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. 2023 Jul 18;16(1):114.
doi: 10.1186/s13068-023-02364-6.

Regulation of acetate tolerance by small ORF-encoded polypeptides modulating efflux pump specificity in Methylomonas sp. DH-1

Seungwoo Cha  1 , Yong-Joon Cho  2 , Jong Kwan Lee  1 , Ji-Sook Hahn  3
Affiliations

Regulation of acetate tolerance by small ORF-encoded polypeptides modulating efflux pump specificity in Methylomonas sp. DH-1

Seungwoo Cha et al. Biotechnol Biofuels Bioprod. .

Abstract

Background: Methanotrophs have emerged as promising hosts for the biological conversion of methane into value-added chemicals, including various organic acids. Understanding the mechanisms of acid tolerance is essential for improving organic acid production. WatR, a LysR-type transcriptional regulator, was initially identified as involved in lactate tolerance in a methanotrophic bacterium Methylomonas sp. DH-1. In this study, we investigated the role of WatR as a regulator of cellular defense against weak organic acids and identified novel target genes of WatR.

Results: By conducting an investigation into the genome-wide binding targets of WatR and its role in transcriptional regulation, we identified genes encoding an RND-type efflux pump (WatABO pump) and previously unannotated small open reading frames (smORFs), watS1 to watS5, as WatR target genes activated in response to acetate. The watS1 to watS5 genes encode polypeptides of approximately 50 amino acids, and WatS1 to WatS4 are highly homologous with one predicted transmembrane domain. Deletion of the WatABO pump genes resulted in decreased tolerance against formate, acetate, lactate, and propionate, suggesting its role as an efflux pump for a wide range of weak organic acids. WatR repressed the basal expression of watS genes but activated watS and WatABO pump genes in response to acetate stress. Overexpression of watS1 increased tolerance to acetate but not to other acids, only in the presence of the WatABO pump. Therefore, WatS1 may increase WatABO pump specificity toward acetate, switching the general weak acid efflux pump to an acetate-specific efflux pump for efficient cellular defense against acetate stress.

Conclusions: Our study has elucidated the role of WatR as a key transcription factor in the cellular defense against weak organic acids, particularly acetate, in Methylomonas sp. DH-1. We identified the genes encoding WatABO efflux pump and small polypeptides (WatS1 to WatS5), as the target genes regulated by WatR for this specific function. These findings offer valuable insights into the mechanisms underlying weak acid tolerance in methanotrophic bacteria, thereby contributing to the development of bioprocesses aimed at converting methane into value-added chemicals.

Keywords: Acetate tolerance; LysR-type regulator; Methanotroph; RND-type efflux pump; Small open reading frame (smORF).

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
The effect of watR deletion in the wild-type and JHM80 strains on weak organic acid tolerance. Wild-type, JHM80, ΔwatR (JHM15), and JHM80 ΔwatR (JHM82) strains were grown in NMS media with 20% (v/v) methane containing 0.3 g/L lactate, 0.5 g/L propionate, 1.0 g/L formate, and 1.2 g/L of acetate with pH neutralization. Two independent experiments were averaged and plotted with standard deviations
Fig. 2
Fig. 2
Identification of genome-wide binding sites of WatR. A Binding consensus sequence of WatR, discovered by ChIP-seq analysis. B Confirmation of WatR binding to the promoters of four selected target genes identified by ChIP-seq analysis. Binding of WatR-Flag to the target promoters in JHM80WF strain was detected by ChIP with anti-Flag antibody and indicated as fold enrichment relative to the binding to a negative control (glgA ORF). Each value represents the average ± standard deviations from three independent experiments. C Expression levels of watR operon genes (watR and smtM) in wild-type and JHM80 strains. The mRNA levels detected by qRT-PCR were normalized to that of mxaF gene and indicated as relative expression levels compared with those of wild type. The glgA gene was used as a negative control. Each value represents the average ± standard deviations from two independent experiments. Significant difference from wild-type strain is shown as *p < 0.1; **p < 0.05. D Binding of WatR to the watR promoter detected through in vitro EMSA assay. EMSA assay was performed by incubating GST-WatR protein with biotin-labeled watR promoter probes with or without TT deletion. WatR-binding sites (arrows), a putative -10 box, and transcription start site (TSS) are indicated. The deleted TT nucleotides in JHM80 are shown in red. E Autoregulation of watR expression. In wild type, WatR binds to its own promoter, repressing the expression. In the JHM80 strain, the TT deletion in the promoter prevents WatR binding, resulting in derepression of the operon. F Repression of gltA1 by WatR. Expression levels of gltA1 in the wild-type, JHM80, and JHM80 ΔwatR (JHM82) strains were detected by qRT-PCR. The relative mRNA levels are indicated compared with those of JHM80. Each value represents the average ± standard deviations from two independent experiments
Fig. 3
Fig. 3
WatR-dependent activation of genes encoding an RND-type efflux pump contributing to organic acid tolerance. A Gene structure and putative functions of watPAB and watO genes regulated by WatR. Side view of the predicted WatABO efflux pump is shown. OM: outer membrane; IM: inner membrane. B WatR-dependent activation of the efflux pump genes. Transcript levels were detected by qRT-PCR in the wild-type, JHM80, and JHM80 ΔwatR (JHM82) strains and indicated as values relative to those of wild type. C The effect of deleting the efflux pump genes on acid tolerance. The JHM80 strain and JHM80 strain lacking the WatABO efflux pump (JHM87) were grown in NMS media with 20% (v/v) methane without or with the indicated weak organic acids. Each value represents the average ± standard deviations from two independent experiments
Fig. 4
Fig. 4
Induction of WatR target genes by acetate but not lactate. The wild-type and ΔwatR (JHM15) strains were grown in NMS media with 20% (v/v) methane until early exponential phase and then treated with 0.15 g/L lactate (A) or 0.6 g/L acetate (B) for 10 min. Transcript levels were detected by qRT-PCR and indicated as values relative to those of untreated wild type. Each value represents the average ± standard deviations from two (for lactate) or three (for acetate) independent experiments. Significant difference from wild-type strain is shown as *p < 0.1; **p < 0.05
Fig. 5
Fig. 5
WatR-dependent regulation of smORF genes upon acetate stress. A WatR-dependent regulation of smORF genes. Locations of five unannotated smORFs (watS1-watS5) are aligned with the WatR-binding peaks detected via ChIP-Seq and transcript levels detected via RNA-seq using the IGV 2.3.72 program. RNA-seq analysis was performed in the wild-type and ΔwatR (JHM15) strains with or without acetate treatment. B The promoter sequences of watS1-watS5 with their expected -35 box, -10 box, and TSS. The conserved WatR-binding sites are shown as inverted arrows. C The homology alignment of amino acid sequences of WatS1 to WatS5. A putative transmembrane domain region conserved in WatS1 to WatS4 is indicated. D WatR-dependent regulation of WatABO efflux pump genes. The gene locations were aligned with the WatR-binding peaks detected via ChIP-seq analysis and transcript levels detected by RNA-seq. E The promoter sequences of the divergently transcribed watPAB and watO genes with their expected -35 box, -10 box, and TSS. The putative WatR-binding sites are shown as inverted arrows
Fig. 6
Fig. 6
Changes in DNA binding affinity of WatR upon acetate stress. A Induction of watP and watS1 gene expression by acetate. The JHM16WF strain expressing watR-Flag from the PEFTu promoter was grown in NMS medium with 20% (v/v) methane and treated with 3.0 g/L of acetate for 10 min. The mRNA expression levels were detected by qRT-PCR and indicated as values relative to those of untreated control. Each value represents the average ± SD of the relative fold enrichment of three independent experiments, normalized to glgA. Significant difference from untreated sample is shown as **p < 0.05. B Changes in WatR DNA binding upon acetate stress. The JHM16WF strain was grown in NMS medium with 20% (v/v) methane and treated with 3.0 g/L of acetate for 10 min. ChIP analysis was performed with anti-Flag antibody and WatR binding to the promoters was detected by qPCR. Each value represents the average ± SD of the relative fold enrichment of two independent experiments, normalized to a negative control (glgA ORF). Significant difference from untreated sample is shown as *p < 0.1; **p < 0.05. C Model for the WatR-dependent transcriptional regulation of watPAB and watO genes. The WatR-binding sites does not overlap with the RNA binding sites, enabling basal transcription. Upon acetate stress, WatR activates transcription, which involves increasing DNA binding affinity. D Model for the WatR-dependent transcriptional regulation of smORFs. The WatR-binding sites overlap with the RNA binding sites, repressing basal transcription. Upon acetate stress, WatR activates transcription possibly by shifting the binding site to expose the RNA polymerase binding site
Fig. 7
Fig. 7
WatS1 controls acetate tolerance only in the presence of WatABO pump. A The effect of overexpressing the watS1 and watS5 genes on acid tolerance. The ΔfliE control strain (JHM16) and strains replacing the fliE gene with watS1 or watS5 overexpression cassette (JHM161 and JHM165) were grown in NMS media with 20% (v/v) methane without or with weak organic acids as indicated. Each value represents the average ± standard deviations from two independent experiments. B The effect of deleting the watS1 gene on acid tolerance. The wild-type and watS1 deletion (JHM17) strains were grown in NMS media with 20% (v/v) methane without or with weak organic acids as indicated. Each value represents the average ± standard deviations from two independent experiments. C The effect of deleting the watS1 and WatABO pump genes. The wild-type, ΔwatS1 (JHM17), ΔwatABO (JHM18), and ΔwatABO ΔwatS1 (JHM182) strains were grown in NMS media with 20% (v/v) methane without or with 0.6 g/L acetate. Each value represents the average ± standard deviations from two independent experiments. D The effect of overexpressing the watS1 gene without WatABO pump genes. The control ΔwatABO strain with fliE deletion (JHM181) and ΔwatABO strain overexpressing watS1 (JHM183) were grown in NMS media with 20% (v/v) methane without or with 0.6 g/L acetate. Each value represents the average ± standard deviations from two independent experiments
Fig. 8
Fig. 8
Model for SEP-dependent regulation of the WatABO pump upon acetate stress. Under normal conditions, the WatABO pump extrude a wide range of weak organic acids including acetate, formate, lactate, and propionate. Upon acetate stress, activated WatR induces transcription of WatABO pump and watS smORF genes. WatS SEP binds to the WatABO pump, increasing the specificity toward acetate for efficient removal of acetate from cells
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