High-efficiency genetic engineering toolkit for virus based on lambda red-mediated recombination.

PURPOSE: Viruses, such as Ebola virus (EBOV), evolve rapidly and threaten the human health. There is a great demand to exploit efficient gene-editing techniques for the identification of virus to probe virulence mechanism for drug development. METHODS: Based on lambda Red recombination in Escherichia coli (E. coli), counter-selection, and in vitro annealing, a high-efficiency genetic method was utilized here for precisely engineering viruses. EBOV trVLPs assay and dual luciferase reporter assay were used to further test the effect of mutations on virus replication. RESULTS: Considering the significance of matrix protein VP24 in EBOV replication, the types of mutations within vp24, including several single-base substitutions, one double-base substitution, two seamless deletions, and one targeted insertion, were generated on the multi-copy plasmid of E. coli. Further, the length of the homology arms for recombination and in vitro annealing, and the amount of DNA cassettes and linear plasmids were optimized to create a more elaborate and cost-efficient protocol than original approach. The effects of VP24 mutations on the expression of a reporter gene (luciferase) from the EBOV minigenome were determined, and results indicated that mutations of key sites within VP24 have significant impacts on EBOV replication. CONCLUSION: This precise mutagenesis method will facilitate effective and simple editing of viral genes in E. coli.

Crosstalk of TetR-like regulator SACE_4839 and a nitrogen regulator for erythromycin biosynthesis.

TetR family transcriptional regulators (TFRs) are widespread in actinomycetes, which exhibit diverse regulatory modes in antibiotic biosynthesis. Nitrogen regulators play vital roles in modulation of primary and secondary metabolism. However, crosstalk between TFR and nitrogen regulator has rarely been reported in actinomycetes. Herein, we demonstrated that a novel TFR, SACE_4839, was negatively correlated with erythromycin yield in Saccharopolyspora erythraea A226. SACE_4839 indirectly suppressed erythromycin synthetic gene eryAI and resistance gene ermE and directly inhibited its adjacent gene SACE_4838 encoding a homologue of nitrogen metabolite repression (NMR) regulator NmrA (herein named NmrR). The SACE_4839-binding sites within SACE_4839-nmrR intergenic region were identified. NmrR positively controlled erythromycin biosynthesis by indirectly stimulating eryAI and ermE and directly repressing SACE_4839. NmrR was found to affect growth viability under the nitrogen source supply. Furthermore, NmrR directly repressed glutamine and glutamate utilization-related genes SACE_1623, SACE_5070 and SACE_5979 but activated nitrate utilization-associated genes SACE_1163, SACE_4070 and SACE_4912 as well as nitrite utilization-associated genes SACE_1476 and SACE_4514. This is the first reported NmrA homolog for modulating antibiotic biosynthesis and nitrogen metabolism in actinomycetes. Moreover, combinatorial engineering of SACE_4839 and nmrR in the high-yield S. erythraea WB resulted in a 68.8% increase in erythromycin A production. This investigation deepens the understanding of complicated regulatory network for erythromycin biosynthesis. KEY POINTS: • SACE_4839 and NmrR had opposite contributions to erythromycin biosynthesis. • NmrR was first identified as a homolog of another nitrogen regulator NmrA. • Cross regulation between SACE_4839 and NmrR was revealed.

Joint engineering of SACE_Lrp and its target MarR enhances the biosynthesis and export of erythromycin in Saccharopolyspora erythraea.

The Lrp and MarR families are two groups of transcriptional regulators widely distributed among prokaryotes. However, the hierarchical-regulatory relationship between the Lrp family and the MarR family remains unknown. Our previous study found that an Lrp (SACE_Lrp) from Saccharopolyspora erythraea indirectly repressed the biosynthesis of erythromycin. In this study, we characterized a novel MarR family protein (SACE_6745) from S. erythraea, which is controlled by SACE_Lrp and plays a direct regulatory role in erythromycin biosynthesis and export. SACE_Lrp directly regulated the expression of marR by specifically binding a precise site OM (5'-CTCCGGGAACCATT-3'). Gene disruption of marR increased the production of erythromycin by 45% in S. erythraea A226. We found that MarR has direct DNA-binding activity for the promoter regions of the erythromycin biosynthetic genes, as well as an ABC exporter SACE_2701-2702 which was genetically proved to be responsible for erythromycin efflux. Disruption of SACE_Lrp in industrial S. erythraea WB was an efficient strategy to enhance erythromycin production. Herein, we jointly engineered SACE_Lrp and its target MarR by deleting marR in WBΔSACE_Lrp, resulting in 20% increase in erythromycin yield in mutant WBΔLrpΔmarR compared to WBΔSACE_Lrp, and 39% to WB. Overall, our findings provide new insights into the hierarchical-regulatory relationship of Lrp and MarR proteins and new avenues for coordinating antibiotic biosynthesis and export by joint engineering regulators in actinomycetes. KEY POINTS: • The hierarchical-regulatory relationship between SACE_Lrp and MarR was identified. • MarR directly controlled the expression of erythromycin biosynthesis and export genes. • Joint engineering of SACE_Lrp-MarR regulatory element enhanced erythromycin production.

The variable oligomeric state of Amuc_1100 from Akkermansia muciniphila.

Akkermansia muciniphila is a beneficial microorganism colonized in the human gut that can reverse many intestinal metabolic-related diseases. Amuc_1100 is an outer-membrane protein of A. muciniphila. Oral administration of Amuc_1100 can reduce fat mass development, insulin resistance, and dyslipidemia in mice and activated the toll-like receptor 2 (TLR2) to regulate the immune response of the host, but the molecular mechanism remains unclear. Here we report the crystal structure of the extramembranous domain of Amuc_1100, which consists of a four-stranded antiparallel ß-sheet and four α-helices. Two C-terminal helices and the four-stranded antiparallel ß-sheet formed two "αßß" motifs and constituted the core domain, which shared a similar fold with type IV pili and type II Secretion system protein. Although the full-length of the extramembranous domain of Amuc_1100 existed as a monomer in solution, they formed trimer in the crystal. Elimination of the N-terminal coiled-coil helix α1 led to dimerization of Amuc_1100 both in solution and in crystal, indicating that the oligomeric state of Amuc_1100 was variable and could be influenced by α1. In addition, we identified that Amuc_1100 could directly bind human TLR2 (hTRL2) in vitro, suggesting that Amuc_1100 may serve as a new ligand for hTLR2. Dimerization of Amuc_1100 improved its hTLR2-binding affinity, suggesting that the α1-truncated Amuc_1100 could be a beneficial candidate for the development of A. muciniphila related drugs.

Transcriptional regulation of a leucine-responsive regulatory protein for directly controlling lincomycin biosynthesis in Streptomyces lincolnensis.

Leucine-responsive regulatory proteins (Lrps) are a family of transcription factors involved in diverse biological processes in bacteria. So far, molecular mechanism of Lrps for regulating antibiotics biosynthesis in actinomycetes remains largely unexplored. This study, for the first time in Streptomyces lincolnensis, identified an Lrp (named as SLCG_Lrp) associated with lincomycin production. SLCG_Lrp was validated to be a positive regulator for lincomycin biosynthesis by directly stimulating transcription of two structural genes (lmbA and lmbV), three resistance genes (lmrA, lmrB and lmrC), and a regulatory gene (lmbU) within the lincomycin biosynthetic gene (lin) cluster. SLCG_Lrp was transcriptionally self-inhibited and triggered the expression of its adjacent gene SLCG_3127 encoding a LysE superfamily protein. Further, the binding site of SLCG_Lrp in the intergenic region of SLCG_3127 and SLCG_Lrp was precisely identified. Inactivation of SLCG_3127 in S. lincolnensis resulted in yield improvement of lincomycin, which was caused by intracellular accumulation of proline and cysteine. Arginine and phenylalanine were identified as specific regulatory ligands, respectively, to reduce and promote DNA-binding affinity of SLCG_Lrp. We further found that SLCG_Lrp was directly repressed by SLCG_2919, the first identified transcription factor outside lin cluster for lincomycin production. Therefore, our findings revealed SLCG_Lrp-mediated transcriptional regulation of lincomycin biosynthesis. This study extends the understanding of molecular mechanisms underlying lincomycin biosynthetic regulation.

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.

TetR-Type Regulator SLCG_2919 Is a Negative Regulator of Lincomycin Biosynthesis in Streptomyces lincolnensis.

Lincomycin A (Lin-A) is a widely used antibacterial antibiotic fermented by Streptomyces lincolnensis However, the transcriptional regulatory mechanisms underlying lincomycin biosynthesis have seldom been investigated. Here, we first identified a TetR family transcriptional regulator (TFR), SLCG_2919, which negatively modulates lincomycin biosynthesis in S. lincolnensis LCGL. SLCG_2919 was found to specifically bind to promoter regions of the lincomycin biosynthetic gene cluster (lin cluster), including 25 structural genes, three resistance genes, and one regulatory gene, and to inhibit the transcription of these genes, demonstrating a directly regulatory role in lincomycin biosynthesis. Furthermore, we found that SLCG_2919 was not autoregulated, but directly repressed its adjacent gene, SLCG_2920, which encodes an ATP/GTP binding protein whose overexpression increased resistance against lincomycin and Lin-A yields in S. lincolnensis The precise SLCG_2919 binding site within the promoter region of SLCG_2920 was determined by a DNase I footprinting assay and by electrophoretic mobility shift assays (EMSAs) based on base substitution mutagenesis, with the internal 10-nucleotide (nt) AT-rich sequence (AAATTATTTA) shown to be essential for SLCG_2919 binding. Our findings indicate that SLCG_2919 is a negative regulator for controlling lincomycin biosynthesis in S. lincolnensis The present study improves our understanding of molecular regulation for lincomycin biosynthesis.IMPORTANCE TetR family transcriptional regulators (TFRs) are generally found to regulate diverse cellular processes in bacteria, especially antibiotic biosynthesis in Streptomyces species. However, knowledge of their function in lincomycin biosynthesis in S. lincolnensis remains unknown. The present study provides a new insight into the regulation of lincomycin biosynthesis through a TFR, SLCG_2919, that directly modulates lincomycin production and resistance. Intriguingly, SLCG_2919 and its adjoining gene, SLCG_2920, which encodes an ATP/GTP binding protein, were extensively distributed in diverse Streptomyces species. In addition, we revealed a new TFR binding motif, in which SLCG_2919 binds to the promoter region of SLCG_2920, dependent on the intervening AT-rich sequence rather than on the flanking inverted repeats found in the binding sites of other TFRs. These insights into transcriptional regulation of lincomycin biosynthesis by SLCG_2919 will be valuable in paving the way for genetic engineering of regulatory elements in Streptomyces species to improve antibiotic production.

Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26.

The newly emergent Middle East respiratory syndrome coronavirus (MERS-CoV) can cause severe pulmonary disease in humans, representing the second example of a highly pathogenic coronavirus, the first being SARS-CoV. CD26 (also known as dipeptidyl peptidase 4, DPP4) was recently identified as the cellular receptor for MERS-CoV. The engagement of the MERS-CoV spike protein with CD26 mediates viral attachment to host cells and virus-cell fusion, thereby initiating infection. Here we delineate the molecular basis of this specific interaction by presenting the first crystal structures of both the free receptor binding domain (RBD) of the MERS-CoV spike protein and its complex with CD26. Furthermore, binding between the RBD and CD26 is measured using real-time surface plasmon resonance with a dissociation constant of 16.7 nM. The viral RBD is composed of a core subdomain homologous to that of the SARS-CoV spike protein, and a unique strand-dominated external receptor binding motif that recognizes blades IV and V of the CD26 ß-propeller. The atomic details at the interface between the two binding entities reveal a surprising protein-protein contact mediated mainly by hydrophilic residues. Sequence alignment indicates, among betacoronaviruses, a possible structural conservation for the region homologous to the MERS-CoV RBD core, but a high variation in the external receptor binding motif region for virus-specific pathogenesis such as receptor recognition.

MiR-106b promotes therapeutic antibody expression in CHO cells by targeting deubiquitinase CYLD.

MicroRNAs (miRNAs) function as important regulators of major cellular processes, such as cell cycle, proliferation, development, and apoptosis. Recently, miRNA engineering of Chinese hamster ovary (CHO) cells has emerged as a promising strategy for enhancing therapeutic antibody production. Previously, we have reported that inhibition of deubiquitinase cylindromatosis (CYLD) remarkably enhanced the therapeutic antibody production in CHO cells. However, the mechanisms regulating CYLD in CHO cells remain elusive. Herein, we demonstrated that miR-106b targets CYLD directly, as shown by a series of bioinformatics analyses and experimental assays. Stable overexpression of miR-106b in CHO cells promoted CHO cell viability and subsequent antibody expression in transient transfection assay. Furthermore, the results in fed-batch culture showed that stable overexpression of miR-106b in a CHO-IgG cell line achieved about 0.66-fold promotion in product titer compared to the parental cells. Meanwhile, overexpression of miR-106b did not affect the quality of antibody. Taken together, our findings highlight the effect of miR-106b inhibition in CYLD synthesis and its function in antibody expression as a new target for improving CHO manufacturing cells.

Characterization and engineering of the Lrp/AsnC family regulator SACE_5717 for erythromycin overproduction in Saccharopolyspora erythraea.

In this work, we found that the Lrp/AsnC family protein SACE_5717 negatively regulated erythromycin biosynthesis in S. erythraea. Disruption of SACE_5717 led to a 27% improvement in the yield of erythromycin in S. erythraea A226. SACE_5717 directly repressed its own gene expression, as well as that of the adjacent gene SACE_5716 by binding to the target sequence 5'-GAACGTTCGCCGTCACGCC-3'. The predicted LysE superfamily protein SACE_5716 directly influenced the export of lysine, histidine, threonine and glycine in S. erythraea. Arginine, tyrosine and tryptophan were characterized as the effectors of SACE_5717 by weakening the binding affinity of SACE_5717. In the industrial S. erythraea WB strain, deletion of SACE_5717 (WBΔSACE_5717) increased erythromycin yield by 20%, and by 36% when SACE_5716 was overexpressed in WBΔSACE_5717 (WBΔSACE_5717/5716). In large-scale 5-L fermentation experiment, erythromycin yield in the engineered strain WBΔSACE_5717/5716 reached 4686 mg/L, a 41% enhancement over 3323 mg/L of the parent WB strain.

Inactivation of deubiquitinase CYLD enhances therapeutic antibody production in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are promising host engineering cells for industry manufacturing of therapeutic antibodies. However, cell death due to apoptosis remains a huge challenge to augment antibody production, and developing CHO cells with enhanced anti-apoptosis and proliferation ability is fundamental for cell line development and high-yielding bioprocesses. Deubiquitinase cylindromatosis (CYLD) has been proved to be a tumor suppressor by negatively regulating NF-κB and Wnt/ß-catenin signaling pathways. Its mutation or deletion is a common chromosome variation in several types of cancers. Here, we engineered CHO CYLD-/- cells by CRISPR-Cas9 editing technology. These cells displayed stronger cell proliferation and anti-apoptosis ability compared to parental cells. Three antibody expression plasmid kits were transiently transfected into these cells. Our data showed that inactivation of CYLD increased the highest titers of rituximab, Herceptin, and one bispecific antibody by 105, 63, and 228%, respectively. Reversely, overexpression of CYLD could promote cell apoptosis, whereas inhibiting cell proliferation and antibody production. Furthermore, inhibition of CYLD in CHO cells stably expressing an IgG antibody (CHO-IgG) achieved about 50% increase in product titer compared to parental cells. Meanwhile, inhibition of CYLD did not affect the quality of antibody. Thus, our data demonstrated that inactivation of CYLD could promote CHO cell proliferation, anti-apoptosis ability, and subsequent antibody production, suggesting that CYLD is a potential functional target for CHO cell engineering.

Complete Genome Sequence of Clostridium kluyveri JZZ Applied in Chinese Strong-Flavor Liquor Production.

Chinese strong-flavor liquor (CSFL), accounting for more than 70% of both Chinese liquor production and sales, was produced by complex fermentation with pit mud. Clostridium kluyveri, an important species coexisted with other microorganisms in fermentation pit mud (FPM), could produce caproic acid, which was subsequently converted to the key CSFL flavor substance ethyl caproate. In this study, we present the first complete genome sequence of C. kluyveri isolated from FPM. Clostridium kluyveri JZZ contains one circular chromosome and one circular plasmid with length of 4,454,353 and 58,581 bp, respectively. 4158 protein-coding genes were predicted and 2792 genes could be assigned with COG categories. It possesses the pathway predicted for biosynthesis of caproic acid with ethanol. Compared to other two C. kluyveri genomes, JZZ consists of longer chromosome with multiple gene rearrangements, and contains more genes involved in defense mechanisms, as well as DNA replication, recombination, and repair. Meanwhile, JZZ contains fewer genes involved in secondary metabolites biosynthesis, transport, and catabolism, including genes encoding Polyketide Synthases/Non-ribosomal Peptide Synthetases. Additionally, JZZ possesses 960 unique genes with relatively aggregating in defense mechanisms and transcription. Our study will be available for further research about C. kluyveri isolated from FPM, and will also facilitate the genetic engineering to increase biofuel production and improve fragrance flavor of CSFL.

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.

Corrections to: Enhanced lincomycin production by co­overexpression of metK1 and metK2 in Streptomyces lincolnensis.

In the online published article, row value "pIB139-metK1-metK2" in table 1 has been processed incorrectly. The correct table is given below.

Engineering of an Lrp family regulator SACE_Lrp improves erythromycin production in Saccharopolyspora erythraea.

Leucine-responsive regulatory proteins (Lrps) are a group of transcriptional regulators that regulate diverse cellular processes in bacteria and archaea. However, the regulatory role of Lrps in antibiotic biosynthesis remains poorly understood. In this study, we show that SACE_5388, an Lrp family regulator named as SACE_Lrp, is an efficient regulator for transporting and catabolizing branched-chain amino acids (BCAAs), playing an important role in regulating erythromycin production in Saccharopolyspora erythraea. SACE_Lrp directly controlled the expression of the divergently transcribed SACE_5387-5386 operon putatively encoding a BCAA ABC transporter by interacting with the intergenic region between SACE_Lrp and SACE_5387 (SACE_Lrp-5387-int), and indirectly controlled the expression of ilvE putatively encoding an aminotransferase catabolizing BCAAs. BCAA catabolism is one source of the precursors for erythromycin biosynthesis. Lysine and arginine promoted the dissociation of SACE_Lrp from SACE_Lrp -5387-int, whereas histidine increased their binding. Gene disruption of SACE_Lrp (ΔSACE_Lrp) in S. erythraea A226 resulted in a 25% increase in erythromycin production, while overexpression of SACE_5387-5386 in A226 enhanced erythromycin production by 36%. Deletion of SACE_Lrp (WBΔSACE_Lrp) in the industrial strain S. erythraea WB enhanced erythromycin production by 19%, and overexpression of SACE_5387-5386 in WBΔSACE_Lrp (WBΔSACE_Lrp/5387-5386) increased erythromycin production by 41% compared to WB. Additionally, supplement of 10mM valine to WBΔSACE_Lrp/5387-5386 culture further increased total erythromycin production up to 48%. In a 5-L fermenter, the erythromycin accumulation in the engineered strain WBΔSACE_Lrp/5387-5386 with 10mM extra valine in the industrial culture media reached 5001mg/L, a 41% increase over 3503mg/L of WB. These insights into the molecular regulation of antibiotic biosynthesis by SACE_Lrp in S. erythraea are instrumental in increasing industrial production of secondary metabolites.

Enhancement of antibiotic productions by engineered nitrate utilization in actinomycetes.

Nitrate is necessary for primary and secondary metabolism of actinomycetes and stimulates the production of a few antibiotics, such as lincomycin and rifamycin. However, the mechanism of this nitrate-stimulating effect was not fully understood. Two putative ABC-type nitrate transporters were identified in Streptomyces lincolnensis NRRL2936 and verified to be involved in lincomycin biosynthesis. With nitrate supplementation, the transcription of nitrogen assimilation genes, nitrate-specific ABC1 transporter genes, and lincomycin exporter gene lmrA was found to be enhanced and positively regulated by the global regulator GlnR, whose expression was also improved. Moreover, heterologous expression of ABC2 transporter genes in Streptomyces coelicolor M145 resulted in an increased actinorhodin production. Further incorporation of a nitrite-specific transporter gene nirC, as in nirC-ABC2 cassette, led to an even higher actinorhodin production. Similarly, the titers of salinomycin, ansamitocin, lincomycin, and geldanamycin were increased with the integration of this cassette to Streptomyces albus BK3-25, Actinosynnema pretiosum ATCC31280, S. lincolnensis LC-G, and Streptomyces hygroscopicus XM201, respectively. Our work expanded the nitrate-stimulating effect to many antibiotic producers by utilizing the nirC-ABC2 cassette for enhanced nitrate utilization, which could become a general tool for titer increase of antibiotics in actinomycetes.

Characterization of an Lrp/AsnC family regulator SCO3361, controlling actinorhodin production and morphological development in Streptomyces coelicolor.

Lrp/AsnC family regulators have been found in many bacteria as crucial regulators controlling diverse cellular processes. By genomic alignment, we found that SCO3361, an Lrp/AsnC family protein from Streptomyces coelicolor, shared the highest similarity to the SACE_Lrp from Saccharopolyspora erythraea. Deletion of SCO3361 led to dramatic reduction in actinorhodin (Act) production and delay in aerial mycelium formation and sporulation on solid media. Dissection of the mechanism underlying the function of SCO3361 in Act production revealed that it altered the transcription of the cluster-situated regulator gene actII-ORF4 by directly binding to its promoter. SCO3361 was an auto-regulator and simultaneously activated the transcription of its adjacent divergently transcribed gene SCO3362. SCO3361 affected aerial hyphae formation and sporulation of S. coelicolor by activating the expression of amfC, whiB, and ssgB. Phenylalanine and cysteine were identified as the effector molecules of SCO3361, with phenylalanine reducing the binding affinity, whereas cysteine increasing it. Moreover, interactional regulation between SCO3361 and SACE_Lrp was discovered for binding to each other's target gene promoter in this work. Our findings indicate that SCO3361 functions as a pleiotropic regulator controlling secondary metabolism and morphological development in S. coelicolor.

Elevated expression of TANK-binding kinase 1 enhances tamoxifen resistance in breast cancer.

Resistance to antiestrogens is one of the major challenges in breast cancer treatment. Although phosphorylation of estrogen receptor α (ERα) is an important factor in endocrine resistance, the contributions of specific kinases in endocrine resistance are still not fully understood. Here, we report that an important innate immune response kinase, the IκB kinase-related TANK-binding kinase 1 (TBK1), is a crucial determinant of resistance to tamoxifen therapies. We show that TBK1 increases ERα transcriptional activity through phosphorylation modification of ERα at the Ser-305 site. Ectopic TBK1 expression impairs the responsiveness of breast cancer cells to tamoxifen. By studying the specimens from patients with breast cancer, we find a strong positive correlation of TBK1 with ERα, ERα Ser-305, and cyclin D1. Notably, patients with tumors highly expressing TBK1 respond poorly to tamoxifen treatment and show high potential for relapse. Therefore, our findings suggest that TBK1 contributes to tamoxifen resistance in breast cancer via phosphorylation modification of ERα.

Identification of Lysine Acetylation in Mycobacterium abscessus Using LC-MS/MS after Immunoprecipitation.

Mycobacterium abscessus (MAB), which manifests in the pulmonary system, is one of the neglected causes of nontuberculous mycobacteria (NTM) infection. Treatment against MAB is difficult, characterized by its intrinsic antibiotic drug resistance. Lysine acetylation can alter the physiochemical property of proteins in living organisms. This study aimed to determine if this protein post-translational modification (PTM) exists in a clinical isolate M. abscessus GZ002. We used the antiacetyl-lysine immunoprecipitation to enrich the low-abundant PTM proteins, followed by the LC-MS/MS analysis. The lysine acetylome of M. abscessus GZ002 was determined. There were 459 lysine acetylation sites found in 289 acetylated proteins. Lysine acetylation occurred in 5.87% of the M. abscessus GZ002 proteome, and at least 25% of them were growth essential. Aerobic respiration and carbohydrate metabolic pathways of M. abscessus GZ002 were enriched with lysine acetylation. Through bioinformatics analysis, we identified four major acetyl motif logos (K(ac)Y, K(ac)F, K(ac)H, and DK(ac)). Further comparison of the reported M. tuberculosis (MTB) acetylomes and that of MAB GZ002 revealed several common features between these two species. The lysine residues of several antibiotic-resistance, virulence, and persistence-related proteins were acetylated in both MAB GZ002 and MTB. There were 51 identical acetylation sites in 37 proteins found in common between MAB GZ002 and MTB. Overall, we demonstrate a profile of lysine acetylation in MAB GZ002 proteome that shares similarities with MTB. Interventions that target at these conserved sections may be valuable as anti-NTM or anti-TB therapies.

Role of the Cys154Arg Substitution in Ribosomal Protein L3 in Oxazolidinone Resistance in Mycobacterium tuberculosis.

We expressed the wild-type rplC and mutated rplC (Cys154Arg) genes, respectively, in Mycobacterium tuberculosis H37Ra and H37Rv in an attempt to delineate the role of rplC (Cys154Arg) regarding oxazolidinone resistance. An increase of the MICs of linezolid (LZD) and sutezolid (PNU-100480, PNU) against the recombinant mycobacteria with overexpressed rplC mutation (Cys154Arg) was found, suggesting the rplC gene is a determinant of bacillary susceptibilities to LZD and PNU.