Swd2/Cps35 determines H3K4 tri-methylation via interactions with Set1 and Rad6.

BACKGROUND: Histone H3K4 tri-methylation (H3K4me3) catalyzed by Set1/COMPASS, is a prominent epigenetic mark found in promoter-proximal regions of actively transcribed genes. H3K4me3 relies on prior monoubiquitination at the histone H2B (H2Bub) by Rad6 and Bre1. Swd2/Cps35, a Set1/COMPASS component, has been proposed as a key player in facilitating H2Bub-dependent H3K4me3. However, a more comprehensive investigation regarding the relationship among Rad6, Swd2, and Set1 is required to further understand the mechanisms and functions of the H3K4 methylation. RESULTS: We investigated the genome-wide occupancy patterns of Rad6, Swd2, and Set1 under various genetic conditions, aiming to clarify the roles of Set1 and Rad6 for occupancy of Swd2. Swd2 peaks appear on both the 5' region and 3' region of genes, which are overlapped with its tightly bound two complexes, Set1 and cleavage and polyadenylation factor (CPF), respectively. In the absence of Rad6/H2Bub, Set1 predominantly localized to the 5' region of genes, while Swd2 lost all the chromatin binding. However, in the absence of Set1, Swd2 occupancy near the 5' region was impaired and rather increased in the 3' region. CONCLUSIONS: This study highlights that the catalytic activity of Rad6 is essential for all the ways of Swd2's binding to the transcribed genes and Set1 redistributes the Swd2 to the 5' region for accomplishments of H3K4me3 in the genome-wide level.

Mycobacterium potentiates protection from colorectal cancer by gut microbial alterations.

Not only are many Mycobacteria pathogens, but they can act as strong non-specific immunopotentiators, generating beneficial effects on the pathogenesis of some diseases. However, there has been no direct evidence of the effect of mycobacterial species on colorectal cancer (CRC). Herein, we showed that there may be a meaningful inverse correlation between the incidence of tuberculosis and CRC based on global statistics and that heat-killed Mycobacterial tuberculosis and live Mycobacterium bovis (Bacillus Calmette-Guérin strain) could ameliorate CRC development. In particular, using a faecal microbiota transplantation and a comparison between separate housing and cohousing, we demonstrated that the gut microbiota is involved in the protective effects. The microbial alterations can be elucidated by the modulation of antimicrobial activities including those of the Reg3 family genes. Furthermore, interleukin-22 production by T helper cells contributed to the anti-inflammatory activity of Mycobacteria. Our results revealed a novel role of Mycobacteria involving gut microbial alterations in dampening inflammation-associated CRC and an immunological mechanism underlying the interaction between microbes and host immunity.

Functional dissection of the phosphotransferase system provides insight into the prevalence of Faecalibacterium prausnitzii in the host intestinal environment.

Faecalibacterium prausnitzii is a dominant member of healthy human colon microbiota, regarded as a beneficial gut bacterium due to its ability to produce anti-inflammatory substances. However, little is known about how F. prausnitzii utilizes the nutrients present in the human gut, influencing its prevalence in the host intestinal environment. The phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS) is a widely distributed and highly efficient carbohydrate transport system found in most bacterial species that catalyses the simultaneous phosphorylation and import of cognate carbohydrates; its components play physiological roles through interaction with other regulatory proteins. Here, we performed a systematic analysis of the 16 genes encoding putative PTS components (2 enzyme I, 2 HPr, and 12 enzyme II components) in F. prausnitzii A2-165. We identified the general PTS components responsible for the PEP-dependent phosphotransfer reaction and the sugar-specific PTS components involved in the transport of two carbohydrates, N-acetylglucosamine and fructose, among five enzyme II complexes. We suggest that the dissection of the functional PTS in F. prausnitzii may help to understand how this species outcompetes other bacterial species in the human intestine.

A large-scale metagenomic study for enzyme profiles using the focused identification of the NGS-based definitive enzyme research (FINDER) strategy.

Excavating the molecular details of many diverse enzymes from metagenomes remains challenging in agriculture, food, health, and environmental fields. We present a versatile method that accelerates metabolic enzyme discovery for highly selective gene capture in metagenomes using next-generation sequencing. Culture-independent enzyme mining of environmental DNA is based on a set of short identifying degenerate sequences specific for a wide range of enzyme superfamilies, followed by multiplexed DNA barcode sequencing. A strategy of 'focused identification of next-generation sequencing-based definitive enzyme research' enabled us to generate targeted enzyme datasets from metagenomes, resulting in minimal hands-on obtention of high-throughput biological diversity and potential function profiles, without being time-consuming. This method also provided a targeted inventory of predicted proteins and molecular features of metabolic activities from several metagenomic samples. We suggest that the efficiency and sensitivity of this method will accelerate the decryption of microbial diversity and the signature of proteins and their metabolism from environmental samples.

Lichenicola cladoniae gen. nov., sp. nov., a member of the family Acetobacteraceae isolated from an Antarctic lichen.

Two Gram-stain-negative, facultative anaerobic, chemoheterotrophic, pink-coloured, rod-shaped and non-motile bacterial strains, PAMC 26568 and PAMC 26569T, were isolated from an Antarctic lichen. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strains PAMC 26568 and PAMC 26569T belong to the family Acetobacteraceae and the most closely related species are Gluconacetobacter takamatsuzukensis (96.1 %), Gluconacetobacter tumulisoli (95.9 %) and Gluconacetobacter sacchari (95.7 %). Phylogenomic and genomic relatedness analyses showed that strains PAMC 26568 and PAMC 26569T are clearly distinguished from other genera in the family Acetobacteraceae by average nucleotide identity values (<72.8 %) and the genome-to-genome distance values (<22.5 %). Genomic analysis revealed that strains PAMC 26568 and PAMC 26569T do not contain genes involved in atmospheric nitrogen fixation and utilization of sole carbon compounds such as methane and methanol. Instead, strains PAMC 26568 and PAMC 26569T possess genes to utilize nitrate and nitrite and certain monosaccharides and disaccharides. The major fatty acids (>10 %) are summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c; 40.3-40.4 %), C18 : 1 2OH (22.7-23.7 %) and summed feature 2 (C14 : 0 3OH and/or C16 : 1 iso I; 12.0 % in PAMC 26568). The major respiratory quinone is Q-10. The genomic DNA G+C content of PAMC 26568 and PAMC 26569T is 64.6 %. Their distinct phylogenetic position and some physiological characteristics distinguish strains PAMC 26568 and PAMC 26569T from other genera in the family Acetobacteraceae supporting the proposal of Lichenicola gen. nov., with the type species Lichenicola cladoniae sp. nov. (type strain, PAMC 26569T=KCCM 43315T=JCM 33604T).

A trans-acting leader RNA from a Salmonella virulence gene.

Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection.

Flavobacterium gilvum sp. nov., isolated from stream water.

A cream-coloured, non-gliding, aerobic Flavobacterium strain, designated EM1308T, was isolated from stream water. 16S rRNA gene sequence analysis indicated that this isolate is closely related to Flavobacterium glycines NBRC 105008T (97.3 % similarity) and Flavobacterium piscis CCUG 60099T (97.2 %). To evaluate the genomic relatedness of the isolate with its neighbours, the whole genome sequences of strain EM1308T and the type strains of F. glycines and F. piscis were determined. Average nucleotide identities revealed that strain EM1308T is independent from other Flavobacterium species. The properties of major cellular fatty acids, polar lipids, menaquinone and DNA G+C content of the isolate were within the general range for the genus Flavobacterium, but many biochemical and physiological characteristics distinguished the isolate from previously known species. Thus, strain EM1308T represents a novel species of the genus Flavobacterium, for which the name Flavobacterium gilvum sp. nov. is proposed. The type strain is EM1308T (=KACC 18113T=JCM 30144T).

Whole genome sequencing analysis of the cutaneous pathogenic yeast Malassezia restricta and identification of the major lipase expressed on the scalp of patients with dandruff.

Malassezia species are opportunistic pathogenic fungi that are frequently associated with seborrhoeic dermatitis, including dandruff. Most Malassezia species are lipid dependent, a property that is compensated by breaking down host sebum into fatty acids by lipases. In this study, we aimed to sequence and analyse the whole genome of Malassezia restricta KCTC 27527, a clinical isolate from a Korean patient with severe dandruff, to search for lipase orthologues and identify the lipase that is the most frequently expressed on the scalp of patients with dandruff. The genome of M. restricta KCTC 27527 was sequenced using the Illumina MiSeq and PacBio platforms. Lipase orthologues were identified by comparison with known lipase genes in the genomes of Malassezia globosa and Malassezia sympodialis. The expression of the identified lipase genes was directly evaluated in swab samples from the scalps of 56 patients with dandruff. We found that, among the identified lipase-encoding genes, the gene encoding lipase homolog MRES_03670, named LIP5 in this study, was the most frequently expressed lipase in the swab samples. Our study provides an overview of the genome of a clinical isolate of M. restricta and fundamental information for elucidating the role of lipases during fungus-host interaction.

Time-resolved pathogenic gene expression analysis of the plant pathogen Xanthomonas oryzae pv. oryzae.

BACKGROUND: Plant-pathogen interactions at early stages of infection are important to the fate of interaction. Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, which is a devastating disease in rice. Although in vivo and in vitro systems have been developed to study rice-Xoo interactions, both systems have limitations. The resistance mechanisms in rice can be better studied by the in vivo approach, whereas the in vitro systems are suitable for pathogenicity studies on Xoo. The current in vitro system uses minimal medium to activate the pathogenic signal (expression of pathogenicity-related genes) of Xoo, but lacks rice-derived factors needed for Xoo activation. This fact emphasizes the need of developing a new in vitro system that allow for an easy control of both pathogenic activation and for the experiment itself. RESULTS: We employed an in vitro system that can activate pathogenicity-related genes in Xoo using rice leaf extract (RLX) and combined the in vitro assay with RNA-Seq to analyze the time-resolved genome-wide gene expression of Xoo. RNA-Seq was performed with samples from seven different time points within 1 h post-RLX treatment and the expression of up- or downregulated genes in RNA-Seq was validated by qRT-PCR. Global analysis of gene expression and regulation revealed the most dramatic changes in functional categories of genes related to inorganic ion transport and metabolism, and cell motility. Expression of many pathogenicity-related genes was induced within 15 min upon contact with RLX. hrpG and hrpX expression reached the maximum level within 10 and 15 min, respectively. Chemotaxis and flagella biosynthesis-related genes and cyclic-di-GMP controlling genes were downregulated for 10 min and were then upregulated. Genes related to inorganic ion uptake were upregulated within 5 min. We introduced a non-linear regression fit to generate continuous time-resolved gene expression levels and tested the essentiality of the transcriptionally upregulated genes by a pathogenicity assay of lesion length using single-gene knock-out Xoo strains. CONCLUSIONS: The in vitro system combined with RNA-Seq generated a genome-wide time-resolved pathogenic gene expression profile within 1 h of initial rice-Xoo interactions, demonstrating the expression order and interaction dependency of pathogenic genes. This combined system can be used as a novel tool to study the initial interactions between rice and Xoo during bacterial blight progression.

EzEditor: a versatile sequence alignment editor for both rRNA- and protein-coding genes.

EzEditor is a Java-based molecular sequence editor allowing manipulation of both DNA and protein sequence alignments for phylogenetic analysis. It has multiple features optimized to connect initial computer-generated multiple alignment and subsequent phylogenetic analysis by providing manual editing with reference to biological information specific to the genes under consideration. It provides various functionalities for editing rRNA alignments using secondary structure information. In addition, it supports simultaneous editing of both DNA sequences and their translated protein sequences for protein-coding genes. EzEditor is, to our knowledge, the first sequence editing software designed for both rRNA- and protein-coding genes with the visualization of biologically relevant information and should be useful in molecular phylogenetic studies. EzEditor is based on Java, can be run on all major computer operating systems and is freely available from http://sw.ezbiocloud.net/ezeditor/.

Profiling bacterial community in upper respiratory tracts.

BACKGROUND: Infection by pathogenic viruses results in rapid epithelial damage and significantly impacts on the condition of the upper respiratory tract, thus the effects of viral infection may induce changes in microbiota. Thus, we aimed to define the healthy microbiota and the viral pathogen-affected microbiota in the upper respiratory tract. In addition, any association between the type of viral agent and the resultant microbiota profile was assessed. METHODS: We analyzed the upper respiratory tract bacterial content of 57 healthy asymptomatic people (17 health-care workers and 40 community people) and 59 patients acutely infected with influenza, parainfluenza, rhino, respiratory syncytial, corona, adeno, or metapneumo viruses using culture-independent pyrosequencing. RESULTS: The healthy subjects harbored primarily Streptococcus, whereas the patients showed an enrichment of Haemophilus or Moraxella. Quantifying the similarities between bacterial populations by using Fast UniFrac analysis indicated that bacterial profiles were apparently divisible into 6 oropharyngeal types in the tested subjects. The oropharyngeal types were not associated with the type of viruses, but were rather linked to the age of the subjects. Moraxella nonliquefaciens exhibited unprecedentedly high abundance in young subjects aged <6 years. The genome of M. nonliquefaciens was found to encode various proteins that may play roles in pathogenesis. CONCLUSIONS: This study identified 6 oropharyngeal microbiome types. No virus-specific bacterial profile was discovered, but comparative analysis of healthy adults and patients identified a bacterium specific to young patients, M. nonliquefaciens.

Preserved structure and function of human serum albumin self-folded in the oxidative cytoplasm of Escherichia coli.

Human serum albumin (HSA), a polypeptide featuring 17 disulfide bonds, acts as a crucial transport protein in human blood plasma. Its extended circulation half-life, mediated by FcRn (neonatal Fc receptor)-facilitated recycling, positions HSA as an excellent carrier for long-acting drug delivery. However, the conventional method of obtaining HSA from human blood faces limitations due to availability and potential contamination risks, such as blood-borne diseases. This study introduced SHuffle, an oxidative Escherichia coli (E. coli) expression system, for the production of recombinant HSA (rHSA) that spontaneously self-folds into its native conformation. This system ensures precise disulfide bond formation and correct folding of cysteine-rich rHSA, eliminating the need for chaperone co-expression or domain fusion of a folding enhancer. The purified rHSA underwent thorough physicochemical characterization, including mass spectrometry, circular dichroism spectroscopy, intrinsic fluorescence spectroscopy, esterase-like activity assay, and size exclusion chromatography, to assess critical quality attributes. Importantly, rHSA maintained native binding affinity to FcRn and the albumin-binding domain. Collectively, our analyses demonstrated a high comparability between rHSA and plasma-derived HSA. The expression of rHSA in E. coli with an oxidizing cytosol provides a secure and cost-effective approach, enhancing the potential of rHSA for diverse medical applications.

Model for end-stage liver disease-3.0 vs. model for end-stage liver disease-sodium: mortality prediction in Korea.

BACKGROUND/AIMS: The model for end-stage liver disease (MELD) serves as an indicator for short-term mortality among patients diagnosed with liver cirrhosis (LC) and is used to prioritize patients for liver transplantation. In 2021, the updated version of MELD, MELD-3.0, was introduced to improve the accuracy of the mortality prediction of MELD. Therefore, this study aimed to compare the efficacy of MELD 3.0 and MELD-Na in predicting mortality among Korean patients with LC. METHODS: A retrospective review was conducted using the medical records of patients diagnosed with LC who were admitted to Konkuk University Hospital From 2011 to 2021. The study calculated the predictive values of MELD-Na and MELD-3.0 for 3- and 6-months mortality using the area under the receiver operating curve (AUROC) and compared the results using the DeLong test. RESULTS: Of the 3,034 patients enrolled in the study, 339 (11.2%) died within 3 months and 421 (14.4%) died within 6 months. The AUROCs values for predicting 3 months mortality were 0.846 for MELD-Na and 0.851 for MELD-3.0. The corresponding AUROC values for predicting 6 months mortality were 0.843 for MELD-Na and 0.848 for MELD-3.0. MELD-3.0 exhibited better discrimination ability than MELD-Na for both 3 (p = 0.03) and 6 months mortality (p = 0.01). CONCLUSION: Our study found a significant difference between the performance of MELD-3.0 and MELD-Na in Korean patients with LC.

A shared mechanism of multidrug resistance in laboratory-evolved uropathogenic Escherichia coli.

The emergence of multidrug-resistant bacteria poses a significant threat to human health, necessitating a comprehensive understanding of their underlying mechanisms. Uropathogenic Escherichia coli (UPEC), the primary causative agent of urinary tract infections, is frequently associated with multidrug resistance and recurrent infections. To elucidate the mechanism of resistance of UPEC to beta-lactam antibiotics, we generated ampicillin-resistant UPEC strains through continuous exposure to low and high levels of ampicillin in the laboratory, referred to as Low AmpR and High AmpR, respectively. Whole-genome sequencing revealed that both Low and High AmpR strains contained mutations in the marR, acrR, and envZ genes. The High AmpR strain exhibited a single additional mutation in the nlpD gene. Using protein modeling and qRT-PCR analyses, we validated the contributions of each mutation in the identified genes to antibiotic resistance in the AmpR strains, including a decrease in membrane permeability, increased expression of multidrug efflux pump, and inhibition of cell lysis. Furthermore, the AmpR strain does not decrease the bacterial burden in the mouse bladder even after continuous antibiotic treatment in vivo, implicating the increasing difficulty in treating host infections caused by the AmpR strain. Interestingly, ampicillin-induced mutations also result in multidrug resistance in UPEC, suggesting a common mechanism by which bacteria acquire cross-resistance to other classes of antibiotics.

Assessment of DNA extraction methods for human gut mycobiome analysis.

The gut mycobiome plays an important role in the health and disease of the human gut, but its exact function is still under investigation. While there is a wealth of information available on the bacterial community of the human gut microbiome, research on the fungal community is still relatively limited. In particular, technical methodologies for mycobiome analysis, especially the DNA extraction method for human faecal samples, varied in different studies. In the current study, two commercial kits commonly used in DNA extraction, the QIAamp® Fast DNA Stool Mini Kit and DNeasy PowerSoil Pro Kit, and one manual method, the International Human Microbiome Standards Protocol Q, were compared. Furthermore, the effectiveness of two different bead-beating machines, the Mini-Beadbeater-16 and FastPrep-24TM 5G, was compared in parallel. A mock fungal community with a known composition of fungal strains was also generated and included to compare different DNA extraction methods. Our results suggested that the method using the DNeasy PowerSoil Pro Kit and Mini-Beadbeater-16 provides the best results to extract DNA from human faecal samples. Based on our data, we propose a standard operating procedure for DNA extraction from human faecal samples for mycobiome analysis.

Functional screening of a metagenomic library reveals operons responsible for enhanced intestinal colonization by gut commensal microbes.

Evidence suggests that gut microbes colonize the mammalian intestine through propagation as an adhesive microbial community. A bacterial artificial chromosome (BAC) library of murine bowel microbiota DNA in the surrogate host Escherichia coli DH10B was screened for enhanced adherence capability. Two out of 5,472 DH10B clones, 10G6 and 25G1, exhibited enhanced capabilities to adhere to inanimate surfaces in functional screens. DNA segments inserted into the 10G6 and 25G1 clones were 52 and 41 kb and included 47 and 41 protein-coding open reading frames (ORFs), respectively. DNA sequence alignments, tetranucleotide frequency, and codon usage analysis strongly suggest that these two DNA fragments are derived from species belonging to the genus Bacteroides. Consistent with this finding, a large portion of the predicted gene products were highly homologous to those of Bacteroides spp. Transposon mutagenesis and subsequent experiments that involved heterologous expression identified two operons associated with enhanced adherence. E. coli strains transformed with the 10a or 25b operon adhered to the surface of intestinal epithelium and colonized the mouse intestine more vigorously than did the control strain. This study has revealed the genetic determinants of unknown commensals (probably resembling Bacteroides species) that enhance the ability of the bacteria to colonize the murine bowel.

Fretibacter rubidus gen. nov., sp. nov., isolated from seawater.

A brick-red-coloured, curved-rod-shaped, prostheca-bearing and non-motile bacterial strain, designated JC2236(T), was isolated from a seawater sample of Jeju Island, Republic of Korea. 16S rRNA gene sequence analysis indicated that this strain belongs to the family Hyphomonadaceae and represents a distinct phyletic line that reflects a novel genus status within a clade containing the genera Litorimonas, Hellea, Robiginitomaculum and Algimonas. The predominant isoprenoid quinone (Q10) and polar lipid profile (phosphatidylglycerol, glucuronopyranosyl diglyceride and monoglycosyl diglyderide) were in line with those of most members of the family. However, the DNA G+C content (49.3 mol%), the abundance of C16 : 0, the requirement of sea salts for growth and absence of cell motility differentiated strain JC2236(T) from other closely related genera. Overall enzyme traits also demonstrated that the novel strain is not closely affiliated with any of the previously described genera. Thus, based on data from the present polyphasic taxonomic study, strain JC2236(T) is considered to represent a novel species of a new genus belonging to the family Hyphomonadaceae, for which the name Fretibacter rubidus gen. nov., sp. nov. is proposed. The type strain of Fretibacter rubidus is JC2236(T) ( = KACC 16935(T) = JCM 15585(T)).

Duplex-specific nuclease efficiently removes rRNA for prokaryotic RNA-seq.

Next-generation sequencing has great potential for application in bacterial transcriptomics. However, unlike eukaryotes, bacteria have no clear mechanism to select mRNAs over rRNAs; therefore, rRNA removal is a critical step in sequencing-based transcriptomics. Duplex-specific nuclease (DSN) is an enzyme that, at high temperatures, degrades duplex DNA in preference to single-stranded DNA. DSN treatment has been successfully used to normalize the relative transcript abundance in mRNA-enriched cDNA libraries from eukaryotic organisms. In this study, we demonstrate the utility of this method to remove rRNA from prokaryotic total RNA. We evaluated the efficacy of DSN to remove rRNA by comparing it with the conventional subtractive hybridization (Hyb) method. Illumina deep sequencing was performed to obtain transcriptomes from Escherichia coli grown under four growth conditions. The results clearly showed that our DSN treatment was more efficient at removing rRNA than the Hyb method was, while preserving the original relative abundance of mRNA species in bacterial cells. Therefore, we propose that, for bacterial mRNA-seq experiments, DSN treatment should be preferred to Hyb-based methods.

Environmental Adaptation of Psychrophilic Bacteria Subtercola spp. Isolated from Various Cryospheric Habitats.

Subtercola boreus K300T is a novel psychrophilic strain that was isolated from permanently cold groundwater in Finland and has also been found in several places in Antarctica including lake, soil, and rocks. We performed genomic and transcriptomic analyses of 5 strains from Antarctica and a type strain to understand their adaptation to different environments. Interestingly, the isolates from rocks showed a low growth rate and smaller genome size than strains from the other isolation sources (lake, soil, and groundwater). Based on these habitat-dependent characteristics, the strains could be classified into two ecotypes, which showed differences in energy production, signal transduction, and transcription in the clusters of orthologous groups of proteins (COGs) functional category. In addition, expression pattern changes revealed differences in metabolic processes, including uric acid metabolism, DNA repair, major facilitator superfamily (MFS) transporters, and xylose degradation, depending on the nutritional status of their habitats. These findings provide crucial insights into the environmental adaptation of bacteria, highlighting genetic diversity and regulatory mechanisms that enable them to thrive in the cryosphere.

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

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.