Presence and expression of terminal oxygen reductases in strictly anaerobic sulfate-reducing bacteria isolated from salt-marsh sediments

Anaerobiosis: Molecular Biology, Genetics & Other Aspects
Volume 14, Issue 3,(June 2008), Pages 145-156

Margarida Santana,
ICAT—Instituto de Cieˆncia Aplicada e Tecnologia
Campo Grande, 1749-016 Lisboa, Portugal

Abstract

In the anaerobic sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough genes were found encoding membrane terminal oxygen reductases of two types: a cytochrome c oxidase and a cytochrome bd oxidase, both enzymes are terminal oxidases typical of facultative or aerobic microorganisms (Heidelberg JF, et al., The genome sequence of the anaerobic, sulfate-reducing bacterium D. vulgaris Hildenborough. Nat Biotechnol 2004; 22: 554–9). To apprehend the presence of both oxidases in other sulfate-reducing bacteria (SRB), several assays were performed on isolates recovered from salt-marsh sediments in Portugal, representative of the different phylogenetic groups identified. Hybridization and PCR experiments for DNA sequencing were performed on the chosen isolates. Primers were selected to amplify conserved regions of cytochrome c oxidases and cytochrome bd oxidases taking into consideration alignment of corresponding subunit I sequences. The results showed that both oxidase genes are present on the chromosome of several isolates characterized as Desulfovibrio. These genes were shown to be transcribed, as demonstrated by Reverse Transcriptase-PCR experiments on total RNA. In order to assess the relative contribution of each oxidase to oxygen consumption, oxygen uptake was measured for each isolate and further characterized by the effect of cyanide on oxygen consumption. It was concluded that cytochrome bd oxidase was the terminal membrane oxygen reductase allowing oxygen consumption. In addition, it was observed that isolates containing cytochrome bd oxidase had higher resistance to air exposure, suggesting an important role of this enzyme in survival to air exposure. The pattern for the presence of oxygen reductase genes was compared to the physiological pattern of substrate use, which was determined for each isolate. Salinity tolerance, pH and temperature growth of each isolate were also analyzed.
Article Outline

1. Introduction
2. Materials and methods
2.1. Isolation and phylogenetic characterization of the SRB
2.2. Growth conditions
2.3. Growth characteristics
2.4. Morphology evaluation
2.5. DNA extraction and PCR amplification
2.6. Sequencing of the PCR fragments
2.7. GenBank accession numbers
2.8. Labeling of DNA probes
2.9. DNA hybridization
2.10. RNA extraction and analysis
2.11. Reverse transcription-PCR reactions
2.12. Measurement of oxygen uptake
2.13. Measurement of sulfide production
2.14. Effect of exposure to air
2.15. Protein and dry weight measurements
3. Results and discussion
3.1. Identification and phylogenetic analysis
3.2. Morphological and physiological characteristics of the isolates
3.3. Detection of cytochrome c oxidase and cytochrome bd oxidase genes
3.4. Transcription analysis of terminal oxygen reductases genes
3.5. Oxygen consumption
3.6. Resistance to air exposure
4. Conclusions
Acknowledgements
References