Direct Selection of IS 903 Transposon Insertions by Use of a Broad-Host-Range Vector: Isolation of Catalase-Deficient Mutants of Actinobacillus actinomycetemcomitans

Abstract

Transposon mutagenesis in bacteria generally requires efficient delivery of a transposon suicide vector to allow the selection of relatively infrequent transposition events. We have developed an IS903-based transposon mutagenesis system for diverse gram-negative bacteria that is not limited by transfer efficiency. The transposon, IS903φkan, carries a cryptic kangene, which can be expressed only after successful transposition. This allows the stable introduction of the transposon delivery vector into the host. Generation of insertion mutants is then limited only by the frequency of transposition. IS903φkan was placed on an IncQ plasmid vector with the transposase gene located outside the transposon and expressed from isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoters. After transposase induction, IS903φkaninsertion mutants were readily selected in Escherichia coliby their resistance to kanamycin. We used IS903φkan to isolate three catalase-deficient mutants of the periodontal pathogenActinobacillus actinomycetemcomitans from a library of random insertions. The mutants display increased sensitivity to hydrogen peroxide, and all have IS903φkan insertions within an open reading frame whose predicted product is closely related to other bacterial catalases. Nucleotide sequence analysis of the catalase gene (designated katA) and flanking intergenic regions also revealed several occurrences of an 11-bp sequence that is closely related to the core DNA uptake signal sequence for natural transformation of Haemophilus influenzae. Our results demonstrate the utility of the IS903φkan mutagenesis system for the study of A. actinomycetemcomitans. Because IS903φkan is carried on a mobilizable, broad-host-range IncQ plasmid, this system is potentially useful in a variety of bacterial species.

Characterization of a quinol peroxidase mutant inAggregatibacter actinomycetemcomitans

“…2a, QPS003 exhibited a decrease of growth in AAGM under aerobic conditions. On the other hand, IKM001 grew normally as reported previously (Thomson et al, 1999).…”

“…Because it is known that catalase (KatA) is expressed at the same time in the cytosol of bacteria as one of the antioxidant enzymes (Thomson et al, 1999), we compared the phenotypes of the QPO null mutant (QPS003) and the KatA transposon insertion mutant (IKM001) to address the unique physiological role of QPO.…”

“…QPS003 (a QPO null mutant from IDH781) and pVJTqpo (a plasmid harboring the full-length sequence of qpo) were generated during a previous study . IKM001 (a KatA transposon insertion mutant) was generated from IDH781 using a method reported previously for pVJT128, the transposon mutagenesis plasmid (Thomson et al, 1999). Aggregatibacter actinomycetemcomitans strains were cultured at 37 1C in an atmosphere containing 5% CO 2 in A. actinomycetemcomitans growth medium (AAGM) (Kachlany et al, 2000) containing appropriate antibiotics (2 mg mL À1 chloramphenicol for the strains harboring pVJTqpo and pVJT128, 50 mg mL À1 of spectinomycin for QPS003, 20 mg mL À1 of kanamycin for IKM001).…”

Novel iron‐regulated and Fur‐regulated small regulatory RNAs in Aggregatibacter actinomycetemcomitans

“…The broad-host-range plasmid pJAK16 was used for fur complementation. The pJAK16 plasmid is an IncQ expression vector that contains a chloramphenicolresistance gene and an IPTG-inducible P tac promoter located upstream from a multiple cloning site (Thomson et al, 1999). Plasmid pJAK-fur was constructed by restriction enzyme digestion of pMfur (Table 1) with KpnI/BamHI, yielding a 506-base pairs (bp) fur fragment which was gel purified and ligated into the KpnI/BamHI sites of pJAK16.…”

“…Each E. coli strain was then used as the donor for conjugation with the HKR. fur::kan mutant strain, using a protocol previously described for rough phenotype strains of A. actinomycetemcomitans (Thomson et al, 1999). Transconjugants were selected on TSBY agar containing chloramphenicol (2 lg ml )1 ) and nalidixic acid (20 lg ml )1 ).…”