequence analysis of nonhemolytic mutants revealed that four mutants had distinct insertion sites in a single genetic locus of 7 kb that was subsequently designated cyl. Eight different open reading frames were identified: cylX, cylD, cylG, acpC, cylZ, cylA, cylB, and cylE, coding for predicted proteins with molecular masses of 11, 33, 26, 11, 15, 35, 32, and 78 kDa, respectively. The deduced amino acid sequence of the protein encoded by cylA harbors a conserved ATP-binding cassette (ABC) motif, and the predicted proteins encoded by cylA and cylB have significant similarities to the nucleotide binding and transmembrane proteins of typical ABC transporter systems. Transcription analysis by reverse transcription-PCR suggests that cylX to cylE are part of an operon. The requirement of acpC and cylZABE for hemolysin production of S. agalactiae was confirmed either by targeted mutagenesis with the vector pGh5, complementation studies with pAT28, or analysis of insertion elements in naturally occurring nonhemolytic mutants.

FIG. 1

Graphic map of the cyl gene cluster. (A) The transcription terminator (tt) upstream of the putative transcription start site is indicated. Primer annealing sites for the RT-PCR experiments are represented by arrows, and the resulting PCR products are indicated. (B) DNA integration sites in nonhemolytic S. agalactiae mutants. Strains were either constructed as described in Materials and Methods or collected from routine clinical specimens. Mutants from the original libraries that were generated through pGh9:ISS1 integration and subsequent excision are indicated as ISS1; the IS1381-like insertion element in naturally occurring nonhemolytic mutants is shown as IS1381. For mutants that were generated through targeted pGh5 integration, the plasmid integration site is marked. (C) Strain BSP1969. The HLY26ex mutant (harboring a copy of the ISS1 element in acpC) was transformed with a copy of the acpC and cylZ gene integrated into the pAT28 plasmid. (D) Genomic structure of the HLY226ex deletion mutant.

FIG. 2

(A) Transcription analysis was performed by RT-PCR. Primers A to D, annealing at nucleotides 5299 to 5276, nucleotides 4355 to 4336, nucleotides 3563 to 3546, and nucleotides 2300 to 2280, respectively, were used for RT. PCR of chromosomal DNA was used as a positive control (lanes DNA). The PCR performed on mRNA subjected to an RT reaction is shown in lanes RT; PCR that was performed on mRNA without prior RT reaction (lanes RNA) served as a negative control. Lane M, molecular mass marker (in nucleotides). (B) RT-PCR analysis of the transcription start site. RT reactions were carried out with a primer annealing at nucleotides 2300 to 2280 of the cyl gene cluster. Subsequent PCRs were performed with forward primers annealing upstream (G) or downstream (E and F) of the putative transcription terminator at nucleotides 35 to 55, nucleotides 352 to 372, and nucleotides 301 to 321, respectively. Positive and negative controls are indicated.

FIG. 3

Hemolytic activity on blood agar of the AC 475 wild-type strain (A), the HLY26ex mutant (B), and the BSP1969 complementation mutant (C). The presence or absence of beta-hemolysis underneath removed colonies is indicated by arrows.

FIG. 4

Pigment production of hemolytic (Hly⁺) wild-type strains, nonhemolytic (Hly⁻) mutants, and nonhemolytic wild-type strains. Bacteria were grown anaerobically at 37°C on THB–5% starch under anaerobic conditions. Isolates: 1, AC 475 (Hly⁺); 2 to 5, clinical isolates (Hly⁻); CYLZ-K1 (Hly⁻); 7, CYLB-K2 (Hly⁻); 8, HLY22 (Hly⁻); 9, O90R wild type (Hly⁺); 10, HLY26ex (Hly⁻); 11, HLY26 (Hly⁻).