Interspecies synergistic interactions mediated by cofactor exchange enhance stress tolerance by inducing biofilm formation.

Metabolic exchange plays a crucial role in shaping microbial community interactions and functions, including the exchange of small molecules such as cofactors. Cofactors are fundamental to enzyme catalytic activities; however, the role of cofactors in microbial stress tolerance is unclear. Here, we constructed a synergistic consortium containing two strains that could efficiently mineralize di-(2-ethylhexyl) phthalate under hyperosmotic stress. Integration of transcriptomic analysis, metabolic profiling, and a genome-scale metabolic model (GEM) facilitated the discovery of the potential mechanism of microbial interactions. Multi-omics analysis revealed that the vitamin B12-dependent methionine-folate cycle could be a key pathway for enhancing the hyperosmotic stress tolerance of synergistic consortium. Further GEM simulations revealed interspecies exchange of S-adenosyl-L-methionine and riboflavin, cofactors needed for vitamin B12 biosynthesis, which was confirmed by in vitro experiments. Overall, we proposed a new mechanism of bacterial hyperosmotic stress tolerance: bacteria might promote the production of vitamin B12 to enhance biofilm formation, and the species collaborate with each other by exchanging cofactors to improve consortium hyperosmotic stress tolerance. These findings offer new insights into the role of cofactors in microbial interactions and stress tolerance and are potentially exploitable for environmental remediation. IMPORTANCE: Metabolic interactions (also known as cross-feeding) are thought to be ubiquitous in microbial communities. Cross-feeding is the basis for many positive interactions (e.g., mutualism) and is a primary driver of microbial community assembly. In this study, a combination of multi-omics analysis and metabolic modeling simulation was used to reveal the metabolic interactions of a synthetic consortium under hyperosmotic stress. Interspecies cofactor exchange was found to promote biofilm formation under hyperosmotic stress. This provides a new perspective for understanding the role of metabolic interactions in microbial communities to enhance environmental adaptation, which is significant for improving the efficiency of production activities and environmental bioremediation.

Triclosan Dioxygenase: A Novel Two-component Rieske Nonheme Iron Ring-hydroxylating Dioxygenase Initiates Triclosan Degradation.

The emerging contaminant triclosan (TCS) is widely distributed both in surface water and in wastewater and poses a threat to aquatic organisms and human health due to its resistance to degradation. The dioxygenase enzyme TcsAB has been speculated to perform the initial degradation of TCS, but its precise catalytic mechanism remains unclear. In this study, the function of TcsAB was elucidated using multiple biochemical and molecular biology methods. Escherichia coli BL21(DE3) heterologously expressing tcsAB from Sphingomonas sp. RD1 converted TCS to 2,4-dichlorophenol. TcsAB belongs to the group IA family of two-component Rieske nonheme iron ring-hydroxylating dioxygenases. The highest amino acid identity of TcsA and the large subunits of other dioxygenases in the same family was only 35.50%, indicating that TcsAB is a novel dioxygenase. Mutagenesis of residues near the substrate binding pocket decreased the TCS-degrading activity and narrowed the substrate spectrum, except for the TcsAF343A mutant. A meta-analysis of 1492 samples from wastewater treatment systems worldwide revealed that tcsA genes are widely distributed. This study is the first to report that the TCS-specific dioxygenase TcsAB is responsible for the initial degradation of TCS. Studying the microbial degradation mechanism of TCS is crucial for removing this pollutant from the environment.

Resting for viability: Gordonia polyisoprenivorans ZM27, a robust generalist for petroleum bioremediation under hypersaline stress.

The intrinsic issue associated with the application of microbes for practical pollution remediation involves maintaining the expected activity of engaged strains or consortiums as effectively as that noted under laboratory conditions. Faced with various stress factors, degraders with dormancy ability are more likely to survive and exhibit degradation activity. In this study, a hydrocarbonoclastic and halotolerant strain, Gordonia polyisoprenivorans ZM27, was isolated via stimulation with resuscitation-promoting factor (Rpf). Long-term exposure to dual stresses of 10% NaCl and starvation induced ZM27 to enter a viable but nonculturable (VBNC)-like state, and ZM27 cells could be resuscitated upon Rpf stimulation. Notable changes in both morphological and physiological characteristics between VBNC-like ZM27 cells and resuscitated cells confirmed the response to Rpf and their robust resistance against harsh environments. Whole-genome sequencing and analysis indicated ZM27 could be a generalist degrader with dormancy ability. Subsequently, VBNC-like ZM27 was applied in a soil microcosm experiment to investigate the practical application potential under harsh conditions. VBNC-like ZM27 combined with Rpf stimulation exhibited the most effective biodegradation performance, and the initial n-hexadecane content (1000 mg kg-1) decreased by 63.29% after 14-day incubation. Based on 16S rRNA amplicon sequencing and analysis, Gordonia exhibited a positive response to Rpf stimulation. The relative abundance of genus Gordonia was negatively correlated with that of Alcanivorax, a genus of obligate hydrocarbon degrader with the greatest abundance during soil incubation. Based on the degradation profile and community analysis, generalist Gordonia may be more efficient in hydrocarbon degradation than specialist Alcanivorax under harsh conditions. The characteristics of ZM27, including its sustainable culturability under long-term stress, response to Rpf and robust performance in soil microcosms, are valuable for the remediation of petroleum pollution under stressful conditions. Our work validated the importance of dormancy and highlighted the underestimated role of low-activity degraders in petroleum remediation.

Comamonas resistens sp. nov. and Pseudomonas triclosanedens sp. nov., two members of the phylum Pseudomonadota isolated from the wastewater treatment system of a pharmaceutical factory.

Five strains of two novel species were isolated from the wastewater treatment systems of a pharmaceutical factory located in Zhejiang province, PR China. Strains ZM22T and Y6 were identified as belonging to a potential novel species of the genus Comamonas, whereas strains ZM23T, ZM24 and ZM25 were identified as belonging to a novel species of the genus Pseudomonas. These strains were characterized by polyphasic approaches including 16S rRNA gene analysis, multi-locus sequence analysis, average nucleotide identity (ANI), in silico DNA-DNA hybridization (isDDH), physiological and biochemical tests, as well as chemotaxonomic analysis. Genome-based phylogenetic analysis further confirmed that strains ZM22T and Y6 form a distinct clade closely related to Comamonas testosteroni ATCC 11996T and Comamonas thiooxydans DSM 17888T. Strains ZM23T, ZM24 and ZM25 were grouped as a separate clade closely related to Pseudomonas nitroreducens DSM 14399T and Pseudomonas nicosulfuronedens LAM1902T. The orthoANI and isDDH results indicated that strains ZM22T and Y6 belong to the same species. In addition, genomic DNA fingerprinting demonstrated that these strains do not originate from a single clone. The same results were observed for strains ZM23T, ZM24 and ZM25. Strains ZM22T and Y6 were resistant to multiple antibiotics, whereas strains ZM23T, ZM24 and ZM25 were able to degrade an emerging pollutant, triclosan. The phylogenetic, physiological and biochemical characteristics, as well as chemotaxonomy, allowed these strains to be distinguished from their genus, and we therefore propose the names Comamonas resistens sp. nov. (type strain ZM22=MCCC 1K08496T=KCTC 82561T) and Pseudomonas triclosanedens sp. nov. (type strain ZM23T=MCCC 1K08497T=JCM 36056T), respectively.

sRNA molecules participate in hyperosmotic stress response regulation in Sphingomonas melonis TY.

Drought and salinity are ubiquitous environmental factors that pose hyperosmotic threats to microorganisms and impair their efficiency in performing environmental functions. However, bacteria have developed various responses and regulatory systems to cope with these abiotic challenges. Posttranscriptional regulation plays vital roles in regulating gene expression and cellular homeostasis, as hyperosmotic stress conditions can lead to the induction of specific small RNA molecules (sRNAs) that participate in stress response regulation. Here, we report a candidate functional sRNA landscape of Sphingomonas melonis TY under hyperosmotic stress, and 18 sRNAs were found with a clear response to hyperosmotic stress. These findings will help in the comprehensive analysis of sRNA regulation in Sphingomonas species. Weighted correlation network analysis revealed a 263 nucleotide sRNA, SNC251, which was transcribed from its own promoter and showed the most significant correlation with hyperosmotic response factors. Deletion of snc251 affected biofilm formation and multiple cellular processes, including ribosome-related pathways, aromatic compound degradation, and the nicotine degradation capacity of S. melonis TY, while overexpression of SNC251 facilitated biofilm formation by TY under hyperosmotic stress. Two genes involved in the TonB system were further verified to be activated by SNC251, which also indicated that SNC251 is a trans-acting sRNA. Briefly, this research reports a landscape of sRNAs participating in the hyperosmotic stress response in S. melonis and reveals a novel sRNA, SNC251, which contributes to the S. melonis TY biofilm formation and thus enhances its hyperosmotic stress response ability.IMPORTANCESphingomonas species play a vital role in plant defense and pollutant degradation and survive extensively under drought or salinity. Previous studies have focused on the transcriptional and translational responses of Sphingomonas under hyperosmotic stress, but the posttranscriptional regulation of small RNA molecules (sRNAs) is also crucial for quickly modulating cellular processes to adapt dynamically to osmotic environments. In addition, the current knowledge of sRNAs in Sphingomonas is extremely scarce. This research revealed a novel sRNA landscape of Sphingomonas melonis and will greatly enhance our understanding of sRNAs' acting mechanisms in the hyperosmotic stress response.

A multicomponent propane monooxygenase catalyzes the initial degradation of methyl tert-butyl ether in Mycobacterium vaccae JOB5.

Methyl tert-butyl ether (MTBE) has been recognized as a groundwater contaminant due to its widespread distribution and potential threat to human health. The limited understanding of the enzymes catalyzing MTBE degradation restricts their application in MTBE bioremediation. In this study, an MTBE-degrading soluble di-iron monooxygenase that clusters phylogenetically with a known propane monooxygenase (PRM) encoded by the prmABCD gene cluster was identified and functionally characterized, revealing their role in MTBE metabolism by Mycobacterium vaccae JOB5. Transcriptome analysis demonstrated that the expression of prmABCD was upregulated when JOB5 was induced by MTBE. Escherichia coli Rosetta heterologously expressing prmABCD from JOB5 could transform MTBE, indicating that the PRM of JOB5 is capable of the initial degradation of MTBE. The loss of the gene encoding the oxygenase α-subunit or ß-subunit, the coupling protein, or the reductase disrupted MTBE transformation by the recombinant E. coli Rosetta. In addition, the catalytic capacity of PRM is likely affected by residue G95 in the active site pocket and residues I84, P165, A269, and V270 in the substrate tunnel structure. Mutation of amino acids in the active site and substrate tunnel resulted in inefficiency or inactivation of MTBE degradation, and the activity in 1,4-dioxane (1,4-D) degradation was diminished less than that in MTBE degradation.IMPORTANCEMulticomponent monooxygenases catalyzing the initial hydroxylation of MTBE are important in MTBE biodegradation. Previous studies of MTBE degradation enzymes have focused on P450s, alkane monooxygenase and MTBE monooxygenase, but the vital role of soluble di-iron monooxygenases has rarely been reported. In this study, we deciphered the essential catalytic role of a PRM and revealed the key residues of the PRM in MTBE metabolism. Our findings provide new insight into the MTBE-degrading gene cluster and enzymes in bacteria. This characterization of the PRM associated with MTBE degradation expands our understanding of MTBE-degrading gene diversity and provides a novel candidate enzyme for the bioremediation of MTBE-contaminated sites.

Granule Dendrobii suppresses chronic atrophic gastritis induced by N-methyl-N'-nitro-N-nitrosoguanidine by modulating the gastrointestinal bacteria in rats.

Chronic atrophic gastritis (CAG) is an important stage in the transformation of the normal gastric mucosa into gastric cancer. Granule Dendrobii (GD), a proprietary Chinese medicine, has proven clinical efficacy in treating CAG. GD might promote the reversal of precancerous lesions by improving them in CAG patients. However, the mechanism of GD in CAG treatment is relatively less understood. Here, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced CAG rats were treated with GD and its efficacy was evaluated by observing the changes in the rats' weight and the pathology of gastric tissues. The potential effect of GD on the bacteria was predicted and verified in the large and small intestines and stomachs of CAG rats using amplicon sequencing and RT-qPCR. The results showed that GD could ameliorate the symptoms of body weight loss in CAG rats. Hematoxylin-Eosin (HE) and Alcian Blue (AB) staining showed that GD significantly improved the pathological state of the gastric mucosa in CAG rats. The relative abundance (RA) of Lactobacillus and Turicibacter significantly decreased after GD intervention compared with that of the model group (P < 0.05), indicating that GD might improve CAG by regulating the RA of Lactobacillus and Turicibacter. These findings revealed that Lactobacillus and Turicibacter as bacteria agents associated with gastritis, have the potential to inhibit gastric cancer, especially Turicibacter maybe another pathogen of CAG besides Helicobacter pylori (HP), which is worthy of further study. Meanwhile, the findings provided new ideas and materials for the research and development of new CAG drugs.

Ribosomal RNA gene operon copy number, a functional trait indicating the hydrocarbon degradation level of bacterial communities.

The lack of universal indicators for predicting microbial biodegradation potential and assessing remediation effects limits the generalization of bioremediation. The community-level ribosomal RNA gene operon (rrn) copy number, an important functional trait, has the potential to serve as a key indicator of the bioremediation of organic pollutants. A meta-analysis based on 1275 samples from 26 hydrocarbon-related studies revealed a positive relationship between the microbial hydrocarbon biodegradation level and the community-level rrn copy number in soil, seawater and culture. Subsequently, a microcosm experiment was performed to decipher the community-level rrn copy number response mechanism during total petroleum hydrocarbon (TPH) biodegradation. The treatment combining straw with resuscitation-promoting factor (Rpf) exhibited the highest community-level rrn copy number and the most effective biodegradation compared with other treatments, and the initial TPH content (20,000 mg kg-1) was reduced by 67.67% after 77 days of incubation. TPH biodegradation rate was positively correlated with the average community-level rrn copy number (p = 0.001, R2 = 0.5781). Both meta and community analyses showed that rrn copy number may reflect the potential of hydrocarbon degradation and microbial dormancy. Our findings provide insight into the applicability of the community-level rrn copy number to assess bacterial biodegradation for pollution remediation.

Characterization of Hsp17, a Novel Small Heat Shock Protein, in Sphingomonas melonis TY under Heat Stress.

Bacteria are constantly exposed to a variety of environmental stresses. Temperature is considered one of the most important environmental factors affecting microbial growth and survival. As ubiquitous environmental microorganisms, Sphingomonas species play essential roles in the biodegradation of organic contaminants, plant protection, and environmental remediation. Understanding the mechanism by which they respond to heat shock will help further improve cell resistance by applying synthetic biological strategies. Here, we assessed the transcriptomic and proteomic responses of Sphingomonas melonis TY to heat shock and found that stressful conditions caused significant changes in functional genes related to protein synthesis at the transcriptional level. The most notable changes observed were increases in the transcription (1,857-fold) and protein expression (11-fold) of Hsp17, which belongs to the small heat shock protein family, and the function of Hsp17 in heat stress was further investigated in this study. We found that the deletion of hsp17 reduced the capacity of the cells to tolerate high temperatures, whereas the overexpression of hsp17 significantly enhanced the ability of the cells to withstand high temperatures. Moreover, the heterologous expression of hsp17 in Escherichia coli DH5α conferred to the bacterium the ability to resist heat stress. Interestingly, its cells were elongated and formed connected cells following the increase in temperature, while hsp17 overexpression restored their normal morphology under high temperature. In general, these results indicate that the novel small heat shock protein Hsp17 greatly contributes to maintaining cell viability and morphology under stress conditions. IMPORTANCE Temperature is generally considered the most important factor affecting metabolic functions and the survival of microbes. As molecular chaperones, small heat shock proteins can prevent damaged protein aggregation during abiotic stress, especially heat stress. Sphingomonas species are widely distributed in nature, and they can frequently be found in various extreme environments. However, the role of small heat shock proteins in Sphingomonas under high-temperature stress has not been elucidated. This study greatly enhances our understanding of a novel identified protein, Hsp17, in S. melonis TY in terms of its ability to resist heat stress and maintain cell morphology under high temperature, leading to a broader understanding of how microbes adapt to environmental extremes. Furthermore, our study will provide potential heat resistance elements for further enhancing cellular resistance as well as the synthetic biological applications of Sphingomonas.

Thiamine-Mediated Microbial Interaction between Auxotrophic Rhodococcus ruber ZM07 and Prototrophic Cooperators in the Tetrahydrofuran-Degrading Microbial Community H-1.

As a crucial growth factor, thiamine can regulate functional microbial communities; however, our current understanding of its effect on bioremediation is lacking. Using metatranscriptome and 16S rRNA gene sequencing, we explored the mechanism of response of an efficient tetrahydrofuran (THF)-degrading microbial culture, designated H-1, to exogenous thiamine. Rhodococcus ruber ZM07, a strain performing the THF degradation function in H-1, is a thiamine-auxotrophic bacterium. Furthermore, thiamine affected the microbial community structure of H-1 by altering resource and niche distributions. A microbial co-occurrence network was constructed to help us identify and isolate the cooperators of strain ZM07 in the microbial community. Based on the prediction of the network, two non-THF-degrading bacteria, Hydrogenophaga intermedia ZM11 and Pigmentiphaga daeguensis ZM12, were isolated. Our results suggest that strain ZM11 is a good cooperator of ZM07, and it might be more competitive than other cooperators (e.g., ZM12) in cocultured systems. Additionally, two dominant strains in our microbial culture displayed a "seesaw" pattern, and they showed completely different responses to exogenous thiamine. The growth of the THF degrader ZM07 was spurred by additional thiamine (with an increased relative abundance and significant upregulation of most metabolic pathways), while the growth of the cooperator ZM11 was obviously suppressed under the same circumstances. This relationship was the opposite without thiamine addition. Our study reveals that exogenous thiamine can affect the interaction patterns between THF- and non-THF-degrading microorganisms and provides new insight into the effects of micronutrients on the environmental microbial community. IMPORTANCE Auxotrophic microorganisms play important roles in the biodegradation of pollutants in nature. Exploring the interspecies relationship between auxotrophic THF-degrading bacteria and other microbes is helpful for the efficient utilization of auxotrophic functional microorganisms. Herein, the thiamine-auxotrophic THF-degrading bacterium ZM07 was isolated from the microbial culture H-1, and the effect of thiamine on the structure of H-1 during THF bioremediation was studied. Thiamine may help ZM07 occupy more niches and utilize more resources, thus improving THF degradation efficiency. This research provides a new strategy to improve the THF or other xenobiotic compound biodegradation performance of auxotrophic functional microorganisms/microbial communities by artificially adding special micronutrients. Additionally, the "seesaw" relationship between the thiamine-auxotrophic strain ZM07 and its prototrophic cooperator ZM11 during THF bioremediation could be changed by exogenous thiamine. This study reveals the effect of micronutrients on microbial interactions and provides an effective way to regulate the pollutant biodegradation efficiency of microbial communities.

Regulation Mechanism of Nicotine Catabolism in Sphingomonas melonis TY by a Dual Role Transcriptional Regulator NdpR.

A gene cluster ndp, responsible for nicotine degradation via a variant of the pyridine and pyrrolidine pathways, was previously identified in Sphingomonas melonis TY, but the regulation mechanism remains unknown. The gene ndpR within the cluster was predicted to encode a TetR family transcriptional regulator. Deletion of ndpR resulted in a notably shorter lag phase, higher maximum turbidity, and faster substrate degradation when cultivated in the presence of nicotine. Real-time quantitative PCR and promoter activity analysis in wild-type TY and TYΔndpR strains revealed that genes in the ndp cluster were negatively regulated by NdpR. However, complementation of ndpR to TYΔndpR did not restore transcription repression, but, instead, the complemented strain showed better growth than TYΔndpR. Promoter activity analysis indicates that NdpR also functions as an activator in the transcription regulation of ndpHFEGD. Further analysis through electrophoretic mobility shift assay and DNase I footprinting assay revealed that NdpR binds five DNA sequences within ndp and that NdpR has no autoregulation. These binding motifs overlap with the -35 or -10 box or are located distal upstream of the corresponding transcriptional start site. Multiple sequence alignment of these five NdpR-binding DNA sequences found a conserved motif, with two of the binding sequences being partially palindromic. 2,5-Dihydroxypyridine acted as a ligand of NdpR, preventing NdpR from binding to the promoter region of ndpASAL, ndpTB, and ndpHFEGD. This study revealed that NdpR binds to three promoters in the ndp cluster and is a dual-role transcriptional regulator in nicotine metabolism. IMPORTANCE Gene regulation is critical for microorganisms in the environment in which they may encounter various kinds of organic pollutants. Our study revealed that transcription of ndpASAL, ndpTB, and ndpHFEGD is negatively regulated by NdpR, and NdpR also exhibits a positive regulatory effect on PndpHFEGD. Furthermore, 2,5-dihydroxypyridine was identified as the effector molecular for NdpR and can both prevent the binding of free NdpR to the promoter and release NdpR from the promoters, which is different from previously reported NicR2. Additionally, NdpR was found to have both negative and positive transcription regulatory effects on the same target, PndpHFEGD, while only one binding site was identified, which is notably different from the previously reported TetR family regulators. Moreover, NdpR was revealed to be a global transcriptional regulator. This study provides new insight into the complex gene expression regulation of the TetR family.

Global transcriptional and translational regulation of Sphingomonas melonis TY in response to hyperosmotic stress.

Hyperosmotic stress is one of the most ubiquitous stress factors in microbial habitats and impairs the efficiency of bacteria performing vital biochemical tasks. Sphingomonas serves as a 'superstar' of plant defense and pollutant degradation, and is widely existed in the environment. However, it is still unclear that how Sphingomonas sp. survives under hyperosmotic stress conditions. In this study, multiomics profiling analysis was conducted with S. melonis TY under hyperosmotic conditions to investigate the intracellular hyperosmotic responses. The transcriptome and proteome revealed that sensing systems, including most membrane protein coding genes were upregulated, genes related to two-component systems were tiered adjusted to reset the whole system, other stress response regulators such as sigma-70 were also significantly tiered upregulated. In addition, transport systems together with compatible solute biosynthesis related genes were significantly upregulated to accumulate intracellular nutrients and compatible solutes. When treated with hyperosmotic stress, redox-stress response systems were triggered and mechanosensitive channels together with ion transporters were induced to maintain cellular ion homeostasis. In addition, cellular concentration of c-di-guanosine monophosphate synthetase (c-di-GMP) was reduced, followed by negative influences on genes involved in flagellar assembly and chemotaxis pathways, leading to severe damage to the athletic ability of S. melonis TY, and causing detachments of biofilms. Briefly, this research revealed a comprehensive response mechanism of S. melonis TY exposure to hyperosmotic stress, and emphasized that flagellar assembly and biofilm formation were vulnerable to hyperosmotic conditions. Importance. Sphingomonas, a genus with versatile functions survives extensively, lauded for its prominent role in plant protection and environmental remediation. Current evidence shows that hyperosmotic stress as a ubiquitous environmental factor, usually threatens the survival of microbes and thus impairs the efficiency of their environmental functions. Thus, it is essential to explore the cellular responses to hyperosmotic stress. Hence, this research will greatly enhance our understanding of the global transcriptional and translational regulation of S. melonis TY in response to hyperosmotic stress, leading to broader perspectives on the impacts of stressful environments.

Degradation of Triclosan in the Water Environment by Microorganisms: A Review.

Triclosan (TCS), a kind of pharmaceuticals and personal care products (PPCPs), is widely used and has had a large production over years. It is an emerging pollutant in the water environment that has attracted global attention due to its toxic effects on organisms and aquatic ecosystems, and its concentrations in the water environment are expected to increase since the COVID-19 pandemic outbreak. Some researchers found that microbial degradation of TCS is an environmentally sustainable technique that results in the mineralization of large amounts of organic pollutants without toxic by-products. In this review, we focus on the fate of TCS in the water environment, the diversity of TCS-degrading microorganisms, biodegradation pathways and molecular mechanisms, in order to provide a reference for the efficient degradation of TCS and other PPCPs by microorganisms.

Nutritional stress induced intraspecies competition revealed by transcriptome analysis in Sphingomonas melonis TY.

Bacteria have developed various mechanisms by which they can compete or cooperate with other bacteria. This study showed that in the cocultures of wild-type Sphingomonas melonis TY and its isogenic mutant TYΔndpD grow with nicotine, the former can outcompete the latter. TYΔndpD undergoes growth arrest after four days when cocultured with wild-type TY, whereas the coculture has just entered a stationary phase and the substrate was nearly depleted, and the interaction between the two related strains was revealed by transcriptomic analysis. Analysis of the differential expression genes indicated that wild-type TY inhibited the growth of TYΔndpD mainly through toxin-antitoxin (TA) systems. The four upregulated antitoxin coding genes belong to type II TA systems in which the bactericidal effect of the cognate toxin was mainly through inhibition of translation or DNA replication, whereas wild-type TY with upregulated antitoxin genes can regenerate cognate immunity protein continuously and thus prevent the lethal action of toxin to itself. In addition, colicin-mediated antibacterial activity against closely related species may also be involved in the competition between wild-type TY and TYΔndpD under nutritional stress. Moreover, upregulation of carbon and nitrogen catabolism related-, stress response related-, DNA repair related-, and DNA replication-related genes in wild-type TY showed that it triggered a series of response mechanisms when facing dual stress of competition from isogenic mutant cells and nutritional limitation. Thus, we proposed that S. melonis TY employed the TA systems and colicin to compete with TYΔndpD under nutritional stress, thereby maximally acquiring and exploiting finite resources. KEY POINTS: • Cross-feeding between isogenic mutants and the wild-type strain. • Nutrition stress caused a shift from cooperation to competition. • TYΔndpD undergo growth arrest by exogenous and endogenous toxins.

Network and meta-omics reveal the cooperation patterns and mechanisms in an efficient 1,4-dioxane-degrading microbial consortium.

1,4-Dioxane is an emerging wastewater contaminant with probable human carcinogenicity. Our current understanding of microbial interactions during 1,4-dioxane biodegradation process in mixed cultures is limited. Here, we applied metagenomic, metatranscriptomic and co-occurrence network analyses to unraveling the microbial cooperation between degrader and non-degraders in an efficient 1,4-dioxane-degrading microbial consortium CH1. A 1,4-dioxane-degrading bacterium, Ancylobacter polymorphus ZM13, was isolated from CH1 and had a potential of being one of the important degraders due to its high relative abundance, highly expressed monooxygenase genes tmoABCDEF and high betweenness centrality of networks. The strain ZM13 cooperated obviously with 6 bacterial genera in the network, among which Xanthobacter and Mesorhizobium could be involved in the intermediates metabolism with responsible genes encoding alcohol dehydrogenase (adh), aldehyde dehydrogenase (aldh), glycolate oxidase (glcDEF), glyoxylate carboligase (gcl), malate synthase (glcB) and 2-isopropylmalate synthase (leuA) differentially high-expressed. Also, 1,4-dioxane facilitated the shift of biodiversity and function of CH1, and those cooperators cooperated with ZM13 in the way of providing amino acids or fatty acids, as well as relieving environmental stresses to promote biodegradation. These results provide new insights into our understandings of the microbial interactions during 1,4-dioxane degradation, and have important implications for predicting microbial cooperation and constructing efficient and stable synthetic 1,4-dioxane-degrading consortia for practical remediation.

Microbial interactions enhanced environmental fitness and expanded ecological niches under dibutyl phthalate and cadmium co-contamination.

Co-contamination of organic pollutants and heavy metals is universal in the natural environment. Dibutyl phthalate (DBP), a typical plasticizer, frequently coexists with cadmium (Cd) in nature. However, little attention has been given to the impacts of co-contamination by DBP and Cd on microbial communities or the responses of microbes. To address this, a microcosm experiment was conducted by supplying the exogenous DBP-degrading bacterium Glutamicibacter nicotianae ZM05 to investigate the interplay among DBP-Cd co-contamination, the exogenous DBP-degrading bacterium G. nicotianae ZM05, and indigenous microorganisms. To adapt to co-contamination stress, microbial communities adjust their diversity, interactions, and functions. The stability of the microbial community decreased under co-contamination, as evidenced by lower diversity, simpler network, and fewer ecological niches. Microbial interactions were strengthened, as evidenced by enriched pathways related to microbial communications. Meanwhile, interactions between microorganisms enhanced the environmental fitness of the exogenous DBP-degrading bacterium ZM05. Based on co-occurrence network prediction and coculture experiments, metabolic interactions between the non-DBP-degrading bacterium Cupriavidus metallidurans ZM16 and ZM05 were proven. Strain ZM16 utilized protocatechuic acid, a DBP downstream metabolite, to relieve acid inhibition and adsorbed Cd to relieve toxic stress. These findings help to explain the responses of bacterial and fungal communities to DBP-Cd co-contamination and provide new insights for the construction of degrading consortia for bioremediation.

A Multicomponent THF Hydroxylase Initiates Tetrahydrofuran Degradation in Cupriavidus metallidurans ZM02.

Tetrahydrofuran (THF) has been recognized as a water contaminant because of its human carcinogenicity, extensive use, and widespread distribution. Previously reported multicomponent monooxygenases (MOs) involved in THF degradation were highly conserved, and all of them were from Gram-positive bacteria. In this study, a novel THF-degrading gene cluster (dmpKLMNOP) encoding THF hydroxylase was identified on the chromosome of a newly isolated Gram-negative THF-degrading bacterium, Cupriavidus metallidurans ZM02, and functionally characterized. Transcriptome sequencing and RT-qPCR demonstrated that the expression of dmpKLMNOP was upregulated during the growth of strain ZM02 on THF or phenol. The deletion of oxygenase alpha or beta subunit or the reductase component disrupted the degradation of THF but did not affect the utilization of its hydroxylated product 2-hydroxytetrahydrofuran. Cupriavidus pinatubonensis JMP134 heterologously expressing dmpKLMNOP from strain ZM02 could grow on THF, indicating that the THF hydroxylase DmpZM02KLMNOP is responsible for the initial degradation of THF. Furthermore, the THF and phenol oxidation activities of crude enzyme extracts were detected, and the highest THF and phenol catalytic activities were 1.38 ± 0.24 µmol min-1 mg-1 and 1.77 ± 0.37 µmol min-1 mg-1, respectively, with the addition of NADPH and Fe2+. The characterization of THF hydroxylase associated with THF degradation enriches our understanding of THF-degrading gene diversity and provides a novel potential enzyme for the bioremediation of THF-containing pollutants. IMPORTANCE Multicomponent MOs catalyzing the initial hydroxylation of THF are vital rate-limiting enzymes in the THF degradation pathway. Previous studies of THF degradation gene clusters have focused on Gram-positive bacteria, and the molecular mechanism of THF degradation in Gram-negative bacteria has rarely been reported. In this study, a novel THF hydroxylase encoded by dmpKLMNOP in strain ZM02 was identified to be involved in both THF and phenol degradation. Our findings provide new insights into the THF-degrading gene cluster and enzymes in Gram-negative bacteria.

Acinetobacter tandoii ZM06 Assists Glutamicibacter nicotianae ZM05 in Resisting Cadmium Pressure to Preserve Dipropyl Phthalate Biodegradation.

Dipropyl phthalate (DPrP) coexists with cadmium as cocontaminants in environmental media. A coculture system including the DPrP-degrading bacterium Glutamicibacter nicotianae ZM05 and the nondegrading bacterium Acinetobacter tandoii ZM06 was artificially established to degrade DPrP under Cd(II) stress. Strain ZM06 relieved the pressure of cadmium on strain ZM05 and accelerated DPrP degradation in the following three ways: first, strain ZM06 adsorbed Cd(II) on the cell surface (as observed by scanning electron microscopy) to decrease the concentration of Cd(II) in the coculture system; second, the downstream metabolites of ZM05 were utilized by strain ZM06 to reduce metabolite inhibition; and third, strain ZM06 supplied amino acids and fatty acids to strain ZM05 to relieve stress during DPrP degradation, which was demonstrated by comparative transcriptomic analysis. This study provides an elementary understanding of how microbial consortia improve the degradation efficiency of organic pollutants under heavy metals contamination.

Efficient electrotransformation of Rhodococcus ruber YYL with abundant extracellular polymeric substances via a cell wall-weakening strategy.

Rhodococcus spp. have broad potential applications related to the degradation of organic contaminants and the transformation or synthesis of useful compounds. However, some Gram-positive bacteria are difficult to manipulate genetically due to low transformation efficiency. In this study, we investigated the effects of chemicals including glycine, isonicotinic acid hydrazide (INH), Tween 80 and penicillin G, as well as cell growth status, competent cell concentration, electroporation field strength, electroporation time and heat shock time, on the electrotransformation efficiency of the tetrahydrofuran-degrading bacterium Rhodococcus ruber YYL with low transformation efficiency. The highest electrotransformation efficiency was 1.60 × 106 CFU/µg DNA after parameter optimization. GmhD (D-glycero-D-manno-heptose 1-phosphate guanosyltransferase) gene, which is important in the biosynthesis of lipopolysaccharide, was deleted via the optimized electrotransformation method. Compared with wild-type strain, YYL ΔgmhD showed extremely high electrotransformation efficiency because the surface of it had no mushroom-like extracellular polymeric substances (EPS). In addition, the results showed that cell wall-weakening reagents might cause some translucent substances like EPS, to detach from the cells, increasing the electrotransformation efficiency of strain YYL. We propose that these results could provide a new strategy for unique bacteria that are rich in EPS, for which genetic manipulation systems are difficult to establish.

Identification and characterization of Nornicotine degrading strain Arthrobacter sp. NOR5.

Nornicotine, the primary nicotine metabolite that is formed through demethylation of nicotine in the genus Nicotiana tabacum L. Nornicotine is not only a precursor of tobacco-specific nitrosamine N-nitrosonornicotine but also have detrimental effects to human health. Till now, information on the biotransformation of nornicotine is limited. Herein, we identified and characterized a bacterium Arthrobacter sp. strain NOR5, utilized nornicotine as the sole of carbon and energy source, and degraded 500 mg/L nornicotine completely within 60 h under the optimum conditions of pH 7.0 and 30 °C. In this study, we not only identified previously reported intermediate metabolites such as 6-OH-nornicotine, 6-OH-mysomine, 6-OH-pseudooxy-nornicotine (6HPONor) but also identified a new intermediate metabolite 2,6-di-OH-pseudooxy-nornicotine (2,6DHPONor) by UV spectroscopy and liquid chromatography coupled with time of flight mass spectrometry. About half of 6HPONor could be transformed into 2,6DHPONor that was identified as a novel catabolic intermediate of nornicotine. By the addition of an electron acceptor 2,6-dichlorophenolindophenol (DCIP), the cell-free extract exhibited inducible 6HPONor dehydrogenase activity at 179 ± 60 mU/mg that could convert 6HPONor to 2,6DHPONor. Our study demonstrated that Arthrobacter sp. strain NOR5 has a high potential to degrade the nornicotine completely.