Article Figures & Data
Figures
- Fig. 1.
Schematic representation of the has locus inS. pyogenes (A), and the homologous regions in S. uberis 0140J (B to E). Open arrows represent the orientations ofhas genes. Filled arrows indicate the locations and orientations of ISS1-encoded transposase (not shown to scale). Hatched bars represent the pGh9 vector sequence (also not shown to scale). Filled arrowheads indicate the orientation of putative open reading frames (orf). ISS1 insertions were mapped to the points indicated by vertical arrows in strains TRF0-6, TRF0-29, and TRF0-30. (B and D) Arrangements before plasmid excision. (C and E) Arrangements after plasmid excision. Partial and horizontal arrows denote key oligonucleotide primer locations (Table 1). H and E,HindIII and EcoRI restriction sites used in the cloning of flanking sequences, respectively. Erres., erythromycin resistant; Ersens., erythromycin sensitive.
- Fig. 2.
Southern analysis of integrants picked randomly from theS. uberis 0140J ISS1 mutant bank. Genomic DNAs from 12 mutant clones (lanes 1 to 12) and the parent strain 0140J (lane 13) were digested using HindIII and probed with DIG-labeled ISS1. Two clones (lanes 3 and 11) showed three bands, one of which corresponded to a 4.6-kb linearized pGh9::ISS1 fragment (arrow) characteristic of tandem pGh9::ISS1 integration.
- Fig. 3.
PCR amplification products from the 3′ end of theS. uberis homologue of hasA demonstrating band shifts due to the insertion of ISS1 in mutants TRF0-6 and TRF0-29, compared to the wild-type (WT) strain 0140J.
- Fig. 4.
Southern analysis of S. uberis 0140J mutants TRF0-6 and TRF0-29. Genomic DNA was digested withHindIII and hybridized with a DIG-labeled ISS1 probe at 65°C. Lane 1, wild type (WT); lane 2, wild-type 0140J transformed with pGh9::ISS1 and grown at 28°C (pG9); lanes 3 and 4, mutant TRF0-6 before (Err) and after (Ers) excision of the plasmid vector sequence, respectively; lanes 5 and 6, mutant TRF0-29 before (Err) and after (Ers) excision of the plasmid vector sequence, respectively. Lane 2 shows the linearized preintergrated free-plasmid form of pGh9::ISS1. Lanes 3 and 5 show that mutant TRF0-6 contained a tandem insertion, whereas mutant TRF0-29 had a single copy of pGh9::ISS1 inserted in the chromosome.
Tables
- Table 1.
Oligonucleotide primer sequences and their applications
Designation Sequence Application Template Annealing temp (°C) ISS1fora 5′-GGAGAGAATGGGTTCTGTTGC ISS1probe pGh9::ISS1 60 ISS1reva 5′-GCTCTAGAGCATTCTCTGGTTC P050 5′-GTTATCGTTCACCGTTCCC S. pyogenes hasA probe S. pyogenes genomic DNA 47 P052 5′-TACGTGTTCCCCATTCCG P064a 5′-AGAACCGAAGAATTCGAACGCTC Amplification of S. uberis DNA and sequencing primers Wild-type and mutant S. uberis 0140J genomic DNAs 50 P067b 5′-GGTGGAGCGTGCTGCTCA P070 5′-CAAAACGAATAGATGTATCAATCC P082a 5′-CCAACAGCGACAATAATCACATC P084c 5′-CAGTCCCGACGGTGTAAAACG P085c 5′-GCTAAAGAGGTCCCTAGACTCT - Table 2.
Putative translation products for mutant S. uberis hasA and hasC homologues
Strain Gene Predicted amino acid sequencea TRF0-6 hasA … LVAFLVIILV LLQSFLISLF * TRF0-29 hasA … LVAFLVIIFI VALCRNVHYM VKHPFAFLLS PFYGLIHRFC CKVF** TRF0-30 hasC … DDLMDITNTV LLQSLKIKYK VYNPSCS* ↵a ISS1-encoded amino acids are denoted by underlining. An asterisk denotes a stop codon.
- Table 3.
Resistance to phagocytosis and capsular status of mutants TRF0-6, TRF0-29, and TRF0-30 compared to wild-type S. uberis 0140J grown in CDM containing casein hydrolysate
Straina Mean (SD)b % Survivalc μg of capsuled Wild-type 0140J 103.02 (15.21) 145.775 (27.89) TRF0-6 Err 0.118 (0.06) 0.325 (0.26) TRF0-6 Ers 0.440 (0.12) 0.625 (0.46) TRF0-29 Err 0.250 (0.15) 0.750 (0.31) TRF0-29 Ers 0.135 (0.05) 0.700 (0.37) Wild-type 0140J 71.83 (33.67) 122.35 (18.02)e TRF0-30 Err 0.16 (0.06) 1.29 (2.00)e TRF0-30 Ers 0.22 (0.02) 1.63 (1.54)e ↵a Mutant strains were tested before (Err) and after (Ers) pGh plasmid excision.
↵b Unless otherwise indicated, data from triplicate samples from two independently grown broth cultures were pooled (n = 6) .
↵c Resistance to phagocytosis is shown as the percent survival of cells relative to cells at time zero.
↵d Capsule status is shown as the amount of hyaluronidase-released N-acetylglucosamine per 1010 bacteria.
↵e Data from quadruplicate samples from two independently grown cultures were pooled (n = 8) .
