Sphingomonas lacusdianchii sp. nov., an attached bacterium inhibited by metabolites from its symbiotic cyanobacterium.

An alpha-proteobacterial strain JXJ CY 53 T was isolated from the cyanosphere of Microcystis sp. FACHB-905 (MF-905) collected from Lake Dianchi, China. JXJ CY 53 T was observed to be an aerobic, Gram-stain-negative, oval shaped, and mucus-secreting bacterium. It had C18:1ω7c and C16:0 as the major cellular fatty acids, Q-10 as the predominant ubiquinone, and sphingoglycolipid, diphosphatidylglycerol, phosphatidylcholine, and phosphatidylmethylethanolamine as the polar lipids. The G + C content of DNA was 65.85%. The bacterium had 16S rRNA gene sequence identities of 98.9% and 98.7% with Sphingomonas panni DSM 15761 T and Sphingomonas hankookensis KCTC 22579 T, respectively, while less than 97.4% identities with other members of the genus. Further taxonomic analysis indicated that JXJ CY 53 T represented a new member of Sphingomonas, and the species epithet was proposed as Sphingomonas lacusdianchii sp. nov. (type strain JXJ CY 53 T = KCTC 72813 T = CGMCC 1.17657 T). JXJ CY 53 T promoted the growth of MF-905 by providing bio-available phosphorus and nitrogen, plant hormones, vitamins, and carotenoids. It could modulate the relative abundances of nonculturable bacteria associated with MF-905 and influence the interactions of MF-905 and other bacteria isolated from the cyanobacterium, in addition to microcystin production characteristics. Meanwhile, MF-905 could provide JXJ CY 53 T dissolved organic carbon for growth, and control the growth of JXJ CY 53 T by secreting specific chemicals other than microcystins. Overall, these results suggest that the interactions between Microcystis and its attached bacteria are complex and dynamic, and may influence the growth characteristics of the cyanobacterium. This study provided new ideas to understand the interactions between Microcystis and its attached bacteria. KEY POINTS: • A novel bacterium (JXJCY 53 T) was isolated from the cyanosphere of Microcystis sp. FACHB-905 (MF-905) • JXJCY 53 T modulated the growth and microcystin production of MF-905 • MF-905 could control the attached bacteria by specific chemicals other than microcystins (MCs).

Complete biodegradation of tetrabromobisphenol A through sequential anaerobic reductive dehalogenation and aerobic oxidation.

Tetrabromobisphenol A (TBBPA), a common brominated flame retardant and a notorious pollutant in anaerobic environments, resists aerobic degradation but can undergo reductive dehalogenation to produce bisphenol A (BPA), an endocrine disruptor. Conversely, BPA is resistant to anaerobic biodegradation but susceptible to aerobic degradation. Microbial degradation of TBBPA via anoxic/oxic processes is scarcely documented. We established an anaerobic microcosm for TBBPA dehalogenation to BPA facilitated by humin. Dehalobacter species increased with a growth yield of 1.5 × 108 cells per µmol Br- released, suggesting their role in TBBPA dehalogenation. We innovatively achieved complete and sustainable biodegradation of TBBPA in sand/soil columns columns, synergizing TBBPA reductive dehalogenation by anaerobic functional microbiota and BPA aerobic oxidation by Sphingomonas sp. strain TTNP3. Over 42 days, 95.11 % of the injected TBBPA in three batches was debrominated to BPA. Following injection of strain TTNP3 cells, 85.57 % of BPA was aerobically degraded. Aerobic BPA degradation column experiments also indicated that aeration and cell colonization significantly increased degradation rates. This treatment strategy provides valuable technical insights for complete TBBPA biodegradation and analogous contaminants.

Metagenomic and proteomic insights into the self-adaptive cell surface hydrophobicity of Sphingomonas sp. strain PAH02 reducing the migration of cadmium-phenanthrene co-pollutant in rice.

Cell surface hydrophobicity (CSH) dominates the interactions between rhizobacteria and pollutants at the soil-water interface, which is critical for understanding the dissipation of pollutants in the rhizosphere microzone of rice. Herein, we explored the effects of self-adaptive CSH of Sphingomonas sp. strain PAH02 on the translocation and biotransformation behaviour of cadmium-phenanthrene (Cd-Phe) co-pollutant in rice and rhizosphere microbiome. We evidenced that strain PAH02 reduced the adsorption of Cd-Phe co-pollutant on the rice root surface while enhancing the degradation of Phe and adsorption of Cd via its self-adaptive CSH in the hydroponic experiment. The significant upregulation of key protein expression levels such as MerR, ARHDs and enoyl-CoA hydratase/isomerase, ensures self-adaptive CSH to cope with the stress of Cd-Phe co-pollutant. Consistently, the bioaugmentation of strain PAH02 promoted the formation of core microbiota in the rhizosphere soil of rice (Oryza sativa L.), such as Bradyrhizobium and Streptomyces and induced gene enrichment of CusA and PobA that are strongly associated with pollutant transformation. Consequently, the contents of Cd and Phe in rice grains at maturity decreased by 17.2% ± 0.2% and 65.7% ± 0.3%, respectively, after the bioaugmentation of strain PAH02. These findings present new opportunities for the implementation of rhizosphere bioremediation strategies of co-contaminants in paddy fields.

Foliar Spraying of Chlorpyrifos Triggers Plant Production of Linolenic Acid Recruiting Rhizosphere Bacterial Sphingomonas sp.

Plants have developed an adaptive strategy for coping with biotic or abiotic stress by recruiting specific microorganisms from the soil pool. Recent studies have shown that the foliar spraying of pesticides causes oxidative stress in plants and leads to changes in the rhizosphere microbiota, but the mechanisms by which these microbiota change and rebuild remain unclear. Herein, we provide for the first-time concrete evidence that rice plants respond to the stress of application of the insecticide chlorpyrifos (CP) by enhancing the release of amino acids, lipids, and nucleotides in root exudates, leading to a shift in rhizosphere bacterial community composition and a strong enrichment of the genus Sphingomonas sp. In order to investigate the underlying mechanisms, we isolated a Sphingomonas representative isolate and demonstrated that it is both attracted by and able to consume linolenic acid, one of the root exudates overproduced after pesticide application. We further show that this strain selectively colonizes roots of treated plants and alleviates pesticide stress by degrading CP and releasing plant-beneficial metabolites. These results indicate a feedback loop between plants and their associated microbiota allowing to respond to pesticide-induced stress.

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.

Cost-Efficient Production of the Sphingan WL Gum by Sphingomonas sp. WG Using Molasses and Sucrose as the Carbon Sources.

The polysaccharide WL gum is produced by the marine microorganism Sphingomonas sp. WG and presents great commercial utility potential in many industries especially in oil industries. However, the high fermentation cost limits its wide application. Therefore, an efficient production system at a lower cost was established using beet molasses to partially replace the commonly used carbon sources. Four different molasses were screened and their composition was investigated. One-factor design and RSM statistical analysis were employed to optimize the WL gum fermentation medium. The effects of molasses on the rheological properties and gene expression of WL gum were also investigated. The results showed that the pretreated beet molasses generated both high broth viscosity and WL gum production (12.94 Pa·s and 11.16 g/L). Heavy metal ions and ash were found to be the key factors in unpretreated and pretreated molasses affecting WL production. The cost-efficient production medium contained (g/L): sucrose 61.79, molasses 9.95, yeast extract 1.23, K2HPO4 1, MgSO4 0.1, ZnSO4 0.1 and the WL gum production reached 40.25 ± 1.15 g/L. The WL gum product WL-molasses showed the higher apparent viscosity, and viscous modulus and elastic modulus than WL-sucrose and WL-mix, which might be related to its highest molecular mass. The higher expressional level of genes such as pgm, ugp, ugd, rmlA, welS, and welG in WL gum synthesis in the mixed carbon source medium caused the high production and broth viscosity. This work provided a cost-efficient method for WL gum production.

Crystal structural analysis and characterization for MlrC enzyme of Sphingomonas sp. ACM-3962 involved in linearized microcystin degradation.

Microcystinase C (MlrC), one key hydrolase of the microcystinase family, plays an important role in linearized microsystin (L-MC) degradation. However, the three-dimensional structure and structural features of MlrC are still unclear. This study obtained high specific activity and high purity of MlrC by heterologous expression, and revealed that MlrC derived from Sphingomonas sp. ACM-3962 (ACM-MlrC) can degrade linearized products of MC-LR, MC-RR and MC-YR to product 3-amino-9-methoxy-2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda), indicating the degradation function and significance in MC-detoxification. More importantly, this study reported the crystal structure of ACM-MlrC at 2.6 Å resolution for the first time, which provides a basis for further understanding the structural characteristics and functions of MlrC. MlrC had a dual-domain feature, namely N and C terminal domain respectively. The N-terminal domain contained a Glutamate-Aspartate-Histidine-Histidine catalytic quadruplex coordinated with zinc ion in each monomer. The importance of zinc ions and their coordinated residues was analyzed by dialysis and site-directed mutagenesis methods. Moreover, the important influence of the N/C-terminal flexible regions of ACM-MlrC was also analyzed by sequence truncation, and then the higher yield and total activity of variants were obtained, which was beneficial to study the better function and application of MlrC.

Mechanistic and evolutionary insights into alkaline phosphatase superfamily through structure-function studies on Sphingomonas alkaline phosphatase.

Alkaline phosphatases (APs), represented by E. coli AP (ECAP), employ an arginine residue to stabilize the phosphoryl group in the active site; whereas, AP from Sphingomonas (SPAP) shows a unique combination of substrate-binding residues; Thr89, Asn110, Lys171, and Arg173. Although such combination has been observed only in SPAP, these residues are present separately in different members of the AP superfamily. Here, we establish the presence of two distinct classes of APs; ECAP-type and SPAP-type. Bioinformatic analyses show that SPAP-type of APs are widely distributed in the bacterial kingdom. The role of active site residues in the catalytic mechanism has been delineated through a set of crystal structures reported here. These structures, representing different stages of the reaction pathway provide wealth of information for the catalytic mechanism. Despite critical differences in the substrate binding residues, SPAP follows a mechanism similar to that of ECAP-type of APs. Structure-based phylogenetic analysis suggests that SPAP and ECAP may have diverged very early during the evolution from a common ancestor. Moreover, it is proposed that the SPAP-type of APs are fundamental members of the AP superfamily and are more closely related to other members of the superfamily as compared to the ECAP-type of APs.

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.

Deciphering chloramphenicol biotransformation mechanisms and microbial interactions via integrated multi-omics and cultivation-dependent approaches.

BACKGROUND: As a widely used broad-spectrum antibiotic, chloramphenicol is prone to be released into environments, thus resulting in the disturbance of ecosystem stability as well as the emergence of antibiotic resistance genes. Microbes play a vital role in the decomposition of chloramphenicol in the environment, and the biotransformation processes are especially dependent on synergistic interactions and metabolite exchanges among microbes. Herein, the comprehensive chloramphenicol biotransformation pathway, key metabolic enzymes, and interspecies interactions in an activated sludge-enriched consortium were elucidated using integrated multi-omics and cultivation-based approaches. RESULTS: The initial biotransformation steps were the oxidization at the C1-OH and C3-OH groups, the isomerization at C2, and the acetylation at C3-OH of chloramphenicol. Among them, the isomerization is an entirely new biotransformation pathway of chloramphenicol discovered for the first time. Furthermore, we identified a novel glucose-methanol-choline oxidoreductase responsible for the oxidization of the C3-OH group in Sphingomonas sp. and Caballeronia sp. Moreover, the subsequent biotransformation steps, corresponding catalyzing enzymes, and the microbial players responsible for each step were deciphered. Synergistic interactions between Sphingomonas sp. and Caballeronia sp. or Cupriavidus sp. significantly promoted chloramphenicol mineralization, and the substrate exchange interaction network occurred actively among key microbes. CONCLUSION: This study provides desirable strain and enzyme resources for enhanced bioremediation of chloramphenicol-contaminated hotspot sites such as pharmaceutical wastewater and livestock and poultry wastewater. The in-depth understanding of the chloramphenicol biotransformation mechanisms and microbial interactions will not only guide the bioremediation of organic pollutants but also provide valuable knowledge for environmental microbiology and biotechnological exploitation. Video Abstract.

Efficient degradation of hydroquinone by a metabolically engineered Pseudarthrobacter sulfonivorans strain.

Pseudarthrobacter sulfonivorans strain Ar51 can degrade crude oil and multi-substituted benzene compounds efficiently at low temperatures. However, it cannot degrade hydroquinone, which is a key intermediate in the degradation of several other compounds of environmental importance, such as 4-nitrophenol, g-hexachlorocyclohexane, 4-hydroxyacetophenone and 4-aminophenol. Here we co-expressed the two subunits of hydroquinone dioxygenase from Sphingomonas sp. strain TTNP3 with different promoters in the strain Ar51. The strain with 2 hdnO promoters exhibited the strongest hydroquinone catabolic activity. However, in the absence of antibiotic selection this ability to degrade hydroquinone was lost due to plasmid instability. Consequently, we constructed a hisD knockout strain, which was unable to synthesise histidine. By introducing the hisD gene onto the plasmid, the ability to degrade hydroquinone in the absence of antibiotic selection was stabilised. In addition, to make the strain more stable for industrial applications, we knocked out the recA gene and integrated the hydroquinone dioxygenase genes at this chromosomal locus. This strain exhibited the strongest activity in catabolizing hydroquinone, up to 470 mg/L in 16 h without antibiotic selection. In addition, this activity was shown to be stable when the strain has cultured in medium without antibiotic selection after 20 passages.

Isolation of Sphingomonas sp. AJ-1 and its enantioselective S-methylation of the triazole fungicide prothioconazole.

Prothioconazole is a widely used chiral triazole fungicide, and its residue pollution has attracted wide attention in recent years. However, little is known about microbial metabolic processes of prothioconazole enantiomers. In this study, a prothioconazole-degrading strain, Sphingomonas sp. AJ-1, was isolated from activated sludge. The optimal temperature and pH for prothioconazole degradation by strain AJ-1 were 30 °C and 6.0, respectively, and the degradation rate of prothioconazole by strain AJ-1 was negatively correlated with the initial concentration. When supplemented with additional carbon source, the degradation rates of 10 mg/L (Rac)-/(S)-/(R)-prothioconazole by strain AJ-1 were 76.0 %, 100.0 % and 64.8 % within 6 d, respectively. The CS bond of prothioconazole was methylated to produce (S)-/(R)-prothioconazole-S-methyl by strain AJ-1, but the degradation rate of prothioconazole by strain AJ-1 with (S)-enantiomer was 2.54-fold of that with (R)-enantiomer. Moreover, the toxicity of (Rac)-prothioconazole-S-methyl was 5.57 times lower than that of (Rac)-prothioconazole to Pseudokirchneriella subcapitata. The results showed that strain AJ-1 had obvious enantioselective metabolism for prothioconazole, and this metabolism was a detoxification process. This study provides new insights into the enantioselective metabolism of the chiral fungicide prothioconazole in microorganisms.

Substrate size-dependent conformational changes of bacterial pectin-binding protein crucial for chemotaxis and assimilation.

Gram-negative Sphingomonas sp. strain A1 exhibits positive chemotaxis toward acidic polysaccharide pectin. SPH1118 has been identified as a pectin-binding protein involved in both pectin chemotaxis and assimilation. Here we show tertiary structures of SPH1118 with six different conformations as determined by X-ray crystallography. SPH1118 consisted of two domains with a large cleft between the domains and substrates bound to positively charged and aromatic residues in the cleft through hydrogen bond and stacking interactions. Substrate-free SPH1118 adopted three different conformations in the open form. On the other hand, the two domains were closed in substrate-bound form and the domain closure ratio was changed in response to the substrate size, suggesting that the conformational change upon binding to the substrate triggered the expression of pectin chemotaxis and assimilation. This study first clarified that the solute-binding protein with dual functions recognized the substrate through flexible conformational changes in response to the substrate size.

Nutritional inter-dependencies and a carbazole-dioxygenase are key elements of a bacterial consortium relying on a Sphingomonas for the degradation of the fungicide thiabendazole.

Thiabendazole (TBZ), is a persistent fungicide/anthelminthic and a serious environmental threat. We previously enriched a TBZ-degrading bacterial consortium and provided first evidence for a Sphingomonas involvement in TBZ transformation. Here, using a multi-omic approach combined with DNA-stable isotope probing (SIP) we verified the key degrading role of Sphingomonas and identify potential microbial interactions governing consortium functioning. SIP and amplicon sequencing analysis of the heavy and light DNA fraction of cultures grown on 13 C-labelled versus 12 C-TBZ showed that 66% of the 13 C-labelled TBZ was assimilated by Sphingomonas. Metagenomic analysis retrieved 18 metagenome-assembled genomes with the dominant belonging to Sphingomonas, Sinobacteriaceae, Bradyrhizobium, Filimonas and Hydrogenophaga. Meta-transcriptomics/-proteomics and non-target mass spectrometry suggested TBZ transformation by Sphingomonas via initial cleavage by a carbazole dioxygenase (car) to thiazole-4-carboxamidine (terminal compound) and catechol or a cleaved benzyl ring derivative, further transformed through an ortho-cleavage (cat) pathway. Microbial co-occurrence and gene expression networks suggested strong interactions between Sphingomonas and a Hydrogenophaga. The latter activated its cobalamin biosynthetic pathway and Sphingomonas its cobalamin salvage pathway to satisfy its B12 auxotrophy. Our findings indicate microbial interactions aligning with the 'black queen hypothesis' where Sphingomonas (detoxifier, B12 recipient) and Hydrogenophaga (B12 producer, enjoying detoxification) act as both helpers and beneficiaries.

Advances in fermentative production, purification, characterization and applications of gellan gum.

Multiple microbial exopolysaccharides have been reported in recent decade with their structural and functional features. Gellan gum (GG) is among these emerging biopolymers with versatile properties. Low production yield, high downstream cost, and abundant market demand have made GG a high cost material. Hence, an understanding on the various possibilities to develop cost-effective gellan gum bioprocess is desirable. This review focuses on details of upstream and downstream process of GG from an industrial perspective. It emphasizes on GG producing Sphingomonas spp., updates on biosynthesis, strain and media engineering, kinetic modeling, bioreactor design and scale-up considerations. Details of the downstream operations with possible modifications to make it cost-effective and environmentally sustainable have been discussed. The updated regulatory criteria for GG as a food ingredient and analytical tools required to validate the same have been briefly discussed. Derivatives of GG and their applications in various industrial segments have also been highlighted.

Enzymatic degradation, antioxidant and rheological properties of a sphingan WL gum from Sphingomonas sp. WG.

A molecular weight (Mw) controllable degradation strategy using the lyase WelR as the efficient tool was established, and the relationship between the Mw and the rheological properties and antioxidant activity of WL gum was systematically investigated. Four different WL samples WL1-WL4 with a gradient Mw change (from 4.70 × 106 to 1.45 × 106 Da) were obtained by controlling the enzymatic reaction conditions. As the Mw decreased, its apparent viscosity, intrinsic viscosity, viscous modulus (G″) and elastic modulus (G') decreased. More interestingly, in contrast to the native WL, the G″ of the degraded WL became higher than G'. Besides, the biodegraded WL samples possessed much higher hydroxyl radicals scavenging activity than the original WL. WL4 with the lowest Mw showed the highest HO radical scavenging activity, about 94.65% at 1 mg/mL. This work provided a useful method to obtain a series of WL samples with controllable Mw and properties, which will broaden the application of sphingans.

Effect of an inorganic nitrogen source (NH4)2SO4 on the production of welan gum from Sphingomonas sp. mutant obtained through UV-ARTP compound mutagenesis.

As one of the most expensive extracellular polysaccharides, welan gum is widely used in biomedicine, food products, and petroleum because of its unique structure and excellent rheological properties. To reduce the cost of welan gum fermentation, together with (NH4)2SO4, which served as the sole nitrogen source, a high-welan-gum-producing mutant, B-8, screened through UV-ARTP compound mutagenesis was used. Under optimum conditions (C:N ratio 25:1, sucrose 50 g/L, (NH4)2SO4 4 g/L, and adding 8 mM NaCl at 32 h fermentation), the yield of welan gum and sucrose conversion were 18.86 g/L and 0.38 g/g, respectively, which were 98.95% and 137.50% higher than those achieved with the parent strain FM01, respectively. After the same treatment process, IN-welan (obtained with (NH4)2SO4) consumed less 95% ethanol, had higher molecular weight, and exhibited better rheological properties than ON-welan (obtained with beef extract). Transcriptome analysis revealed that (NH4)2SO4 could affect the synthetic pathway and monosaccharide content of welan gum by increasing bacterial chemotaxis and the availability of key intermediates. The fermentation performance of Sphingomonas sp. mutants could further be improved by providing several target genes to the mutants through metabolic engineering.

The Novel Amidase PcnH Initiates the Degradation of Phenazine-1-Carboxamide in Sphingomonas histidinilytica DS-9.

Phenazines are an important class of secondary metabolites and are primarily named for their heterocyclic phenazine cores, including phenazine-1-carboxylic acid (PCA) and its derivatives, such as phenazine-1-carboxamide (PCN) and pyocyanin (PYO). Although several genes involved in the degradation of PCA and PYO have been reported so far, the genetic foundations of PCN degradation remain unknown. In this study, a PCN-degrading bacterial strain, Sphingomonas histidinilytica DS-9, was isolated. The gene pcnH, encoding a novel amidase responsible for the initial step of PCN degradation, was cloned by genome comparison and subsequent experimental validation. PcnH catalyzed the hydrolysis of the amide bond of PCN to produce PCA, which shared low identity (only 26 to 33%) with reported amidases. The Km and kcat values of PcnH for PCN were 33.22 ± 5.70 µM and 18.71 ± 0.52 s-1, respectively. PcnH has an Asp-Lys-Cys motif, which is conserved among amidases of the isochorismate hydrolase-like (IHL) superfamily. The replacement of Asp37, Lys128, and Cys163 with alanine in PcnH led to the complete loss of enzymatic activity. Furthermore, the genes pcaA1A2A3A4 and pcnD were found to encode PCA 1,2-dioxygenase and 1,2-dihydroxyphenazine (2OHPC) dioxygenase, which were responsible for the subsequent degradation steps of PCN. The PCN-degradative genes were highly conserved in some bacteria of the genus Sphingomonas, with slight variations in the sequence identities. IMPORTANCE Phenazines have been widely acknowledged as a natural antibiotic for more than 150 years, but their degradation mechanisms are still not completely elucidated. Compared with the studies on the degradation mechanism of PCA and PYO, little is known regarding PCN degradation by far. Previous studies have speculated that its initial degradation step may be catalyzed by an amidase, but no further studies have been conducted. This study identified a novel amidase, PcnH, that catalyzed the hydrolysis of PCN to PCA. In addition, the PCA 1,2-dioxygenase PcaA1A2A3A4 and 2OHPC dioxygenase PcnD were also found to be involved in the subsequent degradation steps of PCN in S. histidinilytica DS-9. And the genes responsible for PCN catabolism are highly conserved in some strains of Sphingomonas. These results deepen our understanding of the PCN degradation mechanism.

[Development of an LB cloning system and its application in expression of fusion genes in Sphingomonas sp. WG].

In order to overcome the challenges of insufficient restriction enzyme sites, and construct a fusion-expression vector with flexible fusion direction, we designed an LB cloning system based on the type IIS and type IIT restriction enzymes LguⅠ and BbvCⅠ. The LB cloning system is constructed by inserting the LB fragment (GCTCTTCCTCAGC) into the multiple cloning site region of the broad-host plasmid pBBR1MCS-3 using PCR. The LB fragment contains partially overlapped recognition sites of LguⅠ and BbvCⅠ. Therefore, the same non-palindromic sequence will be generated by these two restriction endonucleases digestion. This feature can be used to quickly and flexibly insert multiple genes into the expression vector in a stepwise and directed way. In order to verify the efficacy of the cloning system, two glycosyltransferase genes welB and welK of Sphingomonas sp. WG were consecutively fused to the LB cloning vector, and the recombinant plasmid was transferred into Sphingomonas sp. WG by triparental mating. The results showed that gene fusion expression has little effect on sphingan titer, but enhanced the viscosity of sphingan. The viscosity of the sphingan produced by recombinant strain Sphingomonas sp. WG/pBBR1MCS-3-LB-welKB was 24.7% higher than that of the wild strain after fermentation for 84 h, which would be beneficial for its application. In conclusion, the application of LB cloning system were verified using Sphingomonas sp. WG. The LB cloning system may provide an efficient tool for fusion expression of target genes.

Characterization of a polysaccharide hydrogel with high elasticity produced by a mutant strain Sphingomonas sanxanigenens NX03.

Microbial polysaccharides as renewable bioproducts have attracted lots of attention in various industries. Hesan (Highly elastic Sanxan), an exopolysaccharide produced by a plasma mutagenic strain Sphingomonas sanxanigenens NX03, was characterized. It possessed the same monosaccharide composition as the original polysaccharide Sanxan produced from wild-type strain NX02, but significantly reduced acetyl and glyceryl contents. Textural analysis showed the springiness and cohesiveness of Hesan gel was much higher than Sanxan gel, and rheological behaviors indicated it possessed a lower loss factor, and its conformational transition temperatures at different concentrations were obviously lower than Sanxan gel and high-acyl gellan gel, which suggested that Hesan gel was highly elastic and temperature-sensitive. Additionally, Hesan gel could be efficiently produced through micro-aerobic static culture in shallow (10.46 ± 0.30 g/L) and deep liquids (3.21 ± 0.32 g/L), which was significantly different from the fermentation of other water-soluble polysaccharides. In short, this study characterizes a new mutant strain and its polysaccharide products.