DeoR regulates lincomycin production in Streptomyces lincolnensis.

Regulators belonging to the DeoR family are widely distributed among the bacteria. Few studies have reported that DeoR family proteins regulate secondary metabolism of Streptomyces. This study explored the function of DeoR (SLINC_8027) in Streptomyces lincolnensis. Deletion of deoR in NRRL 2936 led to an increase in cell growth. The lincomycin production of the deoR deleted strain ΔdeoR was 3.4-fold higher than that of the wild strain. This trait can be recovered to a certain extent in the deoR complemented strain ΔdeoR::pdeoR. According to qRT-PCR analysis, DeoR inhibited the transcription of all detectable genes in the lincomycin biosynthesis cluster and repressed the expression of glnR, bldD, and SLCG_Lrp, which encode regulators outside the cluster. DeoR also inhibited the transcription of itself, as revealed by the XylE reporter. Furthermore, we demonstrated that DeoR bound directly to the promoter region of deoR, lmbA, lmbC-D, lmbJ-K, lmrA, lmrC, glnR, and SLCG_Lrp, by recognizing the 5'-CGATCR-3' motif. This study found that versatile regulatory factor DeoR negatively regulates lincomycin biosynthesis and cellular growth in S. lincolnensis, which expanded the regulatory network of lincomycin biosynthesis.

Synergistic approach for acne vulgaris treatment using glycerosomes loaded with lincomycin and lauric acid: Formulation, in silico, in vitro, LC-MS/MS skin deposition assay and in vivo evaluation.

This study aims to develop a pharmaceutical formulation that combines the potent antibacterial effect of lincomycin and lauric acid against Cutibacterium acnes (C. acnes), a bacterium implicated in acne. The selection of lauric acid was based on an in silico study, which suggested that its interaction with specific protein targets of C. acnes may contribute to its synergistic antibacterial and anti-inflammatory effects. To achieve our aim, glycerosomes were fabricated with the incorporation of lauric acid as a main constituent of glycerosomes vesicular membrane along with cholesterol and phospholipon 90H, while lincomycin was entrapped within the aqueous cavities. Glycerol is expected to enhance the cutaneous absorption of the active moieties via hydrating the skin. Optimization of lincomycin-loaded glycerosomes (LM-GSs) was conducted using a mixed factorial experimental design. The optimized formulation; LM-GS4 composed of equal ratios of cholesterol:phospholipon90H:Lauric acid, demonstrated a size of 490 ± 17.5 nm, entrapment efficiency-values of 90 ± 1.4 % for lincomycin, and97 ± 0.2 % for lauric acid, and a surface charge of -30.2 ± 0.5mV. To facilitate its application on the skin, the optimized formulation was incorporated into a carbopol hydrogel. The formed hydrogel exhibited a pH value of 5.95 ± 0.03 characteristic of pH-balanced skincare and a shear-thinning non-Newtonian pseudoplastic flow. Skin deposition of lincomycin was assessed using an in-house developed and validated LC-MS/MS method employing gradient elution and electrospray ionization detection. Results revealed that LM-GS4 hydrogel exhibited a two-fold increase in skin deposition of lincomycin compared to lincomycin hydrogel, indicating improved skin penetration and sustained release. The synergistic healing effect of LM-GS4 was evidenced by a reduction in inflammation, bacterial load, and improved histopathological changes in an acne mouse model. In conclusion, the proposed formulation demonstrated promising potential as a topical treatment for acne. It effectively enhanced the cutaneous absorption of lincomycin, exhibited favorable physical properties, and synergistic antibacterial and healing effects. This study provides valuable insights for the development of an effective therapeutic approach for acne management.

Characterization of a TetR-type positive regulator AtrA for lincomycin production in Streptomyces lincolnensis.

AtrA belongs to the TetR family and has been well characterized for its roles in antibiotic biosynthesis regulation. Here, we identified an AtrA homolog (AtrA-lin) in Streptomyces lincolnensis. Disruption of atrA-lin resulted in reduced lincomycin production, whereas the complement restored the lincomycin production level to that of the wild-type. In addition, atrA-lin disruption did not affect cell growth and morphological differentiation. Furthermore, atrA-lin disruption hindered the transcription of regulatory gene lmbU, structural genes lmbA and lmbW inside the lincomycin biosynthesis gene cluster, and 2 other regulatory genes, adpA and bldA. Completement of atrA-lin restored the transcription of these genes to varying degrees. Notably, we found that AtrA-lin directly binds to the promoter region of lmbU. Collectively, AtrA-lin positively modulated lincomycin production via both pathway-specific and global regulators. This study offers further insights into the functional diversity of AtrA homologs and the mechanism of lincomycin biosynthesis regulation.

Baicalin Alleviates Short-Term Lincomycin-Induced Intestinal and Liver Injury and Inflammation in Infant Mice.

The adverse effects of short-term megadose of antibiotics exposure on the gastrointestinal and liver tissue reactions in young children have been reported. Antibiotic-induced intestinal and liver reactions are usually unpredictable and present a poorly understood pathogenesis. It is, therefore, necessary to develop strategies for reducing the adverse effects of antibiotics. Studies on the harm and rescue measures of antibiotics from the perspective of the gut-liver system are lacking. Here, we demonstrate that lincomycin exposure reduced body weight, disrupted the composition of gut microbiota and intestinal morphology, triggered immune-mediated injury and inflammation, caused liver dysfunction, and affected lipid metabolism. However, baicalin administration attenuated the lincomycin-induced changes. Transcriptome analysis showed that baicalin improved immunity in mice, as evidenced by the decreased levels of intestinal inflammatory cytokines and expression of genes that regulate Th1, Th2, and Th17 cell differentiation, and inhibited mucin type O-glycan biosynthesis pathways. In addition, baicalin improved liver function by upregulating the expression of genes involved in bile acid secretion and lipid degradation, and downregulating genes involved in lipid synthesis in lincomycin-treated mice. Bile acids can regulate intestinal immunity and strengthen hepatoenteric circulation. In addition, baicalin also improved anti-inflammatory bacteria abundance (Blautia and Coprobacillus) and reduced pathogenic bacteria abundance (Proteobacteria, Klebsiella, and Citrobacter) in lincomycin-treated mice. Thus, baicalin can ameliorate antibiotic-induced injury and its associated complications such as liver disease.

GUN1 involvement in the redox changes occurring during biogenic retrograde signaling.

Chloroplast biogenesis requires a tight communication between nucleus and plastids. By retrograde signals, plastids transmit information about their functional and developmental state to adjust nuclear gene expression, accordingly. GENOMES UNCOUPLED 1 (GUN1), a chloroplast-localized protein integrating several developmental and stress-related signals, is one of the main players of retrograde signaling. Here, we focused on the interplay between GUN1 and redox regulation during biogenic retrograde signaling, by investigating redox parameters in Arabidopsis wild type and gun1 seedlings. Our data highlight that during biogenic retrograde signaling superoxide anion (O2-) and hydrogen peroxide (H2O2) play a different role in response to GUN1. Under physiological conditions, even in the absence of a visible phenotype, gun1 mutants show low activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX), with an increase in O2- accumulation and lipid peroxidation, suggesting that GUN1 indirectly protects chloroplasts from oxidative damage. In wild type seedlings, perturbation of chloroplast development with lincomycin causes H2O2 accumulation, in parallel with the decrease of ROS-removal metabolites and enzymes. These redox changes do not take place in gun1 mutants which, in contrast, enhance SOD, APX and catalase activities. Our results indicate that in response to lincomycin, GUN1 is necessary for the H2O2-dependent oxidation of cellular environment, which might contribute to the redox-dependent plastid-to nucleus communication.

Genomics analysis of Lactobacillus paracasei SLP16.

Lactobacillus paracasei SLP 16 was obtained from liquor cellar mud, and it was analysed by genome sequencing on Illumina Hiseqq platform. Then the biological information of L. paracasei SLP16 was analysed by ExPasy (website), and the toxin safety of the strain SLP 16 was analysed by PSI/PHI in the virulence factor database VFDB. Through the second-generation DNA sequencing platform technology, the whole genome information of L. paracasei SLP16 was obtained, which showed that the genome size of the strain SLP 16 was 2·65 mol l-1 , and the GC content of the strain SLP 16 was 46·9%. And a total of 3131 genes were detected, including 3067 genes encoding protein and 63 genes encoding RNA. Whole genome analysis showed that L. paracasei SLP16 had five coding genes of F0 F1 -ATPase, four coding genes of Na+ /H+ antiporter and three coding genes of A-ATPase, which were closely related to the acid tolerance of lactic acid bacteria (LAB). Whole genome analysis of L. paracasei SLP16 showed that SLP 16 had only one CFA synthetic coding gene, and no important BSH coding gene; however, it had F0 F1 -ATPase, Na+ /H+ antiporter and several two-component regulatory systems, and which were related to bile salt tolerance of LAB. Safety evaluation in L. paracasei SLP16 showed that it did not have the virulence factor coding gene related to toxin. Common antibiotic sensitivity tests showed that L. paracasei SLP16 was resistant to compounds such as sulfamethoxazole, ciprofloxacin, gentamicin and lincomycin. In summary, L. paracasei SLP16 had coding genes closely related to acid tolerance and bile salt tolerance, and no coding gene of virulence factors related to toxins, and few kinds of resistant antibiotics. Therefore, whole genome analysis showed that L. paracasei SLP16 was a safe probiotic strain that can be safely applied.

Developmental regulator RamRsl controls both morphological development and lincomycin biosynthesis in Streptomyces lincolnensis.

AIMS: Assessing the role of ramRsl , a gene absent in a lincomycin over-producing strain, in the regulation of morphological development and lincomycin biosynthesis in Streptomyces lincolnensis. METHODS AND RESULTS: The gene ramRsl was deleted from the wild-type strain NRRL 2936 and the ΔramR mutant strain was characterized by a slower growth rate and a delayed morphological differentiation compared to the original strain NRRL 2936. Furthermore, the ΔramR produced 2.6-fold more lincomycin than the original strain, and consistently the level of expression of all lincomycin cluster located genes was enhanced at 48 and 96 h in the ΔramR. Complementation of ΔramR with an intact copy of ramRsl restored all wild-type features, whereas the over-expression of ramRsl led to a reduction of 33% of the lincomycin yield. Furthermore, the level of expression of glnR, bldA and SLCG_2919, three of known lincomycin biosynthesis regulators, was lower in the ΔramR than in the original strain at the early stage of fermentation and we demonstrated, using electrophoretic mobility shift assay and XylE reporter assay, that glnR is a novel direct target of RamR. CONCLUSIONS: Altogether, these results indicated that, beyond promoting the morphological development, RamR regulates negatively lincomycin biosynthesis and positively the expression of the nitrogen regulator GlnR. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated that RamR plays a negative role in the regulation of lincomycin biosynthesis in S. lincolnensis. Interestingly, the deletion of this gene in other antibiotic-producing Streptomyces strains might also increase their antibiotic-producing abilities.

Novel strains with superior degrading efficiency for lincomycin manufacturing biowaste.

As the antibiotic pollution source in the environment, a large amount of biowastes generated from antibiotic fermentation manufacture needs proper disposal. Recycling the biowaste as resources and nutrients is of great interest. Besides, degradation or removal of antibiotics is indispensable for the reclamation of antibiotic manufacturing biowaste. To establish environmentally friendly disposal strategies for lincomycin manufacturing biowaste (LMB), we screened the microbial strains that could efficiently degrade lincomycin from the antibiotic wastewater treatment plant. Among them, three novel strains were identified as Bacillus subtilis (strain LMB-A), Rhodotorula mucilaginosa (strain LMB-D) and Penicillium oxalicum (strain LMB-E), respectively. LMB-A and LMB-D could degrade 92.69% and 74.05% of lincomycin with an initial concentration of 1117.55 mg/L in 144 h, respectively. The lincomycin degradation products were formed by the breakage of amide bond or losing N-demethyl/thiomethyl group from the pyrrolidine/pyranose ringcata cata catalyzed by the strains. Moreover, LMB-A could decontaminate LMB, and the decontaminated LMB could be used as a nitrogen source to culture salt-resistant bacteria and other useful microorganisms. LMB-A and LMB-D have the potential to be used for the bioremediation of water and soil polluted by lincomycin and its analogs. LMB-E could degrade 88.20% LMB after 144-h cultivation. In summary, this study gives an insight into the green disposal of LMB, and the established strategy has potential application for biotreatment of other antibiotic fermentation manufacturing biowastes.

Cellulose defects in the Arabidopsis secondary cell wall promote early chloroplast development.

Lincomycin (LIN)-mediated inhibition of protein synthesis in chloroplasts prevents the greening of seedlings, represses the activity of photosynthesis-related genes in the nucleus, including LHCB1.2, and induces the phenylpropanoid pathway, resulting in the production of anthocyanins. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of LIN or other inhibitors of early chloroplast development. In a screen using concentrations of LIN lower than those employed to isolate gun mutants, we have identified happy on lincomycin (holi) mutants. Several holi mutants show an increased tolerance to LIN, exhibiting de-repressed LHCB1.2 expression and chlorophyll synthesis in seedlings. The mutations responsible were identified by whole-genome single-nucleotide polymorphism (SNP) mapping, and most were found to affect the phenylpropanoid pathway; however, LHCB1.2 expression does not appear to be directly regulated by phenylpropanoids, as indicated by the metabolic profiling of mutants. The most potent holi mutant is defective in a subunit of cellulose synthase encoded by IRREGULAR XYLEM 3, and comparative analysis of this and other cell-wall mutants establishes a link between secondary cell-wall integrity and early chloroplast development, possibly involving altered ABA metabolism or sensing.

Developmental regulator BldD directly regulates lincomycin biosynthesis in Streptomyces lincolnensis.

The regulatory mechanism of lincomycin biosynthesis remains largely unknown, although lincomycin and its derivatives have been of great application in pharmaceutical industry. As a global regulator, BldD is widespread in Streptomyces, and functions as an on-off switch to regulate the transition from morphological differentiation to secondary metabolism, inspiring us to explore scarcely regulatory realm of lincomycin biosynthesis. In this work, deletion of bldD gene (SLCG_1664) in Streptomyces lincolnensis blocked the sporulation and nearly abolished lincomycin production, while the morphological phenotype and lincomycin production were restored when introducing a functional bldD gene into the ΔbldD mutant. S. lincolnensis BldD (BldDSL) was validated to bind to upstream regions of lincomycin biosynthetic structural genes lmbA, lmbC-lmbD, lmbE, lmbV-lmbW, resistant genes lmrA, lmrB, lmrC, and regulatory gene lmbU. Disruption of bldD significantly decreased the transcription of genes in lincomycin biosynthetic cluster, thus resulting in the sharply loss of lincomycin production. These findings indicate that BldDSL, similar to Saccharopolyspora erythraea BldD (BldDSE), directly regulates the biosynthesis of lincomycin. What's more, we discovered that BldDSE could bind to upstream regions of lmbA, lmbV-lmbW, lmrA and lmrC. Corresponding to this, S. lincolnensis BldD can bind to upstream region of eryAI-eryBIV, revealing an interactional regulation of the two BldDs. In summary, our data indicated that the developmental regulator BldD played a vital role in directly regulating the biosynthesis of lincomycin, and expanded the knowledge on lincomycin biosynthetic regulation in S. lincolnensis.

Effects of sunlight, microbial activity, and temperature on the declines of antibiotic lincomycin in freshwater and saline aquaculture pond waters and sediments.

The residues of lincomycin (LIN), an antibiotic administered to aquatic animals, are often detected in aquatic environments. This study investigated effects of three environmental factors, sunlight, microbial activity, and temperature, on declines of spiked LIN in waters and sediment slurry samples collected from freshwater tilapia (Oreochromis mossambicus) and marine shrimp (Litopenaeus vannamei) culture ponds. The results showed that sunlight, temperature, and microbial activity all accelerated LIN transformation in the water and slurry samples. In matrixes of all water and slurry samples, LIN transformation was significantly faster under light conditions [half-life (t1/2) = 24-53 days] than under dark conditions (t1/2 = 154-2897 days). Microbial activity also accelerated LIN transformation; the t1/2 of LIN was shorter after nonsterile treatment (t1/2 = 12-809 days) than after sterile treatment (t1/2 = 154-2897 days). Moreover, LIN transformation was faster at 28 °C (t1/2 = 18-38 days) than at 20 and 12 °C (t1/2 = 34 and 462 days, respectively) in both slurry samples. The results revealed that LIN transformation in aquaculture pond water and sediment was either slow or stagnant. Sunlight, microbial activity, and temperature can accelerate LIN transformation to reduce LIN residue levels.

Hydrothermal treatment of lincomycin mycelial residues: Antibiotic resistance genes reduction and heavy metals immobilization.

In this study, fate of antibiotic resistance genes (ARGs - lmrA, lmrB, ermB, lnuA, lnuB and vgaC) and species distribution of heavy metals during lincomycin mycelial residues hydrothermal treatment (HT) process were investigated. The results showed that HT could reduce both ARGs and mobile genetic elements effectively by 1.02 to 4.14 logs. Total bacterial biomass reflecting by 16S rRNA decreased from 1.27 × 109 to 4.47 × 105 copies g-1 dry weight. Moreover, half-lives of these targets varied from 2.4 min (ermB) to 8.9 min (lmrB) in the first 30 min of treatment based on a biphasic first-order kinetic model. After the first 30 min, however, half-lives ranged between 15.4 min (lmrA) and 247.6 min (ISCR1). Complexation and precipitation resulted in the transformation of heavy metals from weakly bounded to relatively stable fraction in HT process. Simultaneously, their environmental risk level decreased by at least one grade.

Effects of co-composting of lincomycin mycelia dregs with furfural slag on lincomycin degradation, degradation products, antibiotic resistance genes and bacterial community.

This study explored the effects of co-composting of lincomycin mycelia dregs (LMDs) with furfural slag on variations in antibiotic resistance genes (ARGs) and the bacterial community. The results showed that more than 99% lincomycin was reduced after composting. Moreover, the total absolute and relative abundance of ARGs increased by 180 and 5 times, respectively. The gene lnuA was detected in the LMDs compost and it was proved to participate in lincomycin biodegradation based on the analysis of Pearson's correlation and the lincomycin degradation byproducts. Redundancy analysis showed the succession of the bacterial community had a greater influence than the environmental parameters (residual lincomycin, C/N, pH and temperature) on the variation of ARGs during composting. Composting was not effective in reducing most of the ARGs and intI1 and thus the LMDs compost is dangerous to the ecological environment.

[Microbial Community Diversity Analysis During Composting of Lincomycin Mycelia Dreg with Manure].

Aerobic composting experiments were conducted using lincomycin mycelia wastes (dreg) and manure (T), using sewage sludge with manure as a control (CK). High performance liquid phase methods and high throughput sequencing were used to determine the concentration of lincomycin residue and to characterize the microbial community. The results showed that lincomycin was reduced significantly, with the concentration decreasing from 1800 mg·kg-1 to 483 mg·kg-1, accounting for 73% degradation. In addition, the bacterial community abundance and diversity indices were all lower than that of sludge-manure at the mesophilic and thermophilic phases, because of the high concentration of lincomycin residue in lincomycin mycelia dreg. By contrast, the fungal community abundance and diversity indices showed the reverse, due to the high content of organic matter and nitrogen in lincomycin mycelia dreg. Therefore, the microbial communities were greatly different between T and CK treatment with the domain genera of Paucisalibacillus, Cerasibacillus, Bacillus, Virgibacillus, Ureibacillus, Paenibacillus, and Sinibacillus in T compost and Truepera, Actinomadura, Pseudosphingobacterium, Pseudomonas, Luteimonas and Ureibacillus in CK compost. However, as the composting continued to a mature phase, most of the lincomycin was reduced, and the differences between the two microbial communities gradually decreased. This showed that composting could make lincomycin mycelia dreg harmless and could be used to turn it into a resource.

Enhanced lincomycin production by co-overexpression of metK1 and metK2 in Streptomyces lincolnensis.

Streptomyces lincolnensis is generally utilized for the production of lincomycin A (Lin-A), a clinically useful antibiotic to treat Gram-positive bacterial infections. Three methylation steps, catalyzed by three different S-adenosylmethionine (SAM)-dependent methyltransferases, are required in the biosynthesis of Lin-A, and thus highlight the significance of methyl group supply in lincomycin production. In this study, we demonstrate that externally supplemented SAM cannot be taken in by cells and therefore does not enhance Lin-A production. Furthermore, bioinformatics and in vitro enzymatic assays revealed there exist two SAM synthetase homologs, MetK1 (SLCG_1651) and MetK2 (SLCG_3830) in S. lincolnensis that could convert L-methionine into SAM in the presence of ATP. Even though we attempted to inactivate metK1 and metK2, only metK2 was deleted in S. lincolnensis LCGL, named as ΔmetK2. Following a reduction of the intracellular SAM concentration, ΔmetK2 mutant exhibited a significant decrease of Lin-A in comparison to its parental strain. Individual overexpression of metK1 or metK2 in S. lincolnensis LCGL either elevated the amount of intracellular SAM, concomitant with 15% and 22% increase in Lin-A production, respectively. qRT-PCR assays showed that overexpression of either metK1 or metK2 increased the transcription of lincomycin biosynthetic genes lmbA and lmbR, and regulatory gene lmbU, indicating SAM may also function as a transcriptional activator. When metK1 and metK2 were co-expressed, Lin-A production was increased by 27% in LCGL, while by 17% in a high-yield strain LA219X.

A possible mechanism for lincomycin induction of secondary metabolism in Streptomyces coelicolor A3(2).

Lincomycin forms cross-links within the peptidyl transferase loop region of the 23S ribosomal RNA (rRNA) of the 50S subunit of the bacterial ribosome, which is the site of peptide bond formation, thereby inhibiting protein synthesis. We have previously reported that lincomycin at concentrations below the minimum inhibitory concentration potentiates the production of secondary metabolites in actinomycete strains, suggesting that activation of these strains by utilizing the dose-dependent response of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. Here, we aimed to elucidate the fundamental mechanisms underlying lincomycin induction of secondary metabolism in actinomycetes. In the present study, the dose-dependent response of lincomycin on gene expression of the model actinomycete Streptomyces coelicolor A3(2) and possible relationships to secondary metabolism were investigated. RNA sequencing analysis indicated that lincomycin produced enormous changes in gene expression profiles. Moreover, reverse transcription PCR and/or comparative proteome analysis revealed that in S. coelicolor A3(2), lincomycin, which was used at concentrations for markedly increased blue-pigmented antibiotic actinorhodin production, rapidly enhanced expression of the gene encoding the lincomycin-efflux ABC transporter, the 23S rRNA methyltransferase, and the ribosome-splitting factor to boost the intrinsic lincomycin resistance mechanisms and to reconstruct the probably stalled 70S ribosomes with lincomycin; and in contrast temporarily but dramatically reduced mRNA levels of housekeeping genes, such as those encoding FoF1 ATP synthase, RNA polymerase, ribosomal proteins, and transcription and translation factors, with an increase in intracellular NTPs. A possible mechanism for lincomycin induction of secondary metabolism in S. coelicolor A3(2) is discussed on the basis of these results.

Characterization and mechanism analysis of lincomycin biodegradation with Clostridium sp. strain LCM-B isolated from lincomycin mycelial residue (LMR).

Lincomycin mycelial residue (LMR) is the restricted resource because it contains residual lincomycin, which is producing potential risks to the environment and human health. In this study, lincomycin-degrading strain LCM-B was isolated and identified as Clostridium sp. in the LMR. Strain LCM-B was able to degrade 62.03% of lincomycin at the initial concentration of 100 mg L-1 after incubation for 10 d, while only 15.61% of lincomycin was removed at the initial concentration of 500 mg L-1. The removal efficiency of lincomycin by strain LCM-B decreased as the initial concentration increased. Gene lnuB (which encodes the nucleotidyl transferase) was detected in the isolated strain, and it was proven to participate in lincomycin biodegradation based on the analysis of degradation products and pathway. The results provide a relatively complete understanding of lincomycin biodegradation mechanism. Strain LCM-B is promising to eliminate lincomycin from the LMR.

Improvement of lincomycin production by mutant selection and metabolic regulation.

Lincomycin is a lincosamide antibiotic produced by Streptomyces lincolnensis. Through mutagenesis by ethylmethansulfonate (EMS) and ultraviolet (UV) irradiation repeatedly, M2 was picked out in plate with glutamine and propylproline orderly. In 50-L stirred bioreactor, the production of lincomycin, fermented by M2, was increased to 8136 u/ml under the optimal condition as compared to original strain S. lincolnensis 07-5 (6634 u/ml). Two-dimensional gel electrophoresis (2-D GE) and mass spectrometry (MS)-shown LmbG, LmbI, and acetohydroxy acid isomeroreductase were remarkably synthesized in M2. The gene lmbG and lmbI are responsible for methylation in the lincomycin biosynthetic cluster, while acetohydroxy acid isomeroreductase contributes to stronger metabolic capability. Finally, we obtained a better strain for industrial production.

Cometabolic degradation of lincomycin in a Sequencing Batch Biofilm Reactor (SBBR) and its microbial community.

Cometabolism technology was employed to degrade lincomycin wastewater in Sequencing Batch Biofilm Reactor (SBBR). In contrast with the control group, the average removal rate of lincomycin increased by 56.0% and Total Organic Carbon (TOC) increased by 52.5% in the cometabolic system with glucose as growth substrate. Under the same condition, Oxidation-Reduction Potential (ORP) was 85.1±7.3mV in cometabolic system and 198.2±8.4mV in the control group, indicating that glucose changed the bulk ORP and created an appropriate growing environment for function bacteria. Functional groups of lincomycin were effectively degraded in cometabolic system proved by FTIR and GC-MS. Meanwhile, results of DGGE and 16S rDNA showed great difference in dominant populations between cometabolic system and the control group. In cometabolic system, Roseovarius (3.35%), Thiothrix (2.74%), Halomonas (2.49%), Ignavibacterium (2.02%), and TM7_genus_incertae_sedis (1.93%) were verified as dominant populations at genus level. Cometabolism may be synergistically caused by different functional dominant bacteria.

Lincomycin at Subinhibitory Concentrations Potentiates Secondary Metabolite Production by Streptomyces spp.

Antibiotics have either bactericidal or bacteriostatic activity. However, they also induce considerable gene expression in bacteria when used at subinhibitory concentrations (below the MIC). We found that lincomycin, which inhibits protein synthesis by binding to the ribosomes of Gram-positive bacteria, was effective for inducing the expression of genes involved in secondary metabolism in Streptomyces strains when added to medium at subinhibitory concentrations. In Streptomyces coelicolor A3(2), lincomycin at 1/10 of its MIC markedly increased the expression of the pathway-specific regulatory gene actII-ORF4 in the blue-pigmented antibiotic actinorhodin (ACT) biosynthetic gene cluster, which resulted in ACT overproduction. Intriguingly, S. lividans 1326 grown in the presence of lincomycin at a subinhibitory concentration (1/12 or 1/3 of its MIC) produced abundant antibacterial compounds that were not detected in cells grown in lincomycin-free medium. Bioassay and mass spectrometry analysis revealed that some antibacterial compounds were novel congeners of calcium-dependent antibiotics. Our results indicate that lincomycin at subinhibitory concentrations potentiates the production of secondary metabolites in Streptomyces strains and suggest that activating these strains by utilizing the dose-response effects of lincomycin could be used to effectively induce the production of cryptic secondary metabolites. In addition to these findings, we also report that lincomycin used at concentrations for markedly increased ACT production resulted in alteration of the cytoplasmic protein (FoF1 ATP synthase α and ß subunits, etc.) profile and increased intracellular ATP levels. A fundamental mechanism for these unique phenomena is also discussed.