Genetic factors that influence moenomycin production in streptomycetes

Moenomycin, a natural phosphoglycolipid product that has a long history of use in animal nutrition, is currently considered an attractive starting point for the development of novel antibiotics. We recently reconstituted the biosynthesis of this natural product in a heterologous host, Streptomyces lividans TK24, but production levels were too low to be useful. We have examined several other streptomycetes strains as hosts and have also explored the overexpression of two pleiotropic regulatory genes, afsS and relA, on moenomycin production. A moenomycin-resistant derivative of S. albus J1074 was found to give the highest titers of moenomycin, and production was improved by overexpressing relA. Partial duplication of the moe cluster 1 in S. ghanaensis also increased average moenomycin production. The results reported here suggest that rational manipulation of global regulators combined with increased moe gene dosage could be a useful technique for improvement of moenomycin biosynthesis.     

RRP20, a component of the 90S preribosome,is required for pre-18S rRNA processing in Saccharomyces cerevisiae

A strain of Saccharomyces cerevisiae, defective in small subunit ribosomal RNA processing, has a mutation in YOR145c ORF that converts Gly235 to Asp. Yor145c is a nucleolar protein required for cell viability and has been reported recently to be present in 90S pre-ribosomal particles. The Gly235Asp mutation in YOR145c is found in a KH-type RNA-binding domain and causes a marked deficiency in 18S rRNA production. Detailed studies by northern blotting and primer extension analyses show that the mutant strain impairs the early pre-rRNA processing cleavage essentially at sites A₁ and A₂, leading to accumulation of a 22S dead-end processing product that is found in only a few rRNA processing mutants. Furthermore, U3, U14, snR10 and snR30 snoRNAs, involved in early pre-rRNA cleavages, are not destabilized by the YOR145c mutation. As the protein encoded by YOR145c is found in pre-ribosomal particles and the mutant strain is defective in ribosomal RNA processing, we have renamed it as RRP20.     

RNase III Is Required for Actinomycin Production in Streptomyces antibioticus

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain.     

Pleiotropic regulatory genes bldA,adpA and absB are implicated in production of phosphoglycolipid antibiotic moenomycin

Unlike the majority of actinomycete secondary metabolic pathways, the biosynthesis of peptidoglycan glycosyltransferase inhibitor moenomycin in Streptomyces ghanaensis does not involve any cluster-situated regulators (CSRs). This raises questions about the regulatory signals that initiate and sustain moenomycin production. We now show that three pleiotropic regulatory genes for Streptomyces morphogenesis and antibiotic production—bldA, adpA and absB—exert multi-layered control over moenomycin biosynthesis in native and heterologous producers. The bldA gene for tRNA^Leu_UAA is required for the translation of rare UUA codons within two key moenomycin biosynthetic genes (moe), moeO5 and moeE5. It also indirectly influences moenomycin production by controlling the translation of the UUA-containing adpA and, probably, other as-yet-unknown repressor gene(s). AdpA binds key moe promoters and activates them. Furthermore, AdpA interacts with the bldA promoter, thus impacting translation of bldA-dependent mRNAs—that of adpA and several moe genes. Both adpA expression and moenomycin production are increased in an absB-deficient background, most probably because AbsB normally limits adpA mRNA abundance through ribonucleolytic cleavage. Our work highlights an underappreciated strategy for secondary metabolism regulation, in which the interaction between structural genes and pleiotropic regulators is not mediated by CSRs. This strategy might be relevant for a growing number of CSR-free gene clusters unearthed during actinomycete genome mining.     

Novel Approach for Improving the Productivity of Antibiotic-Producing Strains by Inducing Combined Resistant Mutations

We developed a novel approach for improving the production of antibiotic from Streptomyces coelicolor A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str^r), gentamicin (Gen^r), or rifampin (Rif^r), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (str gen and str rif) by introducing gentamicin or rifampin resistance into an str mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (str gen rif) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of str mutants revealed that a point mutation occurred within the rpsL gene, which encodes the ribosomal protein S12. rif mutants were found to have a point mutation in the rpoB gene, which encodes the β-subunit of RNA polymerase. Mutation points in gen mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.     

Development of Antibiotic-Overproducing Strains by Site-Directed Mutagenesis of the rpsL Gene in Streptomyces lividans

Certain rpsL (which encodes the ribosomal protein S12) mutations that confer resistance to streptomycin markedly activate the production of antibiotics in Streptomyces spp. These rpsL mutations are known to be located in the two conserved regions within the S12 protein. To understand the roles of these two regions in the activation of silent genes, we used site-directed mutagenesis to generate eight novel mutations in addition to an already known (K88E) mutation that is capable of activating antibiotic production in Streptomyces lividans. Of these mutants, two (L90K and R94G) activated antibiotic production much more than the K88E mutant. Neither the L90K nor the R94G mutation conferred an increase in the level of resistance to streptomycin and paromomycin. Our results demonstrate the efficacy of the site-directed mutagenesis technique for strain improvement.     

Regulation of the AbrA1/A2 Two-Component System in Streptomyces coelicolor and the Potential of Its Deletion Strain as a Heterologous Host for Antibiotic Production

The Two-Component System (TCS) AbrA1/A2 from Streptomyces coelicolor M145 is a negative regulator of antibiotic production and morphological differentiation. In this work we show that it is able to auto-regulate its expression, exerting a positive induction of its own operon promoter, and that its activation is dependent on the presence of iron. The overexpression of the abrA2 response regulator (RR) gene in the mutant ΔabrA1/A2 results in a toxic phenotype. The reason is an excess of phosphorylated AbrA2, as shown by phosphoablative and phosphomimetic AbrA2 mutants. Therefore, non-cognate histidine kinases (HKs) or small phospho-donors may be responsible for AbrA2 phosphorylation in vivo. The results suggest that in the parent strain S. coelicolor M145 the correct amount of phosphorylated AbrA2 is adjusted through the phosphorylation-dephosphorylation activity rate of the HK AbrA1. Furthermore, the ABC transporter system, which is part of the four-gene operon comprising AbrA1/A2, is necessary to de-repress antibiotic production in the TCS null mutant. Finally, in order to test the possible biotechnological applications of the ΔabrA1/A2 strain, we demonstrate that the production of the antitumoral antibiotic oviedomycin is duplicated in this strain as compared with the production obtained in the wild type, showing that this strain is a good host for heterologous antibiotic production. Thus, this genetically modified strain could be interesting for the biotechnology industry.     

Adaptive evolution of Methylotuvimicrobium alcaliphilum to grow in the presence of rhamnolipids improves fatty acid and rhamnolipid production from CH4

Rhamnolipids (RLs) are well-studied biosurfactants naturally produced by pathogenic strains of Pseudomonas aeruginosa. Current methods to produce RLs in native and heterologous hosts have focused on carbohydrates as production substrate; however, methane (CH₄) provides an intriguing alternative as a substrate for RL production because it is low cost and may mitigate greenhouse gas emissions. Here, we demonstrate RL production from CH₄ by Methylotuvimicrobium alcaliphilum DSM19304. RLs are inhibitory to M. alcaliphilum growth (<0.05 g/l). Adaptive laboratory evolution was performed by growing M. alcaliphilum in increasing concentrations of RLs, producing a strain that grew in the presence of 5 g/l of RLs. Metabolomics and proteomics of the adapted strain grown on CH₄ in the absence of RLs revealed metabolic changes, increase in fatty acid production and secretion, alterations in gluconeogenesis, and increased secretion of lactate and osmolyte products compared with the parent strain. Expression of plasmid-borne RL production genes in the parent M. alcaliphilum strain resulted in cessation of growth and cell death. In contrast, the adapted strain transformed with the RL production genes showed no growth inhibition and produced up to 1 μM of RLs, a 600-fold increase compared with the parent strain, solely from CH₄. This work has promise for developing technologies to produce fatty acid-derived bioproducts, including biosurfactants, from CH₄.     

A novel point mutation of acetylcholinesterase in a trichlorfon-resistant strain of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)

Acetylcholinesterase (AChE) is the target enzyme of organophosphorus and carbamate insecticides. We applied trichlorfon to select resistant strains of Bactrocera dorsalis Hendel in the laboratory. Two trichlorfon-resistant strains, the Tri-R₁ strain with 18.23-fold resistance and the Tri-R₂ strain with 69.5-fold resistance, were obtained. Three known mutations, I159V, G433S and Q588R were identified in AChE of two resistant strains, and a novel mutation, G365A, was identified in the more resistant Tri-R₂ strain. The modeled 3-D-structure of AChE showed that G365A and G433S are closely adjacent in the gorge above the catalytic site S235. Mutations of G365A and G433S resulted in a steric hindrance by stronger Van der Waals force between two sites. Such a minor structural change might block insecticides from squeezing through the gorge to reach the active site, but not the natural substrate. Compared with the susceptible strain, the AChE activity of the Tri-R₁ strain and the Tri-R₂ strain was 0.87- and 0.67-fold, the K _m value of the Tri-R₁ strain and the Tri-R₂ strain was 0.11- and 0.10-fold, the V _max value of two resistant strains was 0.26- and 0.15-fold, whereas, the I ₅₀ to trichlorfon significantly increased by 9.07- and 13.19-fold. These results suggested that the novel point mutation G365A of AChE might be involved in increasing resistance to trichlorfon in the resistant strain of oriental fruit fly.     

Stability of Transmembrane Amyloid β-Peptide and Membrane Integrity Tested by Molecular Modeling of Site-Specific Aβ 42 Mutations

Interactions of the amyloid β-protein (Aβ) with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer’s disease. Natural mutations in Aβ ₄₂, such as the Arctic mutation (E22G) have been shown to increase Aβ ₄₂ aggregation and neurotoxicity, leading to the early-onset of Alzheimer’s disease. A correlation between the propensity of Aβ ₄₂ to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ ₄₂ peptide it was further revealed that the aggregation of different Aβ ₄₂ mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ ₄₂ mutation and peptide–membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ ₄₂ variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants) as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ ₄₂ stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ ₄₂ peptide and a decreased stability for D23G compared to wild-type Aβ ₄₂, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.     

Characterization of the A2AR-D2R interface: focus on the role of the C-terminal tail and the transmembrane helices

A single serine point mutation (S374A) in the adenosine A_2A receptor (A_2AR) C-terminal tail reduces A_2AR-D₂R heteromerization and prevents its allosteric modulation of the dopamine D₂ receptor (D₂R). By means of site directed mutagenesis of the A_2AR and synthetic transmembrane (TM) α-helix peptides of the D₂R we further explored the role of electrostatic interactions and TM helix interactions of the A_2AR-D₂R heteromer interface. We found evidence that the TM domains IV and V of the D₂R play a major role in the A_2AR-D₂R heteromer interface since the incubation with peptides corresponding to these domains significantly reduced the ability of A_2AR and D₂R to heteromerize. In addition, the incubation with TM-IV or TM-V blocked the allosteric modulation normally found in A_2AR-D₂R heteromers. The mutation of two negatively charged aspartates in the A_2AR C-terminal tail (D401A/D402A) in combination with the S374A mutation drastically reduced the physical A_2AR-D₂R interaction and lost the ability of antagonistic allosteric modulation over the A_2AR-D₂R interface, suggesting further evidence for the existence of an electrostatic interaction between the C-terminal tail of A_2AR and the intracellular loop 3 (IL3) of D₂R. On the other hand, molecular dynamic model and bioinformatic analysis propose that specific AAR, AQE, and VLS protriplets as an important motive in the A_2AR-D_2LR heteromer interface together with D_2LR TM segments IV/V interacting with A_2AR TM-IV/V or TM-I/VII.     

Mutation in ace1 associated with an insecticide resistant population of Plutella xylostella

Insensitive acetylcholinesterase (AChE) was determined to be involved in an EPN-resistant (ER) strain and a contaminated susceptible (CS) strain of diamondback moth (DBM, Plutella xylostella L.), as estimated by AChE inhibition assay using DDVP as a inhibitor in a nondenaturing electrophoresis gel. The ER strain exhibited very high AChE insensitivity, high resistance ratio, and two point mutations (G324A, A298S) in ace1-type AChE gene (Pxace1). The CS strain showed low AChE insensitivity, low resistance ratio, and it has only one point mutation (G324A). These findings suggest that the A298S mutation, along with reported G324A mutation (Baek et al, 2005), can be important in the development of organophosphate resistance. These results also suggest that the A298S mutation could be a good candidate for a molecular diagnosis marker for resistance monitoring. Three molecular diagnosis methods (Quantitative Sequencing; QS, PCR amplification of specific alleles; PASA and restriction fragment length polymorphism; RFLP) were developed which successfully detected specific resistance associated point mutations. Seven local population DBMs were surveyed and showed high insecticide resistance levels and a A298S mutation in Pxace1. These methods can be used to monitor the resistance allele in field population of DBMs and resistance management strategy.     

Bacteriophage lambda DNA maturation. The functional relationships among the products of genes Nul, A and FI

The FI gene of bacteriophage λ functions in head assembly, but its exact role is not well understood. FI mutants are leaky, producing between 0.1 and 0.5 viable particles per infected cell. In order to investigate the function of the FI product (gpFI) in vivo, mutants of λ were isolated that are able to grow in the absence of gpFI. These mutants, called fin (for FI independence) map in the region of gene Nul and the beginning of gene A.Proteins made in cells infected with the fin mutants were labelled with [³⁵S]methionine and analysed by polyacrylamide gel electrophoresis. In addition, the levels of activity of the A product were measured in the in vitro DNA packaging assay. As a result of these experiments, the fin mutants can be classified in two groups. Upon infection, fin mutants of one group selectively produce three to fivefold more gpA than do wild-type phage fin mutants of the second group do not overproduce any λ late gene product detectable by the autoradiographic technique.gpA overproducers can also be isolated by selecting for λAam Wam phages that can plate on a weak suII cell strain. The mutation responsible for this pseudoreversion is called Aop and maps in the Nu1-A region. Aop is also a fin mutation, since its presence in λFI^? enables it to plate on non-permissive hosts.Therefore, it seems that one condition sufficient for normal growth of FI^? phage is the overproduction of gpA. The nature of the fin mutations that do not result in gpA overproduction is discussed.     

Innovative Approach for Improvement of an Antibiotic-Overproducing Industrial Strain of Streptomyces albus

Working with a Streptomyces albus strain that had previously been bred to produce industrial amounts (10 mg/ml) of salinomycin, we demonstrated the efficacy of introducing drug resistance-producing mutations for further strain improvement. Mutants with enhanced salinomycin production were detected at a high incidence (7 to 12%) among spontaneous isolates resistant to streptomycin (Str^r), gentamicin, or rifampin (Rif^r). Finally, we successfully demonstrated improvement of the salinomycin productivity of the industrial strain by 2.3-fold by introducing a triple mutation. The Str^r mutant was shown to have a point mutation within the rpsL gene (encoding ribosomal protein S12). Likewise, the Rif^r mutant possessed a mutation in the rpoB gene (encoding the RNA polymerase β subunit). Increased productivity of salinomycin in the Str^r mutant (containing the K88R mutation in the S12 protein) may be a result of an aberrant protein synthesis mechanism. This aberration may manifest itself as enhanced translation activity in stationary-phase cells, as we have observed with the poly(U)-directed cell-free translation system. The K88R mutant ribosome was characterized by increased 70S complex stability in low Mg²⁺ concentrations. We conclude that this aberrant protein synthesis ability in the Str^r mutant, which is a result of increased stability of the 70S complex, is responsible for the remarkable salinomycin production enhancement obtained.     

珠江口沉积物来源链霉菌SCSIO 40020中bafilomycins的鉴定及其生物合成基因簇的异源表达

[目的] 从珠江口沉积物来源的菌株SCSIO 40020中分离bafilomycins,并对其生物合成基因簇进行克隆和异源表达研究。[方法] 通过分析菌株SCSIO 40020的16S rRNA基因序列并构建系统发育树以鉴定菌种,以柱层析法和制备色谱法对次级代谢产物进行分离纯化,借助波谱学手段完成单体化合物的结构鉴定,采用生物信息学分析定位bafilomycins的生物合成基因簇,通过筛选菌株SCSIO 40020基因组的细菌人工染色体文库和接合转移将bafilomycins生物合成基因簇导入3种链霉菌进行异源表达,利用高效液相色谱检测异源表达菌株的发酵产物。[结果] 菌株SCSIO 40020被鉴定为链霉菌属菌株,从其发酵产物中分离鉴定了2个单体化合物bafilomycins A₁和D。克隆了链霉菌SCSIO 40020中bafilomycins的生物合成基因簇并推导了其生物合成途径,在3种链霉菌中表达产生了bafilomycins。[结论] 从珠江口环境中获得了一株产生bafilomycins的链霉菌SCSIO 40020,成功建立了该菌株次级代谢产物生物合成基因簇的异源表达体系,并首次在链霉菌Streptomyces lividans SBT18、Streptomyces coelicolor M1154和Streptomyces albus J1074中进行了表达,获得了bafilomycins,为后续bafilomycins结构类似物的生产和链霉菌SCSIO 40020中新结构活性化合物的挖掘奠定了基础。     

Acquisition of photosynthetic capacity by a reaction centre that lacks the QA ubiquinone; possible insights into the evolution of reaction centres?

A photosynthetically impaired strain of Rhodobacter sphaeroides containing reaction centres with an alanine to tryptophan mutation at residue 260 of the M-polypeptide (AM260W) was incubated under photosynthetic growth conditions. This incubation produced photosynthetically competent strains containing suppressor mutations that changed residue M260 to glycine or cysteine. Spectroscopic analysis demonstrated that the loss of the Q_A ubiquinone seen in the original AM260W mutant was reversed in the suppressor mutants. In the mutant where Trp M260 was replaced by Cys, the rate of reduction of the Q_A ubiquinone by the adjacent (H_A) bacteriopheophytin was reduced by three-fold. The findings of the experiment are discussed in light of the X-ray crystal structures of the wild-type and AM260W reaction centres, and the possible implications for the evolution of reaction centres as bioenergetic complexes are considered.