Three new LmbU targets outside lmb cluster inhibit lincomycin biosynthesis in Streptomyces lincolnensis.

BACKGROUND: Antibiotics biosynthesis is usually regulated by the cluster-situated regulatory gene(s) (CSRG(s)), which directly regulate the genes within the corresponding biosynthetic gene cluster (BGC). Previously, we have demonstrated that LmbU functions as a cluster-situated regulator (CSR) of lincomycin. And it has been found that LmbU regulates twenty non-lmb genes through comparative transcriptomic analysis. However, the regulatory mode of CSRs' targets outside the BGC remains unknown. RESULTS: We screened the targets of LmbU in the whole genome of Streptomyces lincolnensis and found fourteen candidate targets, among which, eight targets can bind to LmbU by electrophoretic mobility shift assays (EMSA). Reporter assays in vivo revealed that LmbU repressed the transcription of SLINC_0469 and SLINC_1037 while activating the transcription of SLINC_8097. In addition, disruptions of SLINC_0469, SLINC_1037, and SLINC_8097 promoted the production of lincomycin, and qRT-PCR showed that SLINC_0469, SLINC_1037, and SLINC_8097 inhibited transcription of the lmb genes, indicating that all the three regulators can negatively regulate lincomycin biosynthesis. CONCLUSIONS: LmbU can directly regulate genes outside the lmb cluster, and these genes can affect both lincomycin biosynthesis and the transcription of lmb genes. Our results first erected the cascade regulatory circuit of LmbU and regulators outside lmb cluster, which provides the theoretical basis for the functional research of LmbU family proteins.

Patulibacter defluvii sp. nov., Isolated from a Wastewater Treatment Plant in Guangzhou City, China.

A novel Gram-stain-positive, aerobic, catalase-positive, oxidase-negative, non-motile, and rod-shaped bacterium with ibuprofen-degrading capacity, designated DM4T, was isolated from the sewage of a wastewater treatment plant (WWTP) in Guangzhou city, China. Strain DM4T grew optimally at 0% (w/v) NaCl, pH 5.0-7.0, and 30 °C, forming white colonies on trypticase soy agar. C18:1ω9c, C18:2ω9.12c and C15:1ω10c were the predominant fatty acids. Results of 16S rRNA gene alignment and phylogenetic analysis indicated that strain DM4T belonged to the genus Patulibacter, was closely related to Patulibacter medicamentivorans DSM 25692T (98.5%) and P. brassicae KCTC 39817T (98.1%). Strain DM4T had a genome size of 5.33Mbp, and the DNA G + C content was 75.0%. The average nucleotide identity (ANI), average amino acid identity (AAI), and digital DNA-DNA hybridisation (dDDH) values between strain DM4T and P. medicamentivorans were 85.2%, 83.9%, and 29.0% respectively, while those between strain DM4T and P. brassicae were 78.5%, 71.3%, and 22.2%, respectively. Strain DM4T could significantly degrade ibuprofen by almost 80% after 84 h of incubation, and the degradation kinetics was well fitted with the first-order kinetics. Evidence from phenotypic, phylogenetic and chemotaxonomic analyses support that strain DM4T (= GDMCC 1.4574T = KCTC 59145T) represents a new species of the genus Patulibacter, for which the name Patulibacter defluvii sp. nov. is proposed.

LcbR1, a newly identified GntR family regulator, represses lincomycin biosynthesis in Streptomyces lincolnensis.

The Actinomycetes Streptomyces lincolnensis is the producer of lincosamide-type antibiotic lincomycin, a widely utilized drug against Gram-positive bacteria and protozoans. In this work, through gene knockout, complementation, and overexpression experiments, we identified LcbR1 (SLINC_1595), a GntR family transcriptional regulator, as a repressor for lincomycin biosynthesis. Deletion of lcbR1 boosted lincomycin production by 3.8-fold, without obvious change in morphological development or cellular growth. The homologues of LcbR1 are widely distributed in Streptomyces. Heterologous expression of SCO1410 from Streptomyces coelicolor resulted in the reduction of lincomycin yield, implying that the function of LcbR1 is conserved across different species. Alignment among sequences upstream of lcbR1 and their homologues revealed a conserved 16-bp palindrome (-TTGAACGATCCTTCAA-), which was further proven to be the recognition motif of LcbR1 by electrophoretic mobility shift assays (EMSAs). Via this motif, LcbR1 suppressed the transcription of lcbR1 and SLINC_1596 sharing the same bi-directional promoter. SLINC_1596, one important target of LcbR1, exerted a positive effect on lincomycin production. As detected by quantitative real-time PCR (qRT-PCR) analyses, the expressions of all selected structural (lmbA, lmbC, lmbJ, lmbV, and lmbW), resistance (lmrA and lmrB) and regulatory genes (lmrC and lmbU) from lincomycin biosynthesis cluster were upregulated in deletion strain ΔlcbR1 at 48 h of fermentation, while the mRNA amounts of bldD, glnR, ramR, SLCG_Lrp, and SLCG_2919, previously characterized as the regulators on lincomycin production, were decreased in strain ΔlcbR1, although the regulatory effects of LcbR1 on the above differential expression genes seemed to be indirect. Besides, indicated by EMSAs, the expression of lcbR1 might be regulated by GlnR, SLCG_Lrp, and SLCG_2919, which shows the complexity of the regulatory network on lincomycin biosynthesis. KEY POINTS: • LcbR1 is a novel and conservative GntR family regulator regulating lincomycin production. • LcbR1 modulates the expressions of lcbR1 and SLINC_1596 through a palindromic motif. • GlnR, SLCG_Lrp, and SLCG_2919 can control the expression of lcbR1.

AflQ1-Q2 represses lincomycin biosynthesis via multiple cascades in Streptomyces lincolnensis.

Lincomycin is a broad-spectrum antibiotic and particularly effective against Gram-positive pathogens. Albeit familiar with the biosynthetic mechanism of lincomycin, we know less about its regulation, limiting the rational design for strain improvement. We therefore analyzed two-component systems (TCSs) in Streptomyces lincolnensis, and selected eight TCS gene(s) to construct their deletion mutants utilizing CRISPR/Cas9 system. Among them, lincomycin yield increased in two strains (Δ3900-3901 and Δ5290-5291) while decreased in other four strains (Δ3415-3416, Δ4153-4154, Δ4985, and Δ7949). Considering the conspicuous effect, SLINC_5291-5290 (AflQ1-Q2) was subsequently studied in detail. Its repression on lincomycin biosynthesis was further proved by gene complementation and overexpression. By binding to a 16-bp palindromic motif, the response regulator AflQ1 inhibits the transcription of its encoding gene and the expression of eight operons inside the lincomycin synthetic cluster (headed by lmbA, lmbJ, lmbK, lmbV, lmbW, lmbU, lmrA, and lmrC), as demonstrated by quantitative RT-PCR and electrophoretic mobility shift assays. Besides, the regulatory genes including bldD, glnR, lcbR1, and ramR are also regulated by the TCS. According to the screening towards nitrogen sources, aspartate affects the regulatory behavior of histidine kinase AflQ2. And in return, AflQ1 accelerates aspartate metabolism via ask-asd, asd2, and thrA. In summary, we acquired six novel regulators related to lincomycin biosynthesis, and elucidated the regulatory mechanism of AflQ1-Q2. This highly conserved TCS is a promising target for the construction of antibiotic high-yield strains. KEY POINTS: • AflQ1-Q2 is a repressor for lincomycin production. • AflQ1 modulates the expression of lincomycin biosynthetic and regulatory genes. • Aspartate affects the behavior of AflQ2, and its metabolism is promoted by AflQ1.

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.

AdpAlin regulates lincomycin and melanin biosynthesis by modulating precursors flux in Streptomyces lincolnensis.

Lincomycin is one of the most important antibiotics. However, transcriptional regulation network of secondary metabolism in Streptomyces lincolnensis, the lincomycin producer, remained obscure. AdpA from S. lincolnensis (namely AdpAlin ) has been proved to activate lincomycin biosynthesis. Here we found that both lincomycin and melanin took l-tyrosine as precursor, and AdpAlin activated melanin biosynthesis as well. Three tyrosinases, MelC2, MelD2, and MelE, and one tyrosine peroxygenase, LmbB2, participated in lincomycin and melanin biosynthesis in different ways. For melanin biosynthesis, MelC2 was the only key enzyme required. For lincomycin biosynthesis, MelD2 and LmbB2 were positive factors and were suggested to convert l-tyrosine to l-dihydroxyphenylalanine (l-DOPA). Otherwise, MelC2 and MelE were negative factors for lincomycin biosynthesis and they were supposed to oxidize l-DOPA to generate melanin and certain unknown metabolite, respectively. Based on in silico analysis combined with electrophoretic mobility shift assays (EMSAs), we proved that AdpAlin directly interacted with promoters of melC, melD, and melE by binding to putative AdpA-binding sites in vitro. Moreover, in vivo experiments revealed that AdpAlin positively regulated the transcription of melC and melE, but negatively regulated melD. In conclusion, AdpAlin was the switch of secondary metabolism in S. lincolnensis, and it modulated precursor flux of lincomycin and melanin biosynthesis by directly activating melC, melE, and lmbB1/lmbB2 or repressing melD.

An alternative σ factor σL sl regulates lincomycin production in Streptomyces lincolnensis.

Lincomycin produced by Streptomyces lincolnensis is a critical antibacterial antibiotic in the clinical. To further understand the regulatory mechanism of lincomycin biosynthesis, we identified an alternative σ factor, σL sl , in Streptomyces lincolnensis NRRL 2936. Deletion of sigLsl resulted in an increase in cell growth but a decrease in lincomycin production. σL sl boosted lincomycin biosynthesis by directly stimulating the transcription of four genes (lmbD, lmbV, lmrC, and lmbU) within the lincomycin biosynthetic lmb gene cluster. Besides, σL sl participated in lincomycin biosynthesis by directly stimulating the transcription of mshC, a gene responsible for MSH synthesis. In conclusion, our findings demonstrated that σL sl plays a direct regulatory role in lincomycin biosynthesis. This study extends the understanding of molecular mechanisms of lincomycin biosynthetic regulation.

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.

Microbial community composition and function prediction involved in the hydrolytic bioreactor of coking wastewater treatment process.

The hydrolytic acidification process has a strong ability to conduct denitrogenation and increase the biological oxygen demand/chemical oxygen demand ratio in O/H/O coking wastewater treatment system. More than 80% of the total nitrogen (TN) was removed in the hydrolytic bioreactor, and the hydrolytic acidification process contributed to the provision of carbon sources for the subsequent nitrification process. The structure and diversity of microbial communities were elaborated using high-throughput MiSeq of the 16S rRNA genes. The results revealed that the operational taxonomic units (OTUs) belonged to phyla Bacteroidetes, Betaproteobacteria, and Alphaproteobacteria were the dominant taxa involved in the denitrogenation and degradation of refractory contaminants in the hydrolytic bioreactor, with relative abundances of 22.94 ± 3.72, 29.77 ± 2.47, and 18.23 ± 0.26%, respectively. The results of a redundancy analysis showed that the OTUs belonged to the genera Thiobacillus, Rhodoplanes, and Hylemonella in the hydrolytic bioreactor strongly positively correlated with the chemical oxygen demand, TN, and the removal of phenolics, respectively. The results of a microbial co-occurrence network analysis showed that the OTUs belonged to the phylum Bacteroidetes and the genus Rhodoplanes had a significant impact on the efficiency of removal of contaminants that contained nitrogen in the hydrolytic bioreactor. The potential function profiling results indicate the complementarity of nitrogen metabolism, methane metabolism, and sulfur metabolism sub-pathways that were considered to play a significant role in the process of denitrification. These results provide new insights into the further optimization of the performance of the hydrolytic bioreactor in coking wastewater treatment.

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.

Dietary supplementation with microalgae enhances the zebrafish growth performance by modulating immune status and gut microbiota.

Microalgae are known to be abundant in various habitats around the globe, and are rich in high value-added products such as fatty acids, polysaccharides, proteins, and pigments. Microalgae can be exploited as the basic and primitive food source of aquatic animals. We investigated the effects of dietary supplementation with Schizochytrium sp., Spirulina platensis, Chloroella sorokiniana, Chromochloris zofingiensis, and Dunaliella salina on the growth performance, immune status, and intestinal health of zebrafish (Danio rerio). The results showed that these five microalgae diets could improve the feed conversion rate (FCR), especially the D. salina (FCR = 1.02%) and Schizochytrium sp. (FCR = 1.20%) additive groups. Moreover, the microalgae diets decreased the gene expression level of the pro-inflammatory cytokines IL6, IL8, and IL1ß at a normal physiological state of the intestine, especially the Schizochytrium sp., S. platensis, and D. salina dietary groups. The expression of neutrophil marker b7r was increased in the C. sorokiniana diet group; after, the zebrafish were challenged with Vibrio anguillarum, improving the ability to resist this disease. We also found that microalgae diets could regulate the gut microbiota of fish as well as increase the relative abundance of probiotics. To further explain, Cetobacterium was significantly enriched in the S. platensis additive group and Stenotrophomonas was higher in the Schizochytrium sp. additive group than in the other groups. Conversely, harmful bacteria Mycoplasma reduced in all tested microalgae diet groups. Our study indicated that these microalgae could serve as a food source supplement and benefit the health of fish. KEY POINTS: • Microalgae diets enhanced the growth performance of zebrafish. • Microalgae diets attenuated the intestinal inflammatory responses of zebrafish. • Microalgae diets modulated the gut microbiota composition to improve fish health.

A putative redox-sensing regulator Rex regulates lincomycin biosynthesis in Streptomyces lincolnensis.

Lincomycin is an important antimicrobial agent which is widely used in clinical and animal husbandry. The biosynthetic pathway of lincomycin comes to light in the past 10 years, however, the regulatory mechanism is still unclear. In this study, a redox-sensing regulator Rex from Streptomyces lincolnensis (Rexlin ) was identified and characterized to affect cell growth and lincomycin biosynthesis. Disruption of rex resulted in an increase in cell growth, but a decrease in lincomycin production. The results of quantitative real-time polymerase chain reaction showed that Rexlin can promote transcription of the regulatory gene lmbU and the structural genes lmbA, lmbC, lmbJ, lmbV, and lmbW. However, electrophoretic mobility shift assay analysis demonstrated that Rexlin can not bind to the promoter regions of these genes above. Findings in this study broadened our horizons in the regulatory mechanism of lincomycin production and laid a foundation for strain improvement of antibiotic producers.

The response of polycyclic aromatic hydrocarbon degradation in coking wastewater treatment after bioaugmentation with biosurfactant-producing bacteria Pseudomonas aeruginosa S5.

The polycyclic aromatic hydrocarbons (PAHs) that accumulate during the coking wastewater treatment process are hazardous for the surrounding environment. High molecular weight (HMW) PAHs account for more than 85% of the total PAHs in coking wastewater and sludge, respectively. The degradation of total PAHs increased by 18.97% due to the increased bioavailability of PAHs, after the biosurfactant-producing bacteria Pseudomonas aeruginosa S5 was added. The toxicity of total PAHs to humans was reduced by 26.66% after inoculation with S5. The results suggest biosurfactant-producing bacteria Pseudomonas aeruginosa S5 not only increase the biodegradation of PAHs significantly, but also have a better effect on reducing the human toxicity of PAHs. Kinetic analyses show that PAHs biodegradation fits to first-order kinetics. The degradation rate constant (k) value decreases as the number of PAH rings increases, indicating that HMW PAHs are more difficult to be biodegraded than low molecular weight (LMW) PAHs. The results indicate the bioaugmentation with the biosurfactant-producing strain has significant potential and utility in remediation of PAHs-polluted sites.

Immobilization of Phosphatidylserine by Ethanol and Lysozyme on the Cell Surface for Evaluation of Apoptosis-Like Decay in Activated-Sludge Bacteria.

Accurate determination of microbial viability can be crucial in microbe-dominated biosystems. However, the identification of metabolic decay in bacterial cells can be elaborate and difficult. We sought to identify apoptosis-like bacterial processes by using annexin V-fluorescein isothiocyanate (FITC) (AVF), a probe typically used to stain phosphatidylserine (PS) on exposed cell membranes. The bacterial cell wall provides a barrier that is responsible for low efficiency of direct PS staining of decayed bacterial cells. This can be overcome by pretreatment of the bacteria with 70% ethanol, which fixates the bacteria and preserves the PS status, combined with lysozyme treatment to hydrolyze the cell wall. That treatment improved the efficiency of AVF staining considerably, as shown for pure strains of an Ochrobactrum sp. and a Micrococcus sp. Using this method, decayed bacterial cells (induced by starvation) were more strongly stained, indicating externalization of PS to a greater extent than seen for cells harvested at logarithmic growth. A multispecies microbial sludge was artificially decayed by heat treatment or alternating anoxic-oxic treatment, which also induced increased AVF staining, again presumably via decay-related PS externalization. The method developed proved to be efficient for identification of bacterial decay and has potential for the evaluation of multispecies bacterial samples from sources like soil matrix, bioaerosol, and activated sludge.IMPORTANCE Since the externalization of phosphatidylserine (PS) is considered a crucial characteristic of apoptosis, we sought to identify apoptosis-like decay in bacterial cells by PS staining using AVF. We show that this is possible, provided the bacteria are pretreated with ethanol plus lysozyme to remove a physical staining barrier and preserve the original, decay-related externalization of PS. Our work suggests that PS externalization occurs in starved bacteria and this can be quantified with AVF staining, providing a measure of bacterial decay. Since PS is the common component of the lipid bilayer in bacterial cell membranes, this approach also has potential for evaluation of cell decay of other bacterial species.

Rapid detection of human mastadenovirus species B by recombinase polymerase amplification assay.

BACKGROUND: As an important component of the causative agent of respiratory tract infections, enteric and eye infections, Human mastadenoviruses (HAdVs) species B spread easily in the crowd. In this study, we developed a recombinase polymerase amplification (RPA) assay for rapidly detecting HAdVs species B which was comprised of two different formats (real-time and lateral-flow device). RESULTS: This assay was confirmed to be able to detect 5 different HAdVs species B subtypes (HAdV-B3, HAdV-B7, HAdV-B11, HAdV-B14 and HAdV-B55) without cross-reactions with other subtypes and other respiratory tract pathogens. This RPA assay has not only highly sensitivity with low detection limit of 50 copies per reaction but also short reaction time (< 15 min per detection). Furthermore, the real-time RPA assay has excellent correlation with real-time PCR assay for detection of HAdVs species B presented in clinical samples. CONCLUSIONS: Thus, the RPA assay developed in this study provides an effective and portable approach for the rapid detection of HAdVs species B.

Expression dynamics of gonadotropin-releasing hormone-I and its mutual regulation with luteinizing hormone in chicken ovary and follicles.

Gonadotropin-releasing hormone-I (GnRH-I) has been identified in the ovaries of vertebrate species, and this decapeptide is a key regulator of reproductive functions. However, its biological action and regulatory mechanism in the chicken ovary remain to be characterized. In this study, the expression of GnRH-I gene in chicken hypothalamus and ovaries at different developmental stages and different sizes of follicles was investigated, and the effect of GnRH-I mRNA on chicken follicular cells was analyzed in vitro. The results showed that the expression of GnRH-I was dramatically decreased in the hen ovary compared to that in the hypothalamus after sexual maturation. In the mature ovarian follicles, GnRH-I mRNA levels were significantly higher in theca cells than that in granulosa cells. Overexpression of GnRH-I decreased the expression of luteinizing hormone receptor (LHR) mRNA in theca cells from preovulatory follicles but had no effect on granulosa cells. Treatment of theca cells with different concentrations of luteinizing hormone (LH) significantly increased GnRH-I mRNA expression at low doses (50 ng/ml) but significantly decreased it at higher doses (200 ng/ml). Furthermore, GnRH-I inhibited LH-induced LHR expression at the lower dose of LH (50 ng/ml). These findings provide strong evidence indicating that GnRH-I is an important regulator in the chicken ovary.

Characterization of the bagremycin biosynthetic gene cluster in Streptomyces sp. Tü 4128.

Bagremycin A and bagremycin B isolated from Streptomyces sp. Tü 4128 have activities against Gram-positive bacteria, fungi and also have a weak antitumor activity, which make them have great potential for development of novel antibiotics. Here, we report a draft genome 8,424,112 bp in length of S. sp. Tü 4128 by Illumina Hiseq2000, and identify the bagremycins biosynthetic gene cluster (BGC) by bioinformatics analysis. The putative bagremycins BGC includes 16 open reading frames (ORFs) with the functions of biosynthesis, resistance and regulation. Disruptions of relative genes and HPLC analysis of bagremycins production demonstrated that not all the genes within the BGC are responsible for the biosynthesis of bagremycins. In addition, the biosynthetic pathways of bagremycins are proposed for deeper inquiries into their intriguing biosynthetic mechanism.

Cloning and identification of the Frigocyclinone biosynthetic gene cluster from Streptomyces griseus strain NTK 97.

Frigocyclinone is a novel antibiotic with antibacterial and anticancer activities. It is produced by both Antarctica-derived Streptomyces griseus NTK 97 and marine sponge-associated Streptomyces sp. M7_15. Here, we first report the biosynthetic gene cluster of frigocyclinone in the S. griseus NTK 97. The frigocyclinone gene cluster spans a DNA region of 33-kb which consists of 30 open reading frames (ORFs), encoding minimal type II polyketide synthase, aromatase and cyclase, redox tailoring enzymes, sugar biosynthesis-related enzymes, C-glycosyltransferase, a resistance protein, and three regulatory proteins. Based on the bioinformatic analysis, a biosynthetic pathway for frigocyclinone was proposed. Second, to verify the cloned gene cluster, CRISPR-Cpf1 mediated gene disruption was conducted. Mutant with the disruption of beta-ketoacyl synthase encoding gene frig20 fully loses the ability of producing frigocyclinone, while inactivating the glycosyltransferase gene frig1 leads to the production of key intermediate of anti-MRSA anthraquinone tetrangomycin.

Differential quantitative proteomics reveals the functional difference of two yigP locus products, UbiJ and EsrE.

The yigP (ubiJ) locus has been shown to be associated with many phenotypic changes in Escherichia coli, while the individual function of its two products, EsrE small RNA and UbiJ protein, is still elusive. In this study, we constructed two single-element mutants, EsrE mutant strain Mut and UbiJ mutant strain Ter, on the basis of the base substitution programs. The variable antibiotics resistance and ubiquinone (UQ, coenzyme Q) yield and the similar cell growth between mutants revealed the division of labor and collaboration of EsrE and UbiJ in JM83. Furthermore, we detected the concentration of intracellular proteins of Mut and Ter by stable isotope-labeled quantitative proteomics. The results demonstrate that both EsrE and UbiJ are involved in the aerobic growth of E. coli, while EsrE preferentially contributes to the amino acid-related pathway, and UbiJ is an indispensable factor in the biosynthesis of UQ. Moreover, we uncovered a potential regulatory circuit of d-cycloserine (DCS) that composed of EsrE, GcvA, and GcvB by proteomic analysis.

Simultaneous decarburization, nitrification and denitrification (SDCND) in coking wastewater treatment using an integrated fluidized-bed reactor.

There are two problems in biological treatment of coking wastewater (CWW): incapability of pre-anaerobic treatment to eliminate the toxicity in wastewater, and the lack of carbon source for subsequent denitrification in pre-aerobic treatment. To achieve simultaneous decarburization, nitrification and denitrification (SDCND) in CWW treatment, biological carrier materials was used to build an integrated fluidized-bed reactor (Reactor B, RB). A conventional fluidized-bed reactor (Reactor A, RA) was used as a control reactor under the same condition. The results showed that RB was more advantageous since its removal efficiencies of COD and TN were 90% and 87%, respectively, which were significantly higher than these in RA (82% and 45%), at a hydraulic retention time (HRT) of 60 h. Microelectrode measurement indicated that oxygen transfer was limited inside the carrier where the formation of a dissolved oxygen (DO) concentration gradient was observed. Microbial community analysis showed that the aerobic and anoxic microenvironments in RB promoted the co-existence of a wider variety of bacteria, thus achieving SDCND. These results indicated the integrated fluidized-bed reactor exhibited promising feasibility for simultaneous carbon and nitrogen removal in CWW treatment under the same aeration driven conditions. The SDCND process realized by fluidized-bed reactor provided a reference for the treatment of toxic industrial wastewater with high carbon to nitrogen ratio.