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1.
Microb Cell Fact ; 23(1): 3, 2024 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-38172890

RESUMO

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.


Assuntos
Proteínas de Bactérias , Streptomyces , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lincomicina , Streptomyces/genética , Streptomyces/metabolismo , Transcriptoma , Regulação Bacteriana da Expressão Gênica , Antibacterianos/farmacologia , Antibacterianos/metabolismo
2.
World J Microbiol Biotechnol ; 39(12): 332, 2023 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-37801155

RESUMO

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.


Assuntos
Lincomicina , Streptomyces , Lincomicina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Metabolismo Secundário , Regulação Bacteriana da Expressão Gênica
3.
Appl Microbiol Biotechnol ; 107(24): 7501-7514, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37768348

RESUMO

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.


Assuntos
Streptomyces coelicolor , Streptomyces , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lincomicina , Antibacterianos/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Regulação Bacteriana da Expressão Gênica , Streptomyces coelicolor/genética , Streptomyces coelicolor/metabolismo
4.
Biosci Biotechnol Biochem ; 87(7): 786-795, 2023 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-37076767

RESUMO

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.


Assuntos
Lincomicina , Streptomyces , Lincomicina/farmacologia , Lincomicina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Streptomyces/genética , Streptomyces/metabolismo , Regulação Bacteriana da Expressão Gênica , Antibacterianos/metabolismo
5.
Appl Microbiol Biotechnol ; 107(9): 2933-2945, 2023 May.
Artigo em Inglês | MEDLINE | ID: mdl-36930277

RESUMO

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.


Assuntos
Ácido Aspártico , Proteínas de Bactérias , Ácido Aspártico/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lincomicina , Antibacterianos , Regulação Bacteriana da Expressão Gênica
6.
J Basic Microbiol ; 63(6): 622-631, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36734183

RESUMO

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.


Assuntos
Lincomicina , Melaninas , Melaninas/metabolismo , Metabolismo Secundário , Levodopa/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Tirosina/metabolismo
7.
Biotechnol Appl Biochem ; 70(1): 238-248, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-35419893

RESUMO

Surfactin, which is composed of a ß-hydroxy fatty acid chain and a peptide ring, has drawn considerable attention due to its potential applications in the biomedicine, bioremediation, and petroleum industries. However, the low yield of surfactin from wild strains still restricts its industrial applications. In this study, eight genes relevant to the fatty acid biosynthesis pathway were targeted to enhance surfactin production, and high surfactin-yielding strains with potential industrial applications were obtained. When ldeHA and acc were co-overexpressed, the surfactin yield of recombinant strains TDS8 and TPS8 increased to 1.55- and 1.19-fold of their parental strains, respectively, again proving that the conversion of acetyl-coenzyme A (CoA) to malonyl-CoA is the rate-limiting step in fatty acid biosynthesis. Furthermore, changes in surfactin isoforms of recombinant strain TPS8 suggest that the fatty acid precursor synthesis pathway can be modified to improve the proportion of different isoforms. In addition, the deletion of lpdV, which is responsible for the conversion of α-ketoacyl-CoA precursors, resulted in a sharp decrease in surfactin production, further demonstrating the importance of branched-chain fatty acid biosynthesis in surfactin production. This work will facilitate the design and construction of more efficiently engineered strains for surfactin production and further extend industrial applications.


Assuntos
Bacillus subtilis , Ácidos Graxos , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Ácidos Graxos/metabolismo , Engenharia Genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Lipopeptídeos/genética , Lipopeptídeos/metabolismo , Peptídeos Cíclicos/genética , Peptídeos Cíclicos/metabolismo
8.
J Basic Microbiol ; 63(2): 190-199, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36453540

RESUMO

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.


Assuntos
Lincomicina , Fator sigma , Fator sigma/genética , Proteínas de Bactérias/genética , Antibacterianos
9.
J Appl Microbiol ; 133(2): 400-409, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35384192

RESUMO

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.


Assuntos
Regulação Bacteriana da Expressão Gênica , Streptomyces , Antibacterianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Lincomicina/metabolismo , Streptomyces/genética , Streptomyces/metabolismo
10.
J Basic Microbiol ; 61(9): 772-781, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34313330

RESUMO

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.


Assuntos
Antibacterianos/biossíntese , Vias Biossintéticas/genética , Regulação Bacteriana da Expressão Gênica , Lincomicina/biossíntese , Streptomyces/genética , Streptomyces/metabolismo , Proteínas de Bactérias/genética , Família Multigênica , Oxirredução , Fatores de Transcrição/genética
12.
ACS Chem Biol ; 15(9): 2476-2484, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32786262

RESUMO

The indolizidine alkaloid swainsonine (SW) is a deadly mycotoxin to livestock that can be produced by different plant-associated fungi, including the endophytic entomopathogenic fungi Metarhizium species. The SW biosynthetic gene cluster has been identified but the genetic mechanism of SW biosynthesis remains obscure. To unveil the SW biosynthetic pathway, we performed gene deletions in M. robertsii, heterologous expression of a core biosynthetic gene, substrate feedings, mass spectrometry, and bioassay analyses in this study. It was unveiled that SW is produced via a multibranched pathway by the hybrid nonribosomal peptide-polyketide synthase (NRPS-PKS) gene cluster in M. robertsii. The precursor pipecolic acid can be converted from lysine by both the SW biosynthetic cluster and the unclustered genes such as lysine cyclodeaminase. The hybrid NRPS-PKS enzyme produces three intermediates with and without domain skipping. Intriguingly, the biosynthetic process is coupled with the cis to trans nonenzymatic epimerization of C1-OH for both hydroxyl- and dihydroxyl-indolizidine intermediates. We also found that SW production was dispensable for fungal colonization of plants and infection of insect hosts. Functional characterization of the SW biosynthetic genes in this study may benefit the safe use of Metarhizium fungi as insect biocontrol agents and the management of livestock pastures from SW contamination by genetic manipulation of the toxin-producing fungi.


Assuntos
Micotoxinas/biossíntese , Swainsonina/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metarhizium/genética , Metarhizium/metabolismo , Família Multigênica , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Domínios Proteicos
13.
Synth Syst Biotechnol ; 5(2): 37-48, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32322696

RESUMO

The lincosamide family antibiotic lincomycin is a widely used antibacterial pharmaceutical generated by Streptomyces lincolnensis, and the high-yield strain B48 produces 2.5 g/L lincomycin, approximately 30-fold as the wild-type strain NRRL 2936. Here, the genome of S. lincolnensis B48 was completely sequenced, revealing a ~10.0 Mb single chromosome with 71.03% G + C content. Based on the genomic information, lincomycin-related primary metabolism network was constructed and the secondary metabolic potential was analyzed. In order to dissect the overproduction mechanism, a comparative genomic analysis with NRRL 2936 was performed. Three large deletions (LDI-III), one large inverted duplication (LID), one long inversion and 80 small variations (including 50 single nucleotide variations, 13 insertions and 17 deletions) were found in B48 genome. Then several crucial mutants contributing to higher production phenotype were validated. Deleting of a MarR-type regulator-encoding gene slinc377 from LDI, and the whole 24.7 kb LDII in NRRL 2936 enhanced lincomycin titer by 244% and 284%, respectively. Besides, lincomycin production of NRRL 2936 was increased to 7.7-fold when a 71 kb supercluster BGC33 from LDIII was eliminated. As for the duplication region, overexpression of the cluster situated genes lmbB2 and lmbU, as well as two novel transcriptional regulator-encoding genes (slinc191 and slinc348) elevated lincomycin titer by 77%, 75%, 114% and 702%, respectively. Furthermore, three negative correlation genes (slinc6156, slinc4481 and slinc6011) on lincomycin biosynthesis, participating in regulation were found out. And surprisingly, inactivation of RNase J-encoding gene slinc6156 and TPR (tetratricopeptide repeat) domain-containing protein-encoding gene slinc4481 achieved lincomycin titer equivalent to 83% and 68% of B48, respectively, to 22.4 and 18.4-fold compared to NRRL 2936. Therefore, the comparative genomics approach combined with confirmatory experiments identified that large fragment deletion, long sequence duplication, along with several mutations of genes, especially regulator genes, are crucial for lincomycin overproduction.

14.
J Microbiol Biotechnol ; 30(1): 127-135, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31693839

RESUMO

Small RNAs (sRNAs) are widespread and play major roles in regulation circuits in bacteria. Previously, we have demonstrated that transcription of esrE is under the control of its own promoter. However, the regulatory elements involved in EsrE sRNA expression are still unknown. In this study, we found that different cis-regulatory elements exist in the promoter region of esrE. We then screened and analyzed seven potential corresponding trans-regulatory elements by using pull-down assays based on DNA affinity chromatography. Among these candidate regulators, we investigated the relationship between the ferric uptake regulator (Fur) and the EsrE sRNA. Electrophoresis mobility shift assays (EMSAs) and ß-galactosidase activity assays demonstrated that Fur can bind to the promoter region of esrE, and positively regulate EsrE sRNA expression in the presence of Fe2+.


Assuntos
Proteínas de Bactérias/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regiões Promotoras Genéticas , RNA Bacteriano/genética , Proteínas Repressoras/genética , Transcrição Gênica
15.
Front Microbiol ; 10: 2428, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31708899

RESUMO

Lincomycin is one of the most important antibiotics in clinical practice. To further understand the regulatory mechanism on lincomycin biosynthesis, we investigated a pleiotropic transcriptional regulator AdpAlin in the lincomycin producer Streptomyces lincolnensis NRRL 2936. Deletion of adpA lin (which generated ΔadpA lin ) interrupted lincomycin biosynthesis and impaired the morphological differentiation. We also found that putative AdpA binding sites were unusually scattered in the promoters of all the 8 putative operons in the lincomycin biosynthetic gene cluster (BGC). In ΔadpA lin , transcript levels of structural genes in 8 putative operons were decreased with varying degrees, and electrophoretic mobility shift assays (EMSAs) confirmed that AdpAlin activated the overall putative operons via directly binding to their promoter regions. Thus, we speculated that the entire lincomycin biosynthesis is under the control of AdpAlin. Besides, AdpAlin participated in lincomycin biosynthesis by binding to the promoter of lmbU which encoded a cluster sited regulator (CSR) LmbU of lincomycin biosynthesis. Results of qRT-PCR and catechol dioxygenase activity assay showed that AdpAlin activated the transcription of lmbU. In addition, AdpAlin activated the transcription of the bldA by binding to its promoter, suggesting that AdpAlin indirectly participated in lincomycin biosynthesis and morphological differentiation. Uncommon but understandable, AdpAlin auto-activated its own transcription via binding to its own promoter region. In conclusion, we provided a molecular mechanism around the effect of AdpAlin on lincomycin biosynthesis in S. lincolnensis, and revealed a cascade regulation of lincomycin biosynthesis by AdpAlin, LmbU, and BldA.

16.
J Basic Microbiol ; 59(11): 1125-1133, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31553492

RESUMO

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.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , RNA Bacteriano/metabolismo , RNA não Traduzido/metabolismo , Proteínas de Transporte/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutação , Proteômica , RNA Bacteriano/genética , RNA não Traduzido/genética , Ubiquinona/biossíntese
17.
Sci Rep ; 9(1): 12537, 2019 08 29.
Artigo em Inglês | MEDLINE | ID: mdl-31467306

RESUMO

Prophage enriched the prokaryotic genome, and their transcriptional factors improved the protein expression network of the host. In this study, we uncovered a new prophage-prophage interaction in E. coli JM83. The Rac prophage protein RacR (GenBank accession no. AVI55875.1) directly activated the transcription of φ80dlacZΔM15 prophage lysozyme encoding gene 19 (GenBank accession no. ACB02445.1, renamed it lysN, lysozyme nineteen), resulting in the growth defect of JM83. This phenomenon also occurred in DH5α, but not in BL21(DE3) and MG1655 due to the genotype differences. However, deletion of lysN could not completely rescued JM83 from the growth arrest, indicating that RacR may regulate other related targets. In addition, passivation of RacR regulation was found in the late period of growth of JM83, and it was transmissible to daughter cells. Altogether, our study revealed part of RacR regulatory network, which suggested some advanced genetic strategies in bacteria.


Assuntos
Escherichia coli/crescimento & desenvolvimento , Escherichia coli/virologia , Prófagos/metabolismo , Proteínas Virais/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Interações Hospedeiro-Patógeno , Muramidase/metabolismo , Prófagos/genética , Proteínas Virais/genética
18.
Biochem Mol Biol Educ ; 47(6): 632-637, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31403746

RESUMO

Genomics is a core course for bioscience-majored undergraduates in many universities. However, the teaching of genomics faces many challenges such as the need for rapid updating of related knowledge, content overlap between genomics and molecular biology, lack of motivation of students in the traditional passive classroom-learning model, the difficulty of presenting omics data analysis processes through lectures, and the challenges of developing the research abilities of senior students. Here we report our updated genomics course design and the teaching practices we used to address these issues, including: use of the presentation-assimilation-discussion (PAD) pedagogy and opportunities for students to practice real data analysis and intensive reading of research papers. The resultant positive outcomes and problems are also discussed. © 2019 International Union of Biochemistry and Molecular Biology, 47(6):632-637, 2019.


Assuntos
Genômica/educação , Aprendizagem , Ensino/educação , Currículo , Humanos , Estudantes , Universidades
19.
J Microbiol Biotechnol ; 29(8): 1273-1280, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31337186

RESUMO

Edwardsiella piscicida is the causative agent of edwardsiellosis, which has caused enormous economic losses worldwide. In our previous research, an attenuated live vaccine WED based on the virulent strain E. piscicida EIB202 can effectively protect turbots against edwardsiellosis via intraperitoneal injection, while vaccination by immersion exhibits a weaker effect. During the development of the immersion vaccine, we surprisingly found the counts of ΔpEIB202/ EIB202 colonized on zebrafish were 100 times lower than those of EIB202. However, pEIB202 carries 53 predicted ORFs and has several copies in E. piscicida EIB202, impeding the study of its function. Thus the replication region is located at a 1 980 bp fragment (from 18 837 to 20 816 bp), containing a transcriptional repressor and a replication protein. Moreover, the minimal replication plasmid, named pRep-q77, has low copies in both E. coli and E. piscicida, but is more stable in E. piscicida than in E. coli. This work lays a foundation for further examination of the function of the virulence plasmid pEIB202.


Assuntos
Replicação do DNA , Edwardsiella/genética , Plasmídeos/genética , Plasmídeos/isolamento & purificação , Animais , Proteínas de Bactérias/genética , DNA Bacteriano/isolamento & purificação , Edwardsiella/crescimento & desenvolvimento , Infecções por Enterobacteriaceae , Escherichia coli/genética , Doenças dos Peixes/microbiologia , Brânquias/microbiologia , Análise de Sequência de DNA , Pele/microbiologia , Virulência/genética , Peixe-Zebra/microbiologia
20.
Biosci Biotechnol Biochem ; 83(11): 2082-2089, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31303144

RESUMO

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.


Assuntos
Antraquinonas/metabolismo , Família Multigênica/genética , Streptomyces griseus/genética , Streptomyces griseus/metabolismo , Clonagem Molecular , Glicosiltransferases/genética , Glicosiltransferases/metabolismo , Streptomyces griseus/enzimologia
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