Transcriptional analysis of the rubrerythrin and superoxide dismutase genes of Clostridium perfringens

Abstract

We cloned and sequenced a 2.7-kb fragment of chromosomal DNA from Clostridium perfringens containing the superoxide dismutase-encoding gene, sod. Previously, rubrerythrin from C. perfringens had been isolated and its gene (rbr) had been cloned (Y. Lehmann, L. Meile, and M. Teuber, J. Bacteriol. 178:7152-7158, 1996). Northern blot experiments revealed a length of approximately 800 bases for each transcript of rbr and sod of C. perfringens. Thus, rbr and sod each represent a monocistronic operon. Their transcription start points were located by primer extension analyses. sod transcription was shown to depend on the growth phase, and it reached a maximum during the transition from log phase to stationary phase. Neither sod nor rbr transcription was influenced by oxidative stress.

FIG. 1

Nucleotide sequence of the sod gene of C. perfringens and the predicted amino acid sequence of the encoded SOD and the sod promoter region. The underlined amino acids are highly conserved among SODs and indicate the regions from which oligonucleotides lm16 and lm17 were derived. The overlined nucleotide sequence indicates the complementary sequence of the primer (tg6) used for primer extension analysis. A potential ribosome binding site is marked by asterisks. The consensus promoter sequences (−35 and −10) are in underlined boldface, and the transcription start point is marked by an arrow. Boldface amino acids indicate the N terminus, which is unusually expanded compared to other Mn- and Fe-SODs.

FIG. 2

Northern blot analysis of the C. perfringens sod and rbr genes. sod and rbr represent monocistronic operons of approximately 800 nucleotides each.

FIG. 3

Mapping of the 5′ ends of the sod and rbr transcripts of C. perfringens by primer extension analysis. Primer extension products (pe) are shown to the right of the corresponding sequencing ladder on a 6% (wt/vol) sequencing gel. The transcription start point is boxed for the sod operon (left), and three potential start points are numbered for the rbr operon (right). The numbers assigned to the nucleotides correspond to the numbering of the nucleotides in Fig. 4.

FIG. 4

Nucleotide sequence of the rbr promoter region from C. perfringens (9). The overlined nucleotide sequence indicates the complementary sequence of the primer used for primer extension analysis. A potential ribosome binding site is marked by asterisks. Possible consensus promoter sequences are bold underlined, and the transcription start point is marked by an arrow. A predicted stem-loop structure that might have resulted in a preferred processing site at position −58 is double underlined (+, possible bulges).

FIG. 5

sod (A) and rbr (B) transcripts during log and stationary phases of C. perfringens grown under anaerobic (■) or partially aerobic (□) conditions. Relative intensities were determined by densitometry analysis. Growth was followed by spectrophotometry at 600 nm (+). OD, optical density.