A novel gene from the acidophilic bacterium Leptospirillum sp. CF-1 and its role in oxidative stress and chromate tolerance

Abstract

Background: Acidophilic microorganisms like Leptospirillum sp. CF-1 thrive in environments with extremely low pH and high concentrations of dissolved heavy metals that can induce the generation of reactive oxygen species (ROS). Several hypothetical genes and proteins from Leptospirillum sp. CF-1 are known to be up-regulated under oxidative stress conditions.

Results: In the present work, the function of hypothetical gene ABH19_09590 from Leptospirillum sp. CF-1 was studied. Heterologous expression of this gene in Escherichia coli led to an increase in the ability to grow under oxidant conditions with 5 mM K₂CrO₄ or 5 mM H₂O₂. Similarly, a significant reduction in ROS production in E. coli transformed with a plasmid carrying ABH19_09590 was observed after exposure to these oxidative stress elicitors for 30 min, compared to a strain complemented with the empty vector. A co-transcriptional study using RT-PCR showed that ABH19_09590 is contained in an operon, here named the "och" operon, that also contains ABH19_09585, ABH19_09595 and ABH19_09600 genes. The expression of the och operon was significantly up-regulated in Leptospirillum sp. CF-1 exposed to 5 mM K₂CrO₄ for 15 and 30 min. Genes of this operon potentially encode a NADH:ubiquinone oxidoreductase, a CXXC motif-containing protein likely involved in thiol/disulfide exchange, a hypothetical protein, and a di-hydroxy-acid dehydratase. A comparative genomic analysis revealed that the och operon is a characteristic genetic determinant of the Leptospirillum genus that is not present in other acidophiles.

Conclusions: Altogether, these results suggest that the och operon plays a protective role against chromate and hydrogen peroxide and is an important mechanism required to face polyextremophilic conditions in acid environments.

Keywords: Chromate; Hydrogen peroxide; Hypothetical proteins; Leptospirillum; Och operon; Oxidative stress.

Fig. 1

Growth of E. coli transformed with ABH19_09590 from Leptospirillum sp. CF-1. Cells transformed with plasmid pBADTopo/ABH19_09590 were exposed to A 5 mM K₂CrO₄, or B 5 mM H₂O₂. Cell growth was measured each hour at 600 nm absorbance

Fig. 2

ROS levels of E. coli transformed with ABH19_09590 from Leptospirillum sp. CF-1. Cells were stressed with A 5 mM K₂CrO₄, or B 5 mM H₂O₂ for 30 min. Cytoplasmic ROS content is expressed as relative fluorescence units (RFU) of the activated fluorescent probe H₂DCFDA per mg of protein

Fig. 3

Genetic organization of the och operon of Leptospirillum sp. CF-1. A Schematic map of the och operon. Primers designed in each intergenic region are indicated below the operon. B RT-PCR amplification of intergenic regions. The negative control corresponds to RT-PCR amplification without reverse transcriptase, whilst the positive control corresponds to standard PCR amplification with genomic DNA. Primer pairs used for amplification are indicated above each panel and the amplification product sizes are indicated below each panel

Fig. 4

Effect of chromate exposure on the relative mRNA levels of the och operon of Leptospirillum sp. CF-1. The relative expression of A ABH19_09585, B ABH19_09590, C ABH19_09595 and D ABH19_9600 encoding genes was evaluated in cells treated with 5 mM K₂CrO₄ for 15 and 30 min. Data represent the average of three independent experiments (bars indicate the value range). Statistical analysis was carried out by the ANOVA Test