The response of Sphingopyxis granuli strain TFA to the hostile anoxic condition

Abstract

Sphingomonads comprises a group of interesting aerobic bacteria because of their ubiquity and metabolic capability of degrading many recalcitrant contaminants. The tetralin-degrader Sphingopyxis granuli strain TFA has been recently reported as able to anaerobically grow using nitrate as the alternative electron acceptor and so far is the only bacterium with this ability within the sphingomonads group. To understand how strain TFA thrives under anoxic conditions, a differential transcriptomic analysis while growing under aerobic or anoxic conditions was performed. This analysis has been validated and complemented with transcription kinetics of representative genes of different functional categories. Results show an extensive change of the expression pattern of this strain in the different conditions. Consistently, the most induced operon in anoxia codes for proteases, presumably required for extensive changes in the protein profile. Besides genes that respond to lack of oxygen in other bacteria, there are a number of genes that respond to stress or to damage of macromolecules, including genes of the SOS DNA-damage response, which suggest that anoxic conditions represent a hostile environment for this bacterium. Interestingly, growth under anoxic conditions also resulted in repression of all flagellar and type IV pilin genes, which suggested that this strain shaves its appendages off while growing in anaerobiosis.

Figure 1

Distribution of anaerobically regulated genes of S. granulli strain TFA in COG categories. Genes represented here showed a differential expression of at least 3-fold.

Figure 2

Induction kinetics of genes coding for electron transfer chains components. Anaerobic conditions with 20 mM nitrate in the WT strain are represented by black symbols, anaerobic conditions without nitrate in the WT strain by white symbols and anaerobic conditions with 20 mM nitrate in the ΔnarG mutant by grey symbols. Fold induction of: (a) narG gene; (b) cyoC (squares) and cydA (circles) genes; (c) ccoH gene and (d) aox gene. As a control, expression kinetics of each gene in aerobiosis was performed along the growth curve and expression changed less than 3-fold. Fold change induction of each gene over time with respect to time 0 is shown and graphics represent the mean ± SD of 3–4 technical replicates.

Figure 3

Induction kinetics of the yhbU gene, coding for a protease. Anaerobic conditions with 20 mM nitrate in the WT strain are represented by black squares, anaerobic conditions without nitrate in the WT strain by white squares and anaerobic conditions with nitrate 20 mM in the ΔnarG mutant by grey squares. As a control, expression kinetics in aerobiosis along the growth curve was also performed and expression changed less than 2-fold. Fold change induction of the gene over time with respect to time 0 is shown and graphic represents the mean ± SD of 3–4 technical replicates.

Figure 4

Induction kinetics of genes involved in stress response and detoxification. Anaerobic conditions with 20 mM in the WT strain are represented by black symbols, anaerobic conditions without nitrate in the WT strain by white symbols and anaerobic conditions with nitrate 20 mM in the ΔnarG mutant by grey symbols. (a) Induction of ahpC2 (squares), lsfA (circles) and ectA (triangles) genes. (b) Induction of ytfE. (c) Induction of norB. As a control, expression kinetics of each gene in aerobiosis was performed along the growth curve and expression changed less than 4.5-fold. Fold change induction of each gene over time with respect to time 0 is shown and graphics represent the mean ± SD of 3–4 technical replicates.

Figure 5

Induction kinetics of genes involved in replication, cell cycle control and cell division. (a) Fold induction of ccrM (squares) and ctrA (circles) genes. Anaerobic conditions with 20 mM nitrate in the WT strain are represented by black symbols and aerobic conditions in the WT strain by grey symbols. (b) Fold induction of nrdZ gene. Anaerobic conditions with 20 mM nitrate in the WT strain are represented by black squares, anaerobic conditions without nitrate in the WT strain by white squares and anaerobic conditions with 20 mM nitrate in the ΔnarG mutant by grey squares. As a control, expression kinetics of nrdZ in aerobiosis was performed along the growth curve and expression changed less than 2.5-fold. Fold change induction of each gene over time with respect to time 0 is shown and graphics represent the mean ± SD of 3–4 technical replicates.

Figure 6

Induction kinetics of genes of the SOS DNA repair system. Induction of recA (squares) and imuA (circles) in anaerobic conditions with 20 mM nitrate in the WT strain. As a control, expression kinetics of each gene in aerobiosis was performed along the growth curve and expression changed less than 3-fold. Fold change induction of each gene over time with respect to time 0 is shown and graphic represents the mean ± SD of 3–4 technical replicates.

Figure 7

Regulation kinetics of flagellar and pili genes in TFA. Regulation of fliC (squares) and cpaA (circles) genes. Aerobic conditions in the WT strain are represented by grey symbols and anaerobic conditions with 20 mM nitrate in the WT strain by black symbols. Fold change induction of each gene over time with respect to time 0 is shown and graphic represents the mean ± SD of 3–4 technical replicates.

Figure 8

Swimming motility assays. Graphs represent the evolution of the swimming circles diameter of WT strain (squares) and the spontaneous swimming mutant MPO255 (circles) in aerobic (white) and anaerobic (black) conditions. Graphic represents the mean ± SD of 3–4 biological replicates.