Isolation and Characterization of the Gibberellin Biosynthetic Gene Cluster in Sphaceloma manihoticola

Gibberellins (GAs) are tetracyclic diterpenoid phytohormones that were first identified as secondary metabolites of the fungus Fusarium fujikuroi (teleomorph, Gibberella fujikuroi). GAs were also found in the cassava pathogen Sphaceloma manihoticola, but the spectrum of GAs differed from that in F. fujikuroi. In contrast to F. fujikuroi, the GA biosynthetic pathway has not been studied in detail in S. manihoticola, and none of the GA biosynthetic genes have been cloned from the species. Here, we present the identification of the GA biosynthetic gene cluster from S. manihoticola consisting of five genes encoding a bifunctional ent-copalyl/ent-kaurene synthase (CPS/KS), a pathway-specific geranylgeranyl diphosphate synthase (GGS2), and three cytochrome P450 monooxygenases. The functions of all of the genes were analyzed either by a gene replacement approach or by complementing the corresponding F. fujikuroi mutants. The cluster organization and gene functions are similar to those in F. fujikuroi. However, the two border genes in the Fusarium cluster encoding the GA₄ desaturase (DES) and the 13-hydroxylase (P450-3) are absent in the S. manihoticola GA gene cluster, consistent with the spectrum of GAs produced by this fungus. The close similarity between the two GA gene clusters, the identical gene functions, and the conserved intron positions suggest a common evolutionary origin despite the distant relatedness of the two fungi.     

Characterization of a Loss-of-Function Mutant of Gibberellin Biosynthetic Gene LsGA3ox1 in Lettuce

A previous study generated lettuce (Lactuca sativa) mutant lines tagged by retrotransposon Tnt1 from tobacco (Nicotiana tabacum) and identified a homozygous mutant, Tnt6a, that exhibited severe dwarf phenotype. Here we show that Tnt1 is inserted into the intron of gibberellin biosynthetic gene LsGA3ox1 in Tnt6a mutants. Expression analysis suggests that LsGA3ox1 is nearly knocked out in the Tnt6a mutants.     

Characterization of the prfA Virulence Gene Cluster Insertion Site in Non-Hemolytic Listeria spp.: Probing the Evolution of the Listeria Virulence Gene Island

The prfA virulence gene cluster is present between prs and ldh in the pathogenic L. monocytogenes and L. ivanovii, but absent from the non-pathogenic L. innocua and L. welshimeri. To probe the evolution of this virulence gene cluster, we sequenced the prs-ldh intergenic region in L. welshimeri and L. innocua. Two ORFs (ORFA and ORFB) were found in both species as well as in L. monocytogenes. Another ORF of unknown function (ORFZ) was found in L. monocytogenes and L. innocua, while two unique ORFs were present in L. welshimeri. ORFA and ORFB showed significant functional constraint, suggesting that further investigations in the functions of these genes, including possible roles in horizontal gene transfer or sequence deletion, are warranted. DNA sequences homologous to Tn1545 integration consensus sequences were found downstream of prs and ORFB, thus defining the likely junctions of the virulence gene island and indicating that the prs-ldh intergenic region may represent a Tn insertion hot spot. Our results are consistent with the hypothesis that a combination of horizontal gene transfer and deletion events may have been involved in the evolution of the prfA virulence gene cluster in Listeria. Received: 27 November 2000 / Accepted: 20 February 2001     

Valinomycin Biosynthetic Gene Cluster in Streptomyces: Conservation,Ecology and Evolution

Many Streptomyces strains are known to produce valinomycin (VLM) antibiotic and the VLM biosynthetic gene cluster (vlm) has been characterized in two independent isolates. Here we report the phylogenetic relationships of these strains using both parsimony and likelihood methods, and discuss whether the vlm gene cluster shows evidence of horizontal transmission common in natural product biosynthetic genes. Eight Streptomyces strains from around the world were obtained and sequenced for three regions of the two large nonribosomal peptide synthetase genes (vlm1 and vlm2) involved in VLM biosynthesis. The DNA sequences representing the vlm gene cluster are highly conserved among all eight environmental strains. The geographic distribution pattern of these strains and the strict congruence between the trees of the two vlm genes and the housekeeping genes, 16S rDNA and trpB, suggest vertical transmission of the vlm gene cluster in Streptomyces with no evidence of horizontal gene transfer. We also explored the relationship of the sequence of vlm genes to that of the cereulide biosynthetic genes (ces) found in Bacillus cereus and found them highly divergent from each other at DNA level (genetic distance values≥95.6%). It is possible that the vlm gene cluster and the ces gene cluster may share a relatively distant common ancestor but these two gene clusters have since evolved independently.     

Gibberellin Biosynthetic Pathway inGibberella fujikuroi:Evidence for a Gene Cluster

Differential screening of aGibberella fujikuroicDNA library was used to successfully clone and identify genes involved in the pathway of gibberellin biosynthesis. Several cDNA clones that hybridized preferentially to a cDNA probe prepared from mycelium induced for gibberellin production were isolated and characterized. The deduced amino acid sequences of two (identical) clones contained the conserved heme-binding motif of cytochrome P450 monooxygenases (FXXGXXXCXG). One of these cDNA fragments was used as a homologous probe for the screening of a genomic library. A hybridizing 6.7-kb genomicSalI fragment was cloned into pUC19. The sequencing of this clone revealed that a second cytochrome P450 monooxygenase gene was closely linked to the first one. Since at least four cytochrome P450 monooxygenase-catalyzed steps are involved in the synthesis of gibberellins, chromosome walking was performed to find a further gene of this family or other genes involved in gibberellin pathway. Next to the two P450 monooxygenase genes, a putative geranylgeranyl diphosphate synthase gene, the copalyl diphosphate synthase gene, which is the first specific gene of the gibberellin pathway, and a third P450 monooxygenase gene were identified. These results suggest that at least some of the genes involved in the biosynthesis of gibberellins are closely linked in a gene cluster inG. fujikuroi,as has been recently found for other “dispensable” pathways in fungi.     

Cloning and Heterologous Expression of the Vibrioferrin Biosynthetic Gene Cluster from a Marine Metagenomic Library

A biosynthetic gene cluster of siderophore consisting of five open reading frames (ORFs) was cloned by functional screening of a metagenomic library constructed from tidal-flat sediment. Expression of the cloned biosynthetic genes in Escherichia coli led to the production of vibrioferrin, a siderophore originally reported for the marine bacterium Vibrio parahaemolyticus. To the best of our knowledge, this is the first example of heterologous production of a siderophore by biosynthetic genes cloned from a metagenomic library. The cloned cluster was one of the largest of the clusters obtained by functional screening. In this study, we demonstrated and extended the possibility of function-based metagenomic research.     

Isolation, Characterization, and Heterologous Expression of the Novel Lantibiotic Epicidin 280 and Analysis of Its Biosynthetic Gene Cluster

Epicidin 280 is a novel type A lantibiotic produced by Staphylococcus epidermidis BN 280. During C₁₈ reverse-phase high-performance liquid chromatography two epicidin 280 peaks were obtained; the two compounds had molecular masses of 3,133 ± 1.5 and 3,136 ± 1.5 Da, comparable antibiotic activities, and identical amino acid compositions. Amino acid sequence analysis revealed that epicidin 280 exhibits 75% similarity to Pep5. The strains that produce epicidin 280 and Pep5 exhibit cross-immunity, indicating that the immunity peptides cross-function in antagonization of both lantibiotics. The complete epicidin 280 gene cluster was cloned and was found to comprise at least five open reading frames (eciI, eciA, eciP, eciB, and eciC, in that order). The proteins encoded by these open reading frames exhibit significant sequence similarity to the biosynthetic proteins of the Pep5 operon of Staphylococcus epidermidis 5. A gene for an ABC transporter, which is present in the Pep5 gene cluster but is necessary only for high yields (G. Bierbaum, M. Reis, C. Szekat, and H.-G. Sahl, Appl. Environ. Microbiol. 60:4332–4338, 1994), was not detected. Instead, upstream of the immunity gene eciI we found an open reading frame, eciO, which could code for a novel lantibiotic modification enzyme involved in reduction of an N-terminally located oxopropionyl residue. Epicidin 280 produced by the heterologous host Staphylococcus carnosus TM 300 after introduction of eciIAPBC (i.e., no eciO was present) behaved homogeneously during reverse-phase chromatography.     

Identification and Characterization of a New Erythromycin Biosynthetic Gene Cluster in Actinopolyspora erythraea YIM90600, a Novel Erythronolide-Producing Halophilic Actinomycete Isolated from Salt Field

Erythromycins (Ers) are clinically potent macrolide antibiotics in treating pathogenic bacterial infections. Microorganisms capable of producing Ers, represented by Saccharopolyspora erythraea, are mainly soil-dwelling actinomycetes. So far, Actinopolyspora erythraea YIM90600, a halophilic actinomycete isolated from Baicheng salt field, is the only known Er-producing extremophile. In this study, we have reported the draft genome sequence of Ac. erythraea YIM90600, genome mining of which has revealed a new Er biosynthetic gene cluster encoding several novel Er metabolites. This Er gene cluster shares high identity and similarity with the one of Sa. erythraea NRRL2338, except for two absent genes, eryBI and eryG. By correlating genotype and chemotype, the biosynthetic pathways of 3′-demethyl-erythromycin C, erythronolide H (EH) and erythronolide I have been proposed. The formation of EH is supposed to be sequentially biosynthesized via C-6/C-18 epoxidation and C-14 hydroxylation from 6-deoxyerythronolide B. Although an in vitro enzymatic activity assay has provided limited evidence for the involvement of the cytochrome P450 oxidase EryF^Ac (derived from Ac. erythraea YIM90600) in the catalysis of a two-step oxidation, resulting in an epoxy moiety, the attempt to construct an EH-producing Sa. erythraea mutant via gene complementation was not successful. Characterization of EryK^Ac (derived from Ac. erythraea YIM90600) in vitro has confirmed its unique role as a C-12 hydroxylase, rather than a C-14 hydroxylase of the erythronolide. Genomic characterization of the halophile Ac. erythraea YIM90600 will assist us to explore the great potential of extremophiles, and promote the understanding of EH formation, which will shed new insights into the biosynthesis of Er metabolites.     

黄曲霉素合成相关基因表达与环境因素的关系

简单介绍了黄曲霉毒素的发现、分布、危害和范围,详细叙述了黄曲霉毒素生物合成中相关基因的表达与调控,概述了黄曲霉毒素合成中的关键基因、酶和调控因子重要性,分析了影响黄曲霉毒素合成的环境因素。不仅在基础理论上对黄曲霉毒素合成机理进行了深入探讨,而且在应用研究上为减少粮食和食品受到重金属污染和黄曲霉毒素危害提供了新的思路。     

Degradation of Substituted Phenylurea Herbicides by Arthrobacter globiformis Strain D47 and Characterization of a Plasmid-Associated Hydrolase Gene, puhA

Arthrobacter globiformis D47 was shown to degrade a range of substituted phenylurea herbicides in soil. This strain contained two plasmids of approximately 47 kb (pHRIM620) and 34 kb (pHRIM621). Plasmid-curing experiments produced plasmid-free strains as well as strains containing either the 47- or the 34-kb plasmid. The strains were tested for their ability to degrade diuron, which demonstrated that the degradative genes were located on the 47-kb plasmid. Studies on the growth of these strains indicated that the ability to degrade diuron did not offer a selective advantage to A. globiformis D47 on minimal medium designed to contain the herbicide as a sole carbon source. The location of the genes on a plasmid and a lack of selection would explain why the degradative phenotype, as with many other pesticide-degrading bacteria, can be lost on subculture. A 22-kb EcoRI fragment of plasmid pHRIM620 was expressed in Escherichia coli and enabled cells to degrade diuron. Transposon mutagenesis of this fragment identified one open reading frame that was essential for enzyme activity. A smaller subclone of this gene (2.5 kb) expressed in E. coli coded for the protein that degraded diuron. This gene and its predicted protein sequence showed only a low level of protein identity (25% over ca. 440 amino acids) to other database sequences and was named after the enzyme it encoded, phenylurea hydrolase (puhA gene).     

吸水链霉菌17997格尔德霉素部分生物合成基因簇的克隆和分析

吸水链霉菌17997(Streptomyceshy groscopicus17997)是我所从中国云南土壤中分离到的格尔德霉素(geldanamycin,GDM)产生菌,GDM具有良好的抗肿瘤和抗病毒活性,但其肝毒性和水溶性差的缺点限制了其在临床上的应用。为了实现对GDM结构的生物学改造,首先要获得GDM的生物合成基因。根据GDM后修饰基因——氨甲酰基转移酶基因(gdmN)的保守序列筛选S.hygroscopicus17997的柯斯质粒基因组文库,共获得6个阳性克隆,选择CT-4阳性柯斯质粒进行亚克隆和测序,又通过PCR延伸的方法获得了与CT4连锁的将近5kb的外源序列,共获得28.356kb的外源DNA序列,其中包含了13个可能阅读框架,通过同源比较证实该序列与S.hygroscopicusNRRL3602中的GDM生物合成基因有很高的同源性。为进一步研究GDM生物合成基因的功能,并通过组合生物学的方法改造GDM的结构奠定了基础。     

木霉菌T23胶毒素合成基因的生物信息学分析与克隆

胶毒素是生防木霉菌重要的次生代谢产物之一。本研究以生防木霉菌T23为供试材料,旨在通过生物信息学技术及表达分析,挖掘木霉菌T23中胶毒素合成候选基因,探索木霉菌胶毒素合成的分子调控机制,可为新型生物农药的开发及应用提供理论依据。研究表明,木霉菌T23中胶毒素合成候选基因簇全长28 kb,簇内包含了8个基因,分别与烟曲霉胶毒素合成基因簇内的gliP、gliC、gliN、gliK、gliI、gliG、gliF、gliM高度同源。提取培养2 d、3 d、4 d、5 d的木霉菌T23菌丝的RNA,通过半定量RT-PCR技术探索各候选基因在木霉菌T23不同生长时期的表达情况,显示各基因在不同生长时期均有表达,属于组成型表达基因。成功克隆得到木霉菌T23中的gliP-T23基因并完成基因结构分析,该基因全长6 339 bp,由4个外显子和3个内含子组成,为后续的基因功能验证提供基础。     

The Yersiniabactin Biosynthetic Gene Cluster of Yersinia enterocolitica: Organization and Siderophore-Dependent Regulation

The ability to synthesize and uptake the Yersinia siderophore yersiniabactin is a hallmark of the highly pathogenic, mouse-lethal species Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica 1B. We have identified four genes, irp1, irp3, irp4, and irp5, on a 13-kb chromosomal DNA fragment of Y. enterocolitica O8, WA-314. These genes constitute the yersiniabactin biosynthetic gene cluster together with the previously defined irp2. The irp1 gene consists of 9,486 bp capable of encoding a 3,161-amino-acid high-molecular-weight protein 1 (HMWP1) polypeptide with a predicted mass of 384.6 kDa. The first 3,000 bp of irp1 show similarity to the corresponding regions of the polyketide synthase genes of Bacillus subtilis and Streptomyces antibioticus. The remaining part of irp1 is most similar to irp2, encoding HMWP2, which might be the reason for immunological cross-reactivity of the two polypeptides. Irp4 was found to have 41.7% similarity to thioesterase-like protein of the anguibactin biosynthetic genes of Vibrio anguillarum. Irp5 shows 41% similarity to EntE, the 2,3-dihydroxybenzoic acid-activating enzyme utilized in enterobactin synthesis of Escherichia coli. Irp4 and Irp5 are nearly identical to YbtT and YbtE, recently identified in Y. pestis. irp3 has no similarity to any known gene. Inactivation of either irp1 or irp2 abrogates yersiniabactin synthesis. Mutations in irp1 or fyuA (encoding yersiniabactin/pesticin receptor) result in downregulation of irp2 that can be upregulated by the addition of yersiniabactin. A FyuA-green fluorescent protein translational fusion was downregulated in an irp1 mutant. Upregulation was achieved by addition of yersiniabactin but not desferal, pesticin, or pyochelin, which indicates high specificity of the FyuA receptor and autoregulation of genes involved in synthesis and uptake of yersiniabactin.     

Characterization of a Gene Cluster Involved in 4-Chlorocatechol Degradation by Pseudomonas reinekei MT1

Pseudomonas reinekei MT1 has previously been reported to degrade 4- and 5-chlorosalicylate by a pathway with 4-chlorocatechol, 3-chloromuconate, 4-chloromuconolactone, and maleylacetate as intermediates, and a gene cluster channeling various salicylates into an intradiol cleavage route has been reported. We now report that during growth on 5-chlorosalicylate, besides a novel (chloro)catechol 1,2-dioxygenase, C12O_ccaA, a novel (chloro)muconate cycloisomerase, MCI_ccaB, which showed features not yet reported, was induced. This cycloisomerase, which was practically inactive with muconate, evolved for the turnover of 3-substituted muconates and transforms 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, suggesting that it is a functional intermediate between chloromuconate cycloisomerases and muconate cycloisomerases. The corresponding genes, ccaA (C12O_ccaA) and ccaB (MCI_ccaB), were located in a 5.1-kb genomic region clustered with genes encoding trans-dienelactone hydrolase (ccaC) and maleylacetate reductase (ccaD) and a putative regulatory gene, ccaR, homologous to regulators of the IclR-type family. Thus, this region includes genes sufficient to enable MT1 to transform 4-chlorocatechol to 3-oxoadipate. Phylogenetic analysis showed that C12O_ccaA and MCI_ccaB are only distantly related to previously described catechol 1,2-dioxygenases and muconate cycloisomerases. Kinetic analysis indicated that MCI_ccaB and the previously identified C12O_salD, rather than C12O_ccaA, are crucial for 5-chlorosalicylate degradation. Thus, MT1 uses enzymes encoded by a completely novel gene cluster for degradation of chlorosalicylates, which, together with a gene cluster encoding enzymes for channeling salicylates into the ortho-cleavage pathway, form an effective pathway for 4- and 5-chlorosalicylate mineralization.The aerobic degradation of chloroaromatic compounds usually proceeds via chlorocatechols as central intermediates (20, 47), which in most of the cases reported thus far, are further degraded by enzymes of the chlorocatechol pathway (44). This pathway involves ortho-cleavage by a chlorocatechol 1,2-dioxygenase with high activity for chlorocatechols (12), a chloromuconate cycloisomerase with high activity for chloromuconates (54), a dienelactone hydrolase active with both cis- and trans-dienelactone (4-carboxymethylenebut-2-en-4-olide) (54), and a maleylacetate reductase (MAR) (28).However, it has become evident in recent years that microorganisms have evolved various alternative strategies to mineralize chlorocatechols. Pseudomonas putida GJ31 was found to degrade chlorobenzene rapidly via 3-chlorocatechol using a catechol meta-cleavage pathway (33). Two alternative pathways for 3- and 4-chlorocatechol degradation that involve reactions known from the chlorocatechol, as well as the 3-oxoadipate, pathway have recently been observed in Rhodococcus opacus 1CP (35) and Pseudomonas reinekei MT1 (39). In R. opacus 1CP, 3-chloro- and 2,4-dichloro-cis,cis-muconate (the ring cleavage products of 4-chlorocatechol and 3,5-dichlorocatechol, respectively) are converted to the respective cis-dienelactones (35, 58), similar to the reaction described for proteobacterial chloromuconate cycloisomerases (54). However, proteobacterial chloromuconate cycloisomerase can dehalogenate 2-chloromuconate (the ring cleavage product of 3-chlorocatechol) and transform this compound via 5-chloromuconolactone into trans-dienelactone (54, 65), whereas none of the described chloromuconate cycloisomerases of R. opacus 1CP can catalyze such a dehalogenation, and 5-chloromuconolactone is the product of the cycloisomerization reaction (35, 58). Dehalogenation is achieved by an enzyme with high sequence similarity to muconolactone isomerases (35), which in proteobacteria have been shown to be capable of dehalogenating 5-chloromuconolactone to cis-dienelactone (46).In P. reinekei MT1, a trans-dienelactone hydrolase (trans-DLH) was identified as the key enzyme involved in the degradation of 4- and 5-chlorosalicylate via 4-chlorocatechol as an intermediate (39). In contrast to all previously described dienelactone hydrolases involved in chlorocatechol degradation, which belong to the α/β hydrolase fold enzymes with a catalytic triad consisting of Cys, His, and Asp (10), trans-DLH was shown to be a zinc-dependent hydrolase (8). The function of this enzyme in the 4-chlorocatechol metabolic pathway was to interact with the muconate cycloisomerase (MCI)-mediated transformation of 3-chloromuconate into protoanemonin. By acting on the reaction intermediate 4-chloromuconolactone, trans-DLH prevents the formation of protoanemonin by catalyzing its hydrolysis to maleylacetate (39). Maleylacetate, in turn, is reduced by MAR to 3-oxoadipate.A more detailed genetic and biochemical analysis of the degradation of differently substituted salicylates (7) had shown the presence of two catabolic gene clusters in MT1. An archetype catRBCA gene cluster was shown to be involved in salicylate degradation. The second gene cluster (sal) had a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, clustered with the salCD genes, encoding MCI and catechol 1,2-dioxygenase (C12O), respectively. As these genes were expressed during growth on differently substituted salicylates, it was proposed that the function of the sal gene cluster is to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho-cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways. However, previous analyses had indicated the presence of an additional and thus third (chloro)muconate cycloisomerase in MT1 during growth on chlorosalicylate, which is distinct from both previously described MCIs encoded by the cat cluster (MCI_catB) and the sal cluster (MCI_salC), as it transforms 3-chloromuconate into approximately equal amounts of cis-dienelactone and protoanemonin (39). In the present report, this cycloisomerase is biochemically and genetically described and shown to be located in a third gene cluster involved in the degradation of 5-chlorosalicylate by strain MT1. This cluster comprises genes encoding a third C12O, trans-DLH (8), and a MAR. Evidently, P. reinekei MT1 is the first microorganism in which such a complex net of genes involved in chlorocatechol degradation has been described.