4a), whereas no 4-ABS removal could be observed for RK32(pHG6) (data not shown). Positive control strain PBC(pBBR1MCS-5), on the contrary, exhibited complete removal of 4-ABS. 4-ABS-dependent oxygen uptake was also measured using cell suspension as an indirect measurement of 4-aminobenzenesulfonate 3,4-dioxygenase activity. RK40(pHG5) showed approximately sevenfold higher 4-ABS-dependent oxygen uptake rate than control strain RK40(pBBR1MCS-5) (Fig. 4b). RK40(pHG5) also regained its ability to grow on 4-ABS as sole carbon and nitrogen source in PB medium, albeit, with an additional 96 h of lag phase compared with PBC(pBBR1MCS-5) (Fig. 4c

Ivacaftor clinical trial and d). Study of the 4-ABS metabolic pathway has hitherto been limited to enzymology work focusing on the lower pathway converting 4-sulfocatechol to β-ketoadipate (Contzen et al., 2001; Halak et al., 2006; Halak et selleck chemicals llc al., 2007). In this study, we describe the isolation and characterization of mutants with single insertion in genes affecting 4-ABS degradation of Hydrogenophaga sp. PBC. Several pieces of evidence collected for RK1 point to a mutation in the 4-sulfocatechol 1,2-dioxygenase gene. First, RK1 exhibited no growth with 4-ABS and 4-sulfocatechol as sole carbon source but utilized 4-ABS as sole nitrogen source. Secondly, the secreted brown metabolite was identified as 4-sulfocatechol

through HPLC and TLC comparison with authentic standard. The gene annotation was further supported by the strikingly high sequence identity (99.6%) of the disrupted gene to 4-sulfocatechol 1,2-dioxygenase sequence of H. intermedia S1 (Contzen et al., 2001). As 4-sulfocatechol 1,2-dioxygenase of H. intermedia S1 could oxidize protocatechuate (Contzen et al., 2001), the ability of RK1 to utilize protocatechuate as carbon source was tested. Growth of RK1 on protocatechuate (Table 2) suggests that 4-sulfocatechol

1,2-dioxygenase is not required for protocatechuate utilization and implies the existence of an alternative pathway for the degradation of this phenolic selleck screening library compound. 3-Sulfomuconate cycloisomerase gene is responsible for the conversion of 3-sulfomuconate to 4-sulfomuconolactone in the lower pathway of 4-ABS degradation (Halak et al., 2006). Transposon insertion in the 3-sulfomuconate cycloisomerase gene of RK23 severely impaired its ability to degrade 4-ABS in NB. A similar result was obtained even when it was cultured in minimal media supplemented with protocatechuate as a source of β-ketoadipate, a general inducer of most aromatic compound degradation pathways (data not shown), suggesting that 3-sulfomuconate is a strong repressor and/or its metabolic product, 4-sulfomuconolactone, is an inducer of the 4-ABS biotransformation pathway. The possibility of 3-sulfomuconate being a highly toxic compound, as reported for its analog β-carboxy-cis,cis-muconate (Parke et al.