Exploring functional genes' correlation with (S)-equol concentration and new daidzein racemase identification.

With its estrogenic activity, (S)-equol plays an important role in maintaining host health and preventing estrogen-related diseases. Exclusive production occurs through the transformation of soy isoflavones by intestinal bacteria, but the reasons for variations in (S)-equol production among different individuals and species remain unclear. Here, fecal samples from humans, pigs, chickens, mice, and rats were used as research objects. The concentrations of (S)-equol, along with the genetic homology and evolutionary relationships of (S)-equol production-related genes [daidzein reductase (DZNR), daidzein racemase (DDRC), dihydrodaidzein reductase (DHDR), tetrahydrodaidzein reductase (THDR)], were analyzed. Additionally, in vitro functional verification of the newly identified DDRC gene was conducted. It was found that approximately 40% of human samples contained (S)-equol, whereas 100% of samples from other species contained (S)-equol. However, there were significant variations in (S)-equol content among the different species: rats > pigs > chickens > mice > humans. The distributions of the four genes displayed species-specific patterns. High detection rates across various species were exhibited by DHDR, THDR, and DDRC. In contrast, substantial variations in detection rates among different species and individuals were observed with respect to DZNR. It appears that various types of DZNR may be associated with different concentrations of (S)-equol, which potentially correspond to the regulatory role during (S)-equol synthesis. This enhances our understanding of individual variations in (S)-equol production and their connection with functional genes in vitro. Moreover, the newly identified DDRC exhibits higher potential for (S)-equol synthesis compared to the known DDRC, providing valuable resources for advancing in vitro (S)-equol production. IMPORTANCE: (S)-equol ((S)-EQ) plays a crucial role in maintaining human health, along with its known capacity to prevent and treat various diseases, including cardiovascular diseases, metabolic syndromes, osteoporosis, diabetes, brain-related diseases, high blood pressure, hyperlipidemia, obesity, and inflammation. However, factors affecting individual variations in (S)-EQ production and the underlying regulatory mechanisms remain elusive. This study examines the association between functional genes and (S)-EQ production, highlighting a potential correlation between the DZNR gene and (S)-EQ content. Various types of DZNR may be linked to the regulation of (S)-EQ synthesis. Furthermore, the identification of a new DDRC gene offers promising prospects for enhancing in vitro (S)-EQ production.

Transposon-aided capture (TRACA) of plasmids from the human gut.

The gut microbiota consists of a vast and diverse assemblage of microorganisms that play a pivotal role in maintaining host health. Nevertheless, a significant portion of the human gut microbiota remains uncultivated. Plasmids, a type of MGE, assume a critical function in the biological evolution and adaptation of bacteria to varying environments. To investigate the plasmids present within the gut microbiota community, we used the transposon-aided capture method (TRACA) to explore plasmids derived from the gut microbiota. In this study, fecal samples were collected from two healthy human volunteers and subsequently subjected to the TRACA method for plasmid isolation. Then, the complete sequence of the plasmids was obtained using the genome walking method, and sequence identity was also analyzed. A total of 15 plasmids were isolated. At last, 13 plasmids were successfully sequenced, of which 12 plasmids were highly identical to the plasmids in the National Center for Biotechnology Information (NCBI) database and were all small plasmids. Furthermore, a putative novel plasmid, named pMRPHD, was isolated, which had mobilized elements (oriT and oriV) and a potential type II restriction-modification (R-M) system encoded by DNA cytosine methyltransferase and type II restriction enzyme (Ban I), whose specific functions and applications warrant further exploration.

Streptomyces cynarae sp. nov., a novel actinomycete isolated from the leaves of Cynara scolymus L.

Strain HUAS 13-4T, a novel endophytic actinobacterium, was isolated from the leaves of Cynara scolymus L. collected from Changde City in China and characterized using a polyphasic approach. Based on 16S rRNA gene sequence analysis, strain HUAS 13-4T shared the highest sequence similarities to Streptomyces leeuwenhoekii C34T (98.90%), Streptomyces harenosi PRKS01-65T (98.83%) and Streptomyces glomeratus LMG 19903T (98.76%). Phylogenetic analysis of 16S rRNA gene sequence indicated that strain HUAS 13-4T was clustered together with Streptomyces bluensis ISP 5564T and Streptomyces cavernae SYSU K10008T. Phylogenomic analysis revealed that strain HUAS 13-4T was most closely related to S. glomeratus JCM 9091T. However, the average nucleotide identity and the digital DNA-DNA hybridization values between them were less than 96.7% and 70% cut-off points recommended for delineating species. Based on a comprehensive comparison of the genome sequences and phenotypic characteristics between strain HUAS 13-4T and its relative, strain HUAS 13-4T (= MCCC 1K08364T = JCM 35919T) should evidently represent a novel Streptomyces species, and the name Streptomyces cynarae sp. nov. is proposed.

Proposal of Streptomyces sporoverrucosus Gause et al. 1983 as a later heterotypic synonym of Streptomyces goshikiensis Niida et al. 1966 and an emended description of Streptomyces goshikiensis.

The taxonomic relationship of Streptomyces goshikiensis and Streptomyces sporoverrucosus was re-evaluated using comparative genome analysis. The 16S rRNA gene sequence analysis indicated that S. goshikiensis JCM 4640T and S. sporoverrucosus CGMCC 4.1796T shared 100% sequence similarity. Phylogenetic analysis based on 16S rRNA gene and genomic sequences exhibited that they were closely related to each other. However, the values of average nucleotide identity (ANIb/ANIm) and digital DNA-DNA hybridization (dDDH) between the genomes of two type strains were 98.33%/98.69% and 87.2%, respectively, greater than the two recognized thresholds values of 96.7% ANI and 70% dDDH for species delineation. These results suggested that S. goshikiensis and S. sporoverrucosus should share the same taxonomic position. In addition, this conclusion was further supported by highly similar morphological, cultural, physiological, biochemical and chemotaxonomic characteristics between them. Consequently, it is proposed that S. sporoverrucosus is a later heterotypic synonym of S. goshikiensis.

Complete Genome Sequence of Nocardiopsis exhalans Strain JCM 11759T, Isolated from Indoor Air of a Water-Damaged Private House in Finland.

The genus Nocardiopsis contains pharmaceutically and biotechnologically important species that produce a wide variety of secondary metabolites with a wide range of biological activities. Here, we report the complete genome sequence of Nocardiopsis exhalans JCM 11759T for a better understanding of its metabolic characteristics and toxin synthesis pathway.

Characterization and Identification of a New Daidzein Reductase Involved in (S)-Equol Biosynthesis in Clostridium sp. ZJ6.

(S)-equol (EQ) is an isoflavone with high estrogen-like activity in the human body, and is only produced by some gut bacteria in vivo. It plays an important role in maintaining individual health, however, the dearth of resources associated with (S)-EQ-producing bacteria has seriously restricted the production and application of (S)-EQ. We report here a new functional gene KEC48-07020 (K-07020) that was identified from a chick (S)-EQ-producing bacterium (Clostridium sp. ZJ6, ZJ6). We found that recombinant protein of K-07020 possessed similar function to daidzein reductase (DZNR), which can convert daidzein (DZN) into R/S-dihydrodaidzein (R/S-DHD). Interestingly, K-07020 can reversely convert (R/S)-DHD (DHD oxidase) into DZN even without cofactors under aerobic conditions. Additionally, high concentrations of (S)-EQ can directly promote DHD oxidase but inhibit DZNR activity. Molecular docking and site-directed mutagenesis revealed that the amino acid > Arg75 was the active site of DHD oxidase. Subsequently, an engineered E. coli strain based on K-07020 was constructed and showed higher yield of (S)-EQ than the engineered bacteria from our previous work. Metagenomics analysis and PCR detection surprisingly revealed that K-07020 and related bacteria may be prevalent in the gut of humans and animals. Overall, a new DZNR from ZJ6 was found and identified in this study, and its bidirectional enzyme activities and wide distribution in the gut of humans and animals provide alternative strategies for revealing the individual regulatory mechanisms of (S)-EQ-producing bacteria.

Genomic Island-Mediated Horizontal Transfer of the Erythromycin Resistance Gene erm(X) among Bifidobacteria.

Antibiotic resistance is a serious medical issue driven by antibiotic misuse. Bifidobacteria may serve as a reservoir for antibiotic resistance genes (ARGs) that have the potential risk of transfer to pathogens. The erythromycin resistance gene erm(X) is an ARG with high abundance in bifidobacteria, especially in Bifidobacterium longum species. However, the characteristics of the spread and integration of the gene erm(X) into the bifidobacteria genome are poorly understood. In this study, 10 tetW-positive bifidobacterial strains and 1 erm(X)-positive bifidobacterial strain were used to investigate the transfer of ARGs. Conjugation assays found that the erm(X) gene could transfer to five other bifidobacterial strains. Dimethyl sulfoxide (DMSO) and vorinostat significantly promoted the transfer of the erm(X) from strain Bifidobacterium catenulatum subsp. kashiwanohense DSM 21854 to Bifidobacterium longum subsp. suis DSM 20211. Whole-genome sequencing and comparative genomic analysis revealed that the erm(X) gene was located on the genomic island BKGI1 and that BKGI1 was conjugally mobile and transferable. To our knowledge, this is the first report that a genomic island-mediated gene erm(X) transfer in bifidobacteria. Additionally, BKGI1 is very unstable in B. catenulatum subsp. kashiwanohense DSM 21854 and transconjugant D2TC and is highly excisable and has an intermediate circular formation. In silico analysis showed that the BKGI1 homologs were also present in other bifidobacterial strains and were especially abundant in B. longum strains. Thus, our results confirmed that genomic island BKGI1 was one of the vehicles for erm(X) spread. These findings suggest that genomic islands play an important role in the dissemination of the gene erm(X) among Bifidobacterium species. IMPORTANCE Bifidobacteria are a very important group of gut microbiota, and the presence of these bacteria has many beneficial effects for the host. Thus, bifidobacteria have attracted growing interest owing to their potential probiotic properties. Bifidobacteria have been widely exploited by the food industry as probiotic microorganisms, and some species have a long history of safe use in food and feed production. However, the presence of antibiotic resistance raises the risk of its application. In this study, we analyzed the transfer of the erythromycin resistance gene erm(X) and revealed that the molecular mechanism behind the spread of the gene erm(X) was mediated by genomic island BKGI1. To the best of our knowledge this is the first report to describe the transfer of the gene erm(X) via genomic islands among bifidobacteria. This may be an important way to disseminate the gene erm(X) among bifidobacteria.

Escherichia coli Exopolysaccharides Induced by Ceftriaxone Regulated Human Gut Microbiota in vitro.

A stable intestinal microflora is an essential prerequisite for human health. This study investigated the interaction between Escherichia coli exopolysaccharides (named EPS-m2) and the human gut microbiota (HGM) in vitro. The EPS-m2 was produced by E. coli WM3064 when treated with ceftriaxone. The monosaccharide composition analysis revealed that EPS-m2 is composed of glucuronic acid, glucose, fucose, galactose/N-acetyl glucosamine, arabinose, xylose, and ribose with a molar ratio of approximately 77:44:29:28:2:1:1. The carbohydrates, protein, and uronic acids contents in EPS-m2 was 78.6 ± 0.1%, 4.38 ± 0.11%, and 3.86 ± 0.09%, respectively. In vitro batch fermentation experiments showed that 77% of EPS-m2 could be degraded by human fecal microbiota after 72 h of fermentation. In reverse, 16S rRNA gene sequencing analysis showed that EPS-m2 increased the abundance of Alistipes, Acinetobacter, Alloprevotella, Howardella, and Oxalobacter; GC detection illustrated that EPS-m2 enhanced the production of SCFAs. These findings indicated that EPS-m2 supplementation could regulate the HGM and might facilitate modulation of human health.

Conjugative plasmid-encoded toxin-antitoxin system PrpT/PrpA directly controls plasmid copy number.

Toxin-antitoxin (TA) loci were initially identified on conjugative plasmids, and one function of plasmid-encoded TA systems is to stabilize plasmids or increase plasmid competition via postsegregational killing. Here, we discovered that the type II TA system, Pseudoalteromonas rubra plasmid toxin-antitoxin PrpT/PrpA, on a low-copy-number conjugative plasmid, directly controls plasmid replication. Toxin PrpT resembles ParE of plasmid RK2 while antitoxin PrpA (PF03693) shares no similarity with previously characterized antitoxins. Surprisingly, deleting this prpA-prpT operon from the plasmid does not result in plasmid segregational loss, but greatly increases plasmid copy number. Mechanistically, the antitoxin PrpA functions as a negative regulator of plasmid replication, by binding to the iterons in the plasmid origin that inhibits the binding of the replication initiator to the iterons. We also demonstrated that PrpA is produced at a higher level than PrpT to prevent the plasmid from overreplicating, while partial or complete degradation of labile PrpA derepresses plasmid replication. Importantly, the PrpT/PrpA TA system is conserved and is widespread on many conjugative plasmids. Altogether, we discovered a function of a plasmid-encoded TA system that provides new insights into the physiological significance of TA systems.

Literature-Based Phenotype Survey and In Silico Genotype Investigation of Antibiotic Resistance in the Genus Bifidobacterium.

Bifidobacteria are typical commensals inhabiting the human intestine and are beneficial to the host because of their probiotic properties. One of the risks concerning probiotics is the potential of introducing antibiotic resistance genes (ARGs) to the host gut pathogens. This study was aimed to depict the general antibiotic resistance characteristics of the genus Bifidobacterium by combining the reported phenotype dataset and in silico genotype prediction. Bifidobacteria were mostly reported to be sensitive to beta-lactams, glycopeptides, chloramphenicol, and rifampicin, but resistant to aminoglycosides, polypeptides, quinolones, and mupirocin. Generally, the resistance phenotypes to erythromycin, tetracycline, fusidic acid, metronidazole, clindamycin, and trimethoprim were variable. Besides cmX and tetQ, characterized in bifidobacterial resident plasmids, 3520 putative ARGs were identified from 831 bifidobacterial genomes through BLASTP search. The identified ARGs matched thirty-eight reference ARGs, four of which seemed to be mutant housekeeping genes. The two high-abundant ARGs, tetW and ermX, were found to have different distribution traits. The predicted ARGs reasonably explained most of the corresponding resistant phenotypes in the published literature.

An in vitro evaluation of the effects of different statins on the structure and function of human gut bacterial community.

Statins, a class of drugs that can effectively remove cholesterol from serum, are used to regulate plasma total cholesterol and reduce the risk of cardiovascular diseases, but it is still unclear whether the drug are modulated by gut microbiota or the structures of gut microbiota are shaped by statins. We investigated the interactions between statins and the human gut microbiota during the in vitro fermentation process by 16S rRNA gene sequencing, gas chromatography (GC), and high-performance liquid chromatography (HPLC) analyses. The presence of fluvastatin (FLU2) specifically promoted the growth of Escherichia/Shigella, Ruminococcaceae UCG 014, and Sutterella. However, the composition of the gut bacterial microbiota remained relatively static in samples treated with rosuvastatin (ROS), simvastatin (SIM), and atorvastatin (ATO). The PICRUSt program predicted moderate differences in the functional categories related to the biosynthesis of other secondary metabolites, cellular processes and signaling, and signal transduction in the FLU2 fermentation samples. Our study revealed substantial variation in the structure and function of microbiomes from the FLU2-treated samples. In addition, short-chain fatty acids (SCFAs) were also significantly decreased in FLU2-treated samples compared with the samples treated with other stains. Statins can be degraded by the human gut microbiota in vitro, and the degradation rate was approximately 7%-30% and 19%-48% after fermentation was allowed to proceed for 24 h and 48 h, respectively. Generally, FLU2 could largely shape the composition and function of human gut microbiota, which resulted in changes in the production of SCFAs. In turn, all statins could be degraded or modified by the gut microbiota. Our study paves the way for elucidating statin-gut microbiota interactions in vitro towards the improvement of the host health and personalized medicine.

Effects of an Escherichia coli exopolysaccharide on human and mouse gut microbiota in vitro.

In this study, an exopolysaccharide (EPS) named EPS-RB was produced when the gene cluster ycjD-fabI-yciW-rnb were overexpressed in E. coli. Monosaccharide composition analysis revealed that EPS-RB is a novel EPS that consisted of L-fucose, L-arabinose, D-galactose/N-acetyl glucosamine, D-glucose, D-xylose, D-ribose, and D-glucuronic acid, and their molecular ratio was approximately 80:3:53:69:1:2:64. The content of carbohydrates, protein, and uronic acids in EPS-RB was 90.35 ± 1.35%, 2.62 ± 0.05% and 8.16 ± 1.00%, respectively. The interaction between EPS-RB and gut microbiota was investigated using an in vitro batch fermentation system. The results showed that ~96% of EPS-RB can be degraded by human fecal microbiota after 72 h fermentation, but few can be degraded by mouse cecal microbiota. Furthermore, high-throughput sequencing showed that EPS-RB regulates the human gut microbiota. The genera Collinsella, Butyricimonas, and Hafnia were enriched in group VIR (EPS-RB as a carbon source) when compared with group VI (no carbon source) and VIS (starch as a carbon source). Short-chain fatty acids (SCFAs) production analysis showed that their concentration was significantly higher in group VIR than groups VI and VIS after 72 h fermentation. In summary, an EPS-RB in E. coli was isolated and its regulatory function on gut microbiota was analyzed.

Analysis of Interactions between Endobiotics and Human Gut Microbiota Using In Vitro Bath Fermentation Systems.

Human intestinal microorganisms have recently become an important target of research in promoting human health and preventing diseases. Consequently, investigations of interactions between endobiotics (e.g., drugs and prebiotics) and gut microbiota have become an important research topic. However, in vivo experiments with human volunteers are not ideal for such studies due to bioethics and economic constraints. As a result, animal models have been used to evaluate these interactions in vivo. Nevertheless, animal model studies are still limited by bioethics considerations, in addition to differing compositions and diversities of microbiota in animals vs. humans. An alternative research strategy is the use of batch fermentation experiments that allow evaluation of the interactions between endobiotics and gut microbiota in vitro. To evaluate this strategy, bifidobacterial (Bif) exopolysaccharides (EPS) were used as a representative xenobiotic. Then, the interactions between Bif EPS and human gut microbiota were investigated using several methods such as thin-layer chromatography (TLC), bacterial community compositional analysis with 16S rRNA gene high-throughput sequencing, and gas chromatography of short-chain fatty acids (SCFAs). Presented here is a protocol to investigate the interactions between endobiotics and human gut microbiota using in vitro batch fermentation systems. Importantly, this protocol can also be modified to investigate general interactions between other endobiotics and gut microbiota.

Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system.

OBJECTIVES: To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates. METHODS: Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coli isolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing. RESULTS: In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing. CONCLUSIONS: CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.

Colistin Resistance Gene mcr-1 Mediates Cell Permeability and Resistance to Hydrophobic Antibiotics.

Colistin is considered the last-resort antibiotic used to treat multidrug resistant bacteria-related infections. However, the discovery of the plasmid-mediated colistin resistance gene, mcr-1, threatens the clinical utility of colistin antibiotics. In this study, the physiological function of MCR-1, which encodes an LPS-modifying enzyme, was investigated in E. coli K-12. Specifically, the impact of mcr-1 on membrane permeability and antibiotic resistance of E. coli was assessed by constructing an mcr-1 deletion mutant and by a complementation study. The removal of the mcr-1 gene from plasmid pHNSHP45 not only led to reduced resistance to colistin but also resulted in a significant change in the membrane permeability of E. coli. Unexpectedly, the removal of the mcr-1 gene increased cell viability under high osmotic stress conditions (e.g., 7.0% NaCl) and led to increased resistance to hydrophobic antibiotics. Increased expression of mcr-1 also resulted in decreased growth rate and changed the cellular morphology of E. coli. Collectively, our results revealed that the spread of mcr-1-carrying plasmids alters other physiological functions in addition to conferring colistin resistance.

Antimicrobial Resistance Profile of mcr-1 Positive Clinical Isolates of Escherichia coli in China From 2013 to 2016.

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.

Type II toxin/antitoxin system ParESO /CopASO stabilizes prophage CP4So in Shewanella oneidensis.

Toxin/antitoxin (TA) loci are commonly found in mobile genetic elements such as plasmids and prophages. However, the physiological functions of these TA loci in prophages and cross-regulation among these TA loci remain largely unexplored. Here, we characterized a newly discovered type II TA pair, ParESO /CopASO , in the CP4So prophage in Shewanella oneidensis. We demonstrated that ParESO /CopASO plays a critical role in the maintenance of CP4So in host cells after its excision. The toxin ParESO inhibited cell growth, resulting in filamentous growth and eventually cell death. The antitoxin CopASO neutralized the toxicity of ParESO through direct protein-protein interactions and repressed transcription of the TA operon by binding to a DNA motif in the promoter region containing two inverted repeats [5'-GTANTAC (N)3 GTANTAC-3']. CopASO also repressed transcription of another TA system PemKSO /PemISO in megaplasmid pMR-1 of S. oneidensis through binding to a highly similar DNA motif in its promoter region. CopASO homologs are widely spread in Shewanella and other Proteobacteria, either as a component of a TA pair or as orphan antitoxins. Our study thus illustrated the cross-regulation of the TA systems in different mobile genetic elements and expanded our understanding of the physiological function of TA systems.

Biofilm Formation and Heat Stress Induce Pyomelanin Production in Deep-Sea Pseudoalteromonas sp. SM9913.

Pseudoalteromonas is an important bacterial genus present in various marine habitats. Many strains of this genus are found to be surface colonizers on marine eukaryotes and produce a wide range of pigments. However, the exact physiological role and mechanism of pigmentation were less studied. Pseudoalteromonas sp. SM9913 (SM9913), an non-pigmented strain isolated from the deep-sea sediment, formed attached biofilm at the solid-liquid interface and pellicles at the liquid-air interface at a wide range of temperatures. Lower temperatures and lower nutrient levels promoted the formation of attached biofilm, while higher nutrient levels promoted pellicle formation of SM9913. Notably, after prolonged incubation at higher temperatures growing planktonically or at the later stage of the biofilm formation, we found that SM9913 released a brownish pigment. By comparing the protein profile at different temperatures followed by qRT-PCR, we found that the production of pigment at higher temperatures was due to the induction of melA gene which is responsible for the synthesis of homogentisic acid (HGA). The auto-oxidation of HGA can lead to the formation of pyomelanin, which has been shown in other bacteria. Fourier Transform Infrared Spectrometer analysis confirmed that the pigment produced in SM9913 was pyomelanin-like compound. Furthermore, we demonstrated that, during heat stress and during biofilm formation, the induction level of melA gene was significantly higher than that of the hmgA gene which is responsible for the degradation of HGA in the L-tyrosine catabolism pathway. Collectively, our results suggest that the production of pyomelanin of SM9913 at elevated temperatures or during biofilm formation might be one of the adaptive responses of marine bacteria to environmental cues.

Pyomelanin from Pseudoalteromonas lipolytica reduces biofouling.

Members of the marine bacterial genus Pseudoalteromonas are efficient producers of antifouling agents that exert inhibitory effects on the settlement of invertebrate larvae. The production of pigmented secondary metabolites by Pseudoalteromonas has been suggested to play a role in surface colonization. However, the physiological characteristics of the pigments produced by Pseudoalteromonas remain largely unknown. In this study, we identified and characterized a genetic variant that hyperproduces a dark-brown pigment and was generated during Pseudoalteromonas lipolytica biofilm formation. Through whole-genome resequencing combined with targeted gene deletion and complementation, we found that a point mutation within the hmgA gene, which encodes homogentisate 1,2-dioxygenase, is solely responsible for the overproduction of the dark-brown pigment pyomelanin. In P. lipolytica, inactivation of the hmgA gene led to the formation of extracellular pyomelanin and greatly reduced larval settlement and metamorphosis of the mussel Mytilus coruscus. Additionally, the extracted pyomelanin from the hmgA deletion mutant and the in vitro-synthesized pyomelanin also reduced larval settlement and metamorphosis of M. coruscus, suggesting that extracellular pyomelanin released from marine Pseudoalteromonas biofilm can inhibit the settlement of fouling organisms.

MqsR/MqsA Toxin/Antitoxin System Regulates Persistence and Biofilm Formation in Pseudomonas putida KT2440.

Bacterial toxin/antitoxin (TA) systems have received increasing attention due to their prevalence, diverse structures, and important physiological functions. In this study, we identified and characterized a type II TA system in a soil bacterium Pseudomonas putida KT2440. This TA system belongs to the MqsR/MqsA family. We found that PP_4205 (MqsR) greatly inhibits cell growth in P. putida KT2440 and Escherichia coli, the antitoxin PP_4204 (MqsA) neutralizes the toxicity of the toxin MqsR, and the two genes encoding them are co-transcribed. MqsR and MqsA interact with each other directly in vivo and MqsA is a negative regulator of the TA operon through binding to the promoter. Consistent with the MqsR/MqsA pair in E. coli, the binding of the toxin MqsR to MqsA inhibits the DNA binding ability of MqsA in P. putida KT2440. Disruption of the mqsA gene which induces mqsR expression increases persister cell formation 53-fold, while overexpressing mqsA which represses mqsR expression reduces persister cell formation 220-fold, suggesting an important role of MqsR in persistence in P. putida KT2440. Furthermore, both MqsR and MqsA promote biofilm formation. As a DNA binding protein, MqsA can also negatively regulate an ECF sigma factor AlgU and a universal stress protein PP_3288. Thus, we revealed an important regulatory role of MqsR/MqsA in persistence and biofilm formation in P. putida KT2440.