Induction of the cydAB Operon Encoding the bd Quinol Oxidase Under Respiration-Inhibitory Conditions by the Major cAMP Receptor Protein MSMEG_6189 in Mycobacterium smegmatis

Abstract

The respiratory electron transport chain (ETC) of Mycobacterium smegmatis is terminated with two terminal oxidases, the aa ₃ cytochrome c oxidase and the cytochrome bd quinol oxidase. The bd quinol oxidase with a higher binding affinity for O₂ than the aa ₃ oxidase is known to play an important role in aerobic respiration under oxygen-limiting conditions. Using relevant crp1 (MSMEG_6189) and crp2 (MSMEG_0539) mutant strains of M. smegmatis, we demonstrated that Crp1 plays a predominant role in induction of the cydAB operon under ETC-inhibitory conditions. Two Crp-binding sequences were identified upstream of the cydA gene, both of which are necessary for induction of cydAB expression under ETC-inhibitory conditions. The intracellular level of cAMP in M. smegmatis was found to be increased under ETC-inhibitory conditions. The crp2 gene was found to be negatively regulated by Crp1 and Crp2, which appears to lead to significantly low cellular abundance of Crp2 relative to Crp1 in M. smegmatis. Our RNA sequencing analyses suggest that in addition to the SigF partner switching system, Crp1 is involved in induction of gene expression in M. smegmatis exposed to ETC-inhibitory conditions.

Keywords: Crp; Mycobacterium; aa3 cytochrome c oxidase; cAMP; electron transport chain; regulation of gene expression; respiration.

Figure 1

Expression of the cydAB operon in the WT, Δaa₃, and Δaa₃ΔsigF mutant strains of M. smegmatis. The WT, Δaa₃, and Δaa₃ΔsigF mutant strains of M. smegmatis harboring the cydA::lacZ translational fusion plasmid pNCIIcydA were grown aerobically to an OD₆₀₀ of 0.45–0.5 in 7H9-glucose medium. Cell crude extracts were used to determined β-galactosidase activity. All values provided are the averages of the results from three biological replicates. The error bars indicate the standard deviations.

Figure 2

Expression of the cydAB operon in the WT, Δcrp1, and Δcrp2 mutant strains of M. smegmatis. (A) The relative transcript level of cydA in the WT, Δcrp1, and Δcrp2 mutant strains. (B) Complementation of the Δcrp1 mutant. For complementation of the Δcrp1 mutant, pMV306crp (a pMV306-derived plasmid carrying the intact crp1 gene and its own promoter) was introduced into the mutant. As control strains, the WT and Δcrp1 strains with the empty vector pMV306 were used in the experiment. All the strains were grown aerobically to an OD₆₀₀ of 0.45–0.5 and treated with 100 μM KCN for 15 min. The expression level of cydA was quantitatively determined by qRT-PCR and normalized to sigA (the gene for the principal sigma factor) expression. The expression level of cydA in the KCN-treated WT strain is set at 1, and the relative values are expressed for the mutant strains. All values provide are the average of the results from three biological replicates. The error bars indicate the standard deviations. Statistical significance was determined by two-tailed Student's t-test. *p < 0.05.

Figure 3

Expression levels of two Crp paralogs in M. smegmatis. (A) Transcript levels of crp1 and crp2 were extrapolated from the RPKM values obtained from RNA sequencing analysis on the WT strain of M. smegmatis (Lee et al., 2018). (B) Expressed protein levels of Crp1 and Crp2. To determine the protein levels of Crp1 and Crp2 expressed in M. smegmatis, the Δcrp1 and Δcrp2 mutant strains complemented with pMV306crp1_2B8 and pMV306crp2_2B8, respectively, were grown aerobically to the indicated OD₆₀₀ in 7H9-glucose medium, and their crude extracts (30 μg for detection of Crp1 and Crp2; 5 μg for GroEL detection) were subjected to Western blotting analyses using a 2B8 antibody and an Hsp65 antibody to detect the C-terminally 2B8-tagged Crp and GroEL, respectively. The protein level of GroEL was determined as a loading control.

Figure 4

Identification of the Crp-binding sites in the upstream region of cydA. (A) DNase I Footprinting analysis of the cydA upstream region bound by Crp1. The DNA fragments containing the non-coding strands labeled with TAMRA at their 5′ ends were incubated with increasing concentrations of purified Crp1 in the absence or presence of 200 μM cAMP and then subjected to DNase I footprinting reactions. The concentrations of Crp1 used are given above the lanes. The Crp-binding sites (CBS1 and CBS2) are marked by the thick lines on the right. Lanes G, A, T, and C represent the sequence ladders. (B) The upstream sequence of the cydA gene. The Crp-binding sites (CBS1 and CBS2) are marked by the two head-facing arrows above the sequence. The nucleotide reported to be the TSP (+1) of cydA is shaded in gray, and the putative −10 and −35 elements extrapolated from +1 are boxed (Aung et al., 2014). The start codon of cydA is underlined and the arrow above it indicates the transcriptional direction. The numbers on the left of the sequences indicate the positions of the leftmost nucleotides relative to the cydA gene. The concentration of Crp1 refers to that of the Crp1 monomer.

Figure 5

Binding of Crp1 to the cydA control region. (A) EMSA showing the effect of cAMP on the binding of Crp1 to the upstream region of cydA. The mixtures of 99-bp DNA fragments (70 fmol) containing two Crp-binding sites (CBS1 and CBS2) upstream of cydA and 80-bp non-specific DNA fragments without the Crp-binding site (100 fmol) were incubated with increasing concentrations of purified Crp1 in the presence and absence of 200 μM cAMP. (B) 99-bp DNA fragments (70 fmol) containing either WT or mutated Crp-binding sites (M1, M2, and M3), as well as 80-bp non-specific DNA fragments without the Crp-binding site (100 fmol), were mixed with increasing concentrations of purified Crp1 in the presence of 200 μM cAMP. The Crp1-DNA reaction mixtures were subjected to native PAGE. The concentrations of Crp1 used in EMSA are given above the lanes. The concentration of Crp1 refers to that of the Crp1 monomer.

Figure 6

Effects of deletions and mutations in the Crp-binding sites (CBS1 and CBS2) on expression of the cydAB operon. (A) The cydA promoter activity was determined using the pNCIIcydA-derived cydA::lacZ translational fusions containing serial deletions of the cydA upstream region (pNCIISD1, pNCIISD2, pNCIISD3, and pNCIISD4). (B) The cydA promoter activity was determined using several pNCIIcydA derivatives containing mutations in the Crp-binding sites (pNCIIM1, pNCIIM2, and pNCIIM3). The mutations in the Crp-binding sites of the cydA::lacZ translational fusions are presented on the left. The mutations within CBS1 and CBS2 are indicated by underlines. The WT and Δaa₃ mutant strains of M. smegmatis harboring the cydA::lacZ translational fusion plasmids were grown aerobically to an OD₆₀₀ of 0.45–0.5 in 7H9-glucose medium. Cell crude extracts were used to determined β-galactosidase activity. All values provided are the averages of the results from three biological replicates. The error bars indicate the standard deviations. Statistical significance was determined by two-tailed Student's t-test. *p < 0.05. Con, pNCIIcydA; SD1-SD4, pNCIISD1- pNCIISD4; M1-M3, pNCIIM1-pNCIIM3.

Figure 7

Overlap of the SigF and Crp1 regulons with the genes induced in the Δaa₃ mutant strain of M. smegmatis. (A) Volcano plot showing the DEGs in the Δaa₃ mutant strain relative to the WT strain. RNA sequencing was performed using RNA prepared from three independent replicate cultures of the WT and Δaa₃ mutant strains grown aerobically in 7H9-glucose medium to an OD₆₀₀ of 0.45–0.5. The x-axis displays the log₂ fold change of gene expression (log₂FC) in the Δaa₃ mutant relative to the WT strain, and the y-axis represents -log₁₀ p-value. The horizontal dotted line on the graph indicates the border line indicating the p-value of 0.05, and the vertical dotted lines indicate the border lines indicating the log₂FC values of−2 and +2. One hundred and three genes, whose expression is increased by more than log₂FC ≥ 2 with p < 0.05, are depicted by black filled circles. Among the 103 DEGs, the genes belonging to the SigF regulon are denoted by blue filled circles (Singh et al., 2015), and the genes, whose expression is reduced by FC ≥ 1.5 with p < 0.05 in the Δcrp1 mutant strain relative to the WT strain, are indicated by red filled circles. (B) The nucleotide sequences and locations of the Crp-binding sites in the upstream regions of cydA and MSMEG_3680 and the expression levels of the genes in the WT and Δcrp1 mutant strains of M. smegmatis. The Crp-binding sites are shown in bold. The previously reported TSPs of cydA and MSMEG_3680 are denoted by +1 (Aung et al., ; Martini et al., 2019). The start codons of cydA and MSMEG_3680 are underlined, and the arrows above them indicate the transcriptional direction. The expression levels of cydA and MSMEG_3680 in the WT and Δcrp1 mutant strains were quantitatively determined by qRT-PCR. The strains were aerobically grown to an OD₆₀₀ of 0.45–0.5 and treated with 100 μM KCN for 15 min (+KCN). As controls, the WT and Δcrp1 mutant strains grown aerobically without KCN treatment were included in the experiment (-KCN). The expression levels of cydA and MSMEG_3680 determined by qRT-PCR were normalized to that of sigA. The expression level of each gene in the KCN-untreated WT strain is set at 1, and the relative values are expressed for the other strains. All values provide are the averages of the results from three independent experiments. The error bars indicate the standard deviations.

Figure 8

Intracellular levels of cAMP in M. smegmatis under respiration-inhibitory conditions. (A) Intracellular levels of cAMP in the WT and Δaa₃ mutant strains of M. smegmatis. The WT and Δaa₃ mutant strains of M. smegmatis were grown aerobically to an OD₆₀₀ of 0.45–0.5 in 7H9-glucose medium. (B) Effect of KCN treatment on intracellular levels of cAMP in M. smegmatis. The WT strain of M. smegmatis was grown aerobically to an OD₆₀₀ of 0.45–0.5 in 7H9-glucose medium, and the cultures were treated with 100 μM KCN for 15 min (+KCN). As a control, the WT strain without treatment of KCN was used in the experiment (-KCN). cAMP quantification was performed using a DetectX Direct Cyclic AMP Enzyme Immunoassay kit (Arbor Assays) as described in Materials and Methods. All values provided are the average of the results from three biological replicates. The error bars indicate the standard deviations. Statistical significance was determined by two-tailed Student's t-test. *p < 0.05.

Figure 9

Expression of the crp2 gene in the WT, Δcrp1, Δcrp2, and Δaa₃ mutant strains of M. smegmatis. (A) Determination of the crp2 transcript level in the WT, Δcrp1, and Δcrp2 mutant strains by means of qRT-PCR. The positions of the identified Crp-binding site and the TSP (+1) relative to the start codon of crp2 are shown above the graph (Martini et al., 2019). The start codon of crp2 is underlined and the arrow above it indicates the transcriptional direction. The strains were grown aerobically to an OD₆₀₀ of 0.45–0.5 and treated with 100 μM KCN for 15 min. The expression level of crp2 in the KCN-treated WT strain is set at 1, and the relative values are expressed for the mutant strains. (B) Determination of the crp2 transcript level in the WT and Δaa₃ mutant strains using qRT-PCR. The strains were grown aerobically to an OD₆₀₀ of 0.45–0.5. The expression level of crp2 in the WT strain is set at 1, and the relative values are expressed for the Δaa₃ mutant. All values provide are the average of the results from three biological replicates. The error bars indicate the standard deviations. Statistical significance was determined by two-tailed Student's t-test. *p < 0.05.