ChnA and -B expression in the native and transconjugant F. heparinum strains. Amounts of fractions (0.15 OD₆₀₀ and 0.05 OD⁶⁰⁰ units of soluble fractions from wild-type and transconjugate strains, respectively) were loaded onto SDS-7.5% PAGE reducing gels. (A) Coomassie blue R-250-stained gel. (B and C) Western blots of triplicates of gel A transferred to nitrocellulose membrane filters and developed with polyclonal antibodies against ChnA (B) or ChnB (C). Lanes: M, molecular weight markers; 1 and 4, wild-type F. heparinum; 2 and 5, strain FIBX6 (cslA_HU); 3 and 6, strain FIBX7 (cslB_HU); 7, purified native ChnA (0.5 μg) from wild-type F. heparinum; and 8, purified native ChnB (0.5 μg) from wild-type F. heparinum. lanes 1, 2, and 3 represent strains grown in MA medium, while lanes 4, 5, and 6 represent strains grown in MH medium.

Transcriptional start site determination of hepA using an A.L.F. sequencer. Primer extension was carried with 40 μg of total RNA from the FIBX5 and FIBX6 strains and with 120 μg of total RNA from wild-type F. heparinum and Cy5-labeled primers. The sequencing reactions of pIB17 and pIB30 were used as markers. The transcriptional start site, indicated by a line, was determined by comparing the retention time of the primer extension products with those of the products of the sequencing reactions. (A) Primer extension products from total RNA of strain FIBX6 (top) and plasmid DNA of pIB30 (bottom) run on an A.L.F. sequencer. (B) B1, primer extension products from total RNA of strain FIBX5 grown in heparin-only medium; B2, primer extension products from total RNA of strain FIBX5 grown in glucose-only medium; and B3, primer extension products from total RNA of wild-type F. heparinum grown in heparin-only medium. Bottom, primer extension products from plasmid pIB17.

HepI, -II, and -III expression in native and transconjugant F. heparinum strains grown in MH medium. Coomassie blue R-250-stained gels were used. Numbers of kilodaltons are given on left of gels. (A) SDS-10% PAGE gel. Lanes: 1, 0.5 μg of purified native HepI; 2, 0.125 OD₆₀₀ units of soluble extract from native F. heparinum; 3, same amount of extract from FIBX5; and 4, 0.025 OD₆₀₀ units of soluble extract from FIBX5. (B) SDS-7.5% PAGE gel. Lanes: 1, 0.5 μg of purified native HepII; 2, 0.125 OD₆₀₀ units of soluble extract from native F. heparinum; 3, same amount of extract from FIBX3; and 4, 0.0125 OD₆₀₀ units of soluble extract from FIBX3. (C) SDS-7.5% PAGE gel. Lanes: 1, 0.5 μg of purified native HepIII; 2, 0.125 OD₆₀₀ units of soluble extract from native F. heparinum; 3, same amount of extract from FIBX4; and 4, 0.0125 OD₆₀₀ units of soluble extract from FIBX4. Western blot analysis was performed. (D) Results were transferred from an SDS-10% PAGE gel. Lane 1, 0.2 μg of purified native HepI; lane 2, 0.05 OD₆₀₀ units of soluble extract from native F. heparinum; lane 3, same amount of extract from FIBX5; and lane 4, 0.01 OD₆₀₀ units of soluble extract from FIBX5. (E) Results were transferred from an SDS-7.5% PAGE gel. Lane 1, 0.2 μg of purified native HepII; lane 2, 0.05 OD₆₀₀ units of soluble extract from native F. heparinum; lane 3, same amount of extract from FIBX3; and lane 4, 0.01 OD₆₀₀ units of soluble extract from FIBX3. (F) Results were transferred from an SDS-7.5% PAGE gel. Lane 1, 0.2 μg of purified native HepIII; lane 2, 0.05 OD₆₀₀ units of soluble extract from native F. heparinum; lane 3, same amount of extract from FIBX4; and lane 4, 0.01 OD₆₀₀ units of soluble extract from FIBX4.

Analysis of the hepA regulatory region. The activity was measured as chondroitin sulfate A-degrading activity as shown in Table 2. “+” denotes that the activity measured was the same as in Table 2. “−” denotes the absence of any measurable activity. The strains were grown in MH medium as described in Materials and Methods. The sequence of the hepA upstream region was published previously (27), and an NdeI site was incorporated for strain FIBX6. The transcriptional start site is indicated, and the −10 and −35 regions are underlined.

Coomassie blue R-250 analysis of HepI, -II, and -III and ChnA and -B purified from either the native or transconjugant F. heparinum strain. (A) SDS-10% PAGE Coomassie blue R-250-stained gel. Lane 1, 1 μg of purified native HepI; lane 4, native HepII; and lane 7, native HepIII. Lane 2, 0.1 OD₆₀₀ units of soluble FIBX5; lane 5, same amount of FIBX3; and lane 8, FIBX4 cell extract. Lane 3, 1 μg of purified tHepI; lane 6, same amount of tHepII; and lane 9, same amount of tHepIII. (B) SDS-7.5% PAGE Coomassie blue R-250-stained gel. Lane M, molecular weight marker; lane 1, 1 μg of purified native ChnA; lane 4, 1 μg of native ChnB; lane 2, 0.1 OD₆₀₀ units of soluble FIBX6; lane 5, same amount of FIBX7; lane 3, 1 μg of purified tChnA; and lane 6, same amount of tChnB.