Use of an antisense RNA strategy to investigate the functional significance of Mn-catalase in the extreme thermophile Thermus thermophilus

Abstract

The expression of an antisense RNA revealed that an Mn-catalase was required in Thermus thermophilus for aerobic but not for anaerobic growth. The antisense system is based on the constitutive expression of a "bicistronic" transcript consisting of the kanamycin resistance gene mRNA followed by the antisense RNA against the selected target.

FIG. 1.

The pMK18cat plasmid. The 0.9-kb DNA fragment codifying the Mn-catalase from T. thermophilus HB8 was cloned into pMK18r to obtain plasmid pMK18cat. The relative positions of the ONdecat and kat2 primers used for RT-PCR are indicated. H, HindIII; E, EcoRI.

FIG. 2.

Deleterious effect of pMK18cat. (A) Cell cultures of T. thermophilus HB8 or HB27::nar were transformed with identical amounts of pMK18r and pMK18cat, and the selection plates were grown aerobically (white bars) or anaerobically (stripped bars). The percentages of transformants with pMK18cat with respect to those obtained with pMK18r are represented for each condition and strain. (B) Oligonucleotides ONdecat and kat2 were used for RT-PCR on purified RNA isolated from anaerobically grown cells of T. thermophilus HB8 transformed with plasmid pMK18cat (Lane 2) or pMK18r (Lane 3). Negative controls were carried out with the sample of lane 2 in the absence of the RT step (Lane 1). The sizes (in kilobase pairs) of HindIII-digested lambda phage DNA fragments (M) and of the RT-PCR-amplified fragment are indicated.

FIG. 3.

Role of Mn-catalase during aerobic growth. (A) Identical amounts of anaerobically grown cells of T. thermophilus HB27::nar transformed with plasmid pMK18r (black circles) or pMK18cat (white circles) were inoculated in 12-ml tubes containing 3 ml of TB medium in the presence of the indicated concentrations of H₂O₂. The percentage of the OD₅₅₀ reached after 10 h of incubation at 70°C (microaerobic growth) with respect to untreated cultures compared to the H₂O₂ concentration is shown. (B) RT-PCR to detect the cat gene on total RNA from exponential cultures of T. thermophilus HB8 grown aerobically or after 7 h of anaerobic growth by nitrate respiration (anaerobic). The amounts of total RNA used were 4 μg (lane 1), 2 μg (lane 2), and 1 μg (lane 3). Lane M, size markers in kilobase pairs.