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.

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.

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.

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.

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.

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.

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.

Investigation of the interaction between five alkaloids and human hemoglobin by fluorescence spectroscopy and molecular modeling.

This work studied the interaction of human hemoglobin (HHb) with aminophylline, acefylline, caffeine, theophylline and diprophylline systematically by UV-vis absorption spectroscopy and fluorescence spectroscopy in combination with molecular modeling. Five alkaloids caused the fluorescence quenching of HHb by the formation of alkaloids-HHb complex. The binding constants and thermodynamic parameters were obtained. The hydrophobic and electrostatic interactions were the predominant intermolecular forces to stabilize these complexes. Results of thermodynamic analysis and molecular modeling showed that aminophylline was the strongest quencher and diprophylline was the weakest quencher.

Spectroscopic study on the interaction of Trypsin with Bicyclol and analogs.

The interactions between Trypsin and Bicyclol or analogs (Bifendate, I, II and III) were investigated by spectrophotometric methods. It was found that Bicyclol or analogs had strong ability to quench the intrinsic fluorescence of Trypsin by a static quenching procedure. The binding constants were obtained at three temperatures. The thermodynamics parameters reveal that the hydrophobic and electrostatic interactions play an important role in the interaction. Results showed that the microenvironments of tryptophan residue of Trypsin were disturbed by the analogs. Results indicated that Bifendate was the strongest quencher among five compounds.