An approach for obtaining perfect hybridization probes for unknown polyketide synthase genes: a search for the epothilone gene cluster

An approach is described for obtaining 'perfect probes' for type I modular polyketide synthase (PKS) gene clusters that in turn enables the identification of all such gene clusters in a genome. The approach involves sequencing small fragments of a random genomic DNA library containing one or more modular PKS gene clusters, and identifying which fragments emanate from PKS genes. Knowing the approximate sizes of the genome and the target gene cluster, one can predict the the frequency that a PKS gene fragment will be present in the library sequenced. Computer simulations of the approach were applied to the known PKS and non-ribosomal peptide synthetase (NRPS) gene clusters in the Bacillus subtilus genome. The approach was then used to identify PKS gene fragments in a strain of Sorangium cellulosum that produces epothilone. In addition to identifying fragments of the epothilone gene cluster, we obtained 11 unique fragments from other PKS gene clusters; the results suggest that there may be six to eight PKS gene clusters in this organism. In addition, we identified four unique fragments of NRPS genes, demonstrating that the approach is also applicable for identification of these modular gene clusters.     

Hox clusters of the bichir (Actinopterygii, Polypterus senegalus) highlight unique patterns of sequence evolution in gnathostome phylogeny

Teleost fishes have extra Hox gene clusters owing to shared or lineage-specific genome duplication events in rayfinned fish (actinopterygian) phylogeny. Hence, extrapolating between genome function of teleosts and human or even between different fish species is difficult. We have sequenced and analyzed Hox gene clusters of the Senegal bichir (Polypterus senegalus), an extant representative of the most basal actinopterygian lineage. Bichir possesses four Hox gene clusters (A, B, C, D); phylogenetic analysis supports their orthology to the four Hox gene clusters of the gnathostome ancestor. We have generated a comprehensive database of conserved Hox noncoding sequences that include cartilaginous, lobe-finned, and ray-finned fishes (bichir and teleosts). Our analysis identified putative and known Hox cis-regulatory sequences with differing depths of conservation in Gnathostoma. We found that although bichir possesses four Hox gene clusters, its pattern of conservation of noncoding sequences is mosaic between outgroups, such as human, coelacanth, and shark, with four Hox gene clusters and teleosts, such as zebrafish and pufferfish, with seven or eight Hox gene clusters. Notably, bichir Hox gene clusters have been invaded by DNA transposons and this trend is further exemplified in teleosts, suggesting an as yet unrecognized mechanism of genome evolution that may explain Hox cluster plasticity in actinopterygians. Taken together, our results suggest that actinopterygian Hox gene clusters experienced a reduction in selective constraints that surprisingly predates the teleost-specific genome duplication.     

Comparative analysis of the complete genome sequence of the plant growth-promoting bacterium Bacillus amyloliquefaciens FZB42

Bacillus amyloliquefaciens FZB42 is a Gram-positive, plant-associated bacterium, which stimulates plant growth and produces secondary metabolites that suppress soil-borne plant pathogens. Its 3,918-kb genome, containing an estimated 3,693 protein-coding sequences, lacks extended phage insertions, which occur ubiquitously in the closely related Bacillus subtilis 168 genome. The B. amyloliquefaciens FZB42 genome reveals an unexpected potential to produce secondary metabolites, including the polyketides bacillaene and difficidin. More than 8.5% of the genome is devoted to synthesizing antibiotics and siderophores by pathways not involving ribosomes. Besides five gene clusters, known from B. subtilis to mediate nonribosomal synthesis of secondary metabolites, we identified four giant gene clusters absent in B. subtilis 168. The pks2 gene cluster encodes the components to synthesize the macrolactin core skeleton.     

Detecting anomalous gene clusters and pathogenicity islands in diverse bacterial genomes.

A gene in a genome is defined as putative alien (pA) if its codon usage difference from the average gene exceeds a high threshold and codon usage differences from ribosomal protein genes, chaperone genes and protein-synthesis-processing factors are also high. pA gene clusters in bacterial genomes are relevant for detecting genomic islands (GIs), including pathogenicity islands (PAIs). Four other analyses appropriate to this task are G+C genome variation (the standard method); genomic signature divergences (dinucleotide bias); extremes of codon bias; and anomalies of amino acid usage. For example, the cagA domain of Helicobacter pylori is highly deviant in its genome signature and codon bias from the rest of the genome. Using these methods we can detect two potential PAIs in the Neisseria meningitidis genome, which contain hemagglutinin and/or hemolysin-related genes. Additionally, G+C variation and genome signature differences of the Mycobacterium tuberculosis genome indicate two pA gene clusters.     

Interacting gene clusters and the evolution of the vertebrate immune system

Unraveling the "code" of genome structure is an important goal of genomics research. Colocalization of genes in eukaryotic genomes may facilitate preservation of favorable allele combinations between epistasic loci or coregulation of functionally related genes. However, the presence of interacting gene clusters in the human genome has remained unclear. We systematically searched the human genome for evidence of closely linked genes whose protein products interact. We find 83 pairs of interacting genes that are located within 1 Mbp in the human genome or 37 if we exclude hub proteins. This number of interacting gene clusters is significantly more than expected by chance and is not the result of tandem duplications. Furthermore, we find that these clusters are significantly more conserved across vertebrate (but not chordate) genomes than other pairs of genes located within 1 Mbp in the human genome. In many cases, the genes are both present but not clustered in older vertebrate lineages. These results suggest gene cluster creation along the human lineage. These clusters are not enriched for housekeeping genes, but we find a significant contribution from genes involved in "response to stimulus." Many of these genes are involved in the immune response, including, but not limited to, known clusters such as the major histocompatibility complex. That these clusters were formed contemporaneously with the origin of adaptive immunity within the vertebrate lineage suggests that novel evolutionary and regulatory constraints were associated with the operation of the immune system.     

Differential evolution of the 13 Atlantic salmon Hox clusters

Hox cluster organization represents a valuable marker to study the effects of recent genome duplication in salmonid fish (25-100 Mya). Using polymerase chain reaction amplification of cDNAs, BAC library screening, and genome walking, we reconstructed 13 Hox clusters in the Atlantic salmon containing 118 Hox genes including 8 pseudogenes. Hox paralogs resulting from the genome duplication preceding the radiation of ray-finned fish have been much better preserved in salmon than in other model teleosts. The last genome duplication in the salmon lineage has been followed by the loss of 1 of the 4 HoxA clusters. Four rounds of genome duplication after the vertebrate ancestor salmon Hox clusters display the main organizational features of vertebrate Hox clusters, with Hox genes exclusively that are densely packed in the same orientation. Recently, duplicated Hox clusters have engaged a process of divergence, with several cases of pseudogenization or asymmetrical evolution of Hox gene duplicates, and a marked erosion of identity in noncoding sequences. Strikingly, the level of divergence attained strongly depends on the Hox cluster pairs rather than on the Hox genes within each cluster. It is particularly high between both HoxBb clusters and both HoxDa clusters, whereas both HoxBa clusters remained virtually identical. Positive selection on the Hox protein-coding sequences could not be detected.     

链霉菌Streptomyces antibioticus NRRL 8167中Naphthgeranine A生物合成基因簇的分析鉴定

【背景】微生物来源的天然产物是小分子药物或药物先导物的重要来源。对链霉菌Streptomyces antibioticus NRRL 8167的基因组分析显示,其包含多个次级代谢产物的生物合成基因簇,具有产生多种新化合物的潜力。【目的】对链霉菌S. antibioticus NRRL 8167中次级代谢产物进行研究,以期发现结构新颖或生物活性独特的化合物,并对相应产物的生物合成基因簇和生物合成途径进行解析。【方法】利用HPLC图谱结合特征性紫外吸收和LC-MS方法,排除S. antibioticus NRRL 8167产生的已知化合物,确定具有特殊紫外吸收的化合物作为挖掘对象,然后利用正、反相硅胶柱色谱、高效液相色谱等技术对次级代谢产物进行分离纯化,分离化合物。利用质谱及核磁共振光谱技术对化合物结构进行解析和鉴定;提取链霉菌S. antibioticus NRRL 8167基因组DNA,利用PacBio测序平台进行基因组测序;利用生物信息学对基因组进行注释,并对合成该化合物的基因簇进行定位分析,推导其生物合成途径。【结果】确定这个化合物是NaphthgeranineA,属于聚酮类化合物。全基因组序列分析发现S.antibioticusNRRL8167基因组含有28个次级代谢产物生物合成基因簇,其中基因簇20可能负责Naphthgeranine A的生物合成,并对其生物合成途径进行了推导。【结论】基于紫外吸收光谱和质谱特征,从S. antibioticus NRRL 8167菌株的发酵提取物中分离鉴定了一个聚酮类化合物Naphthgeranine A。该菌株的全基因组测序为其生物合成基因簇的鉴定提供了前提,对Naphthgeranine A生物合成基因簇和生物合成途径的推测为进一步研究这个化合物的生物合成机制奠定了基础。     

东乡野生稻内生放线菌Streptomyces sp. PRh5的全基因组测序及序列分析

【目的】Streptomyces sp. PRh5是从东乡野生稻(Oryza rufipogon Griff.)中分离获得的一株对细菌和真菌都具有较强抗菌活性的内生放线菌。为深入研究PRh5菌株抗菌机制及挖掘次级代谢产物基因资源,有必要解析PRh5菌株的基因组序列信息。【方法】采用高通量测序技术对PRh5菌株进行全基因组测序,然后使用相关软件对测序数据进行基因组组装、基因预测与功能注释、直系同源簇(COG)聚类分析、共线性分析及次级代谢产物合成基因簇预测等。【结果】基因组组装获得290 contigs,整个基因组大小约11.1 Mb,GC含量为71.1%,序列已提交至GenBank数据库,登录号为JABQ00000000。同时,预测得到50个次级代谢产物合成基因簇。【结论】将为Streptomyces sp. PRh5的功能基因组学研究及相关次级代谢产物的生物合成途径与异源表达研究提供基础。     

Genome neighborhood network reveals insights into enediyne biosynthesis and facilitates prediction and prioritization for discovery

The enediynes are one of the most fascinating families of bacterial natural products given their unprecedented molecular architecture and extraordinary cytotoxicity. Enediynes are rare with only 11 structurally characterized members and four additional members isolated in their cycloaromatized form. Recent advances in DNA sequencing have resulted in an explosion of microbial genomes. A virtual survey of the GenBank and JGI genome databases revealed 87 enediyne biosynthetic gene clusters from 78 bacteria strains, implying that enediynes are more common than previously thought. Here we report the construction and analysis of an enediyne genome neighborhood network (GNN) as a high-throughput approach to analyze secondary metabolite gene clusters. Analysis of the enediyne GNN facilitated rapid gene cluster annotation, revealed genetic trends in enediyne biosynthetic gene clusters resulting in a simple prediction scheme to determine 9- versus 10-membered enediyne gene clusters, and supported a genomic-based strain prioritization method for enediyne discovery.     

Recent advances in genome mining of secondary metabolite biosynthetic gene clusters and the development of heterologous expression systems in Aspergillus nidulans

Fungi are prolific producers of secondary metabolites (SMs) that show a variety of biological activities. Recent advances in genome sequencing have shown that fungal genomes harbor far more SM gene clusters than are expressed under conventional laboratory conditions. Activation of these “silent” gene clusters is a major challenge, and many approaches have been taken to attempt to activate them and, thus, unlock the vast treasure chest of fungal SMs. This review will cover recent advances in genome mining of SMs in Aspergillus nidulans. We will also discuss current updates in gene annotation of A. nidulans and recent developments in A. nidulans as a molecular genetic system, both of which are essential for rapid and efficient experimental verification of SM gene clusters on a genome-wide scale. Finally, we will describe advances in the use of A. nidulans as a heterologous expression system to aid in the analysis of SM gene clusters from other fungal species that do not have an established molecular genetic system.     

植物次生代谢基因簇研究进展

类似于原核生物的操纵子,在真核生物（如酵母、真菌、昆虫等）基因组中也出现了彼此功能相关的非同源基因成簇存在的现象。这些基因形成基因簇,可参与多种次生代谢途径。近年来,植物中也发现了越来越多的参与次生代谢产物合成的基因簇,它们已成为植物生物学研究的热点。本文总结并分析了植物中已鉴定的次生代谢基因簇。这些基因簇存在于玉米（Zea mays L.）、水稻（Oryza sativa L.）、拟南芥（Arabidopsis thaliana（L.） Heynh.）、番茄（Solanum lycopersicum L.）等植物的基因组中,分别参与合成苯并噁唑嗪酮类、萜类和生物碱类等次生代谢产物。本文通过解析这些基因簇的组成及结构特点,对其特征进行总结,探讨了基因簇形成的分子机理及其调控机制,对植物次生代谢基因簇在合成生物学及代谢工程学中的研究方向和应用前景进行了展望。     

Microsatellite motifs with moderate GC content are clustered around genes on Arabidopsis thaliana chromosome 2

Microsatellites, arrays of 1-6 bp sequences, are abundant in almost all the eukaryotic genomes. Their distribution in the genome is widely accepted to be differential and non random along the axis of the chromosomes. Arabidopsis thaliana genome is dominated by mononucleotide repeats, (A)n being the most abundant motif. In total, 39 microsatellite motifs extended to more than 100 bp in length. Of these, 8 loci are devoid of any gene in their proximity. (AG)n is the most abundant motif among longer repeats. The non-random distribution of microsatellite in the genome is reflected as occurrence of microsatellite clusters in the genome. In total, 3400 microsatellite clusters have been identified in the Arabidopsis genome. Chromosome 2, which is 19.7 Mb long, harbors 550 clusters accommodating 29% of all the microsatellites present on this chromosome. Further, 409 of the 6239 genes on chromosome 2 are associated with 323 microsatellite clusters. Motifs like (AGG)n and (ACT)n, show preferential accommodation in clusters that overlap with genes. Among all the microsatellite clusters that show an overlap with genes, 80% of the clusters show an overlap in such a way that the cluster ends beyond the 3'-end of the gene or starts before the 5'-end of a gene. Genes with diverse functions show association with the clusters. However, not all members of a gene family show similar associations.     

Histone gene clusters of the newt notophthalmus are separated by long tracts of satellite DNA

The genomic organization of the histone genes of the newt Notophthalmus viridescens is described. Genes for the five proteins are clustered on a 9.0 kb segment of cloned DNA which is part of a homogeneous family of sequences containing 600–800 members per haploid genome. The 9.0 kb histone gene clusters are not adjacent in the genome, but are separated from neighboring clusters by up to 50 kb or more of cluster spacer sequences; some or all of these spacer sequences are members of a predominantly centromeric satellite DNA with a 225 bp repeating unit.     

大肠杆菌基因组基因无痕敲除的优化方法

目的：优化大肠杆菌基因组基因无痕敲除的方法,提高无痕敲除的效率。方法：以无痕敲除大肠杆菌nanKETA基因簇为模型,利用Red同源重组系统和核酸内切酶I-SceI的筛选作用,通过两步连续同源重组无痕敲除大肠杆菌CLM37基因组中的nanKETA基因,优化无痕敲除时同源DNA长度与诱导用于筛选阳性克隆I-SceI表达的诱导剂浓度。通过比较敲除nanKETA基因前后菌株的生长曲线,研究大肠杆菌CLM37缺失nanKETA基因后的生长状态。结果：成功无痕敲除大肠杆菌CLM37基因组中的nanKETA基因,并在无痕化处理时,通过延长与基因组同源DNA的长度,由通常使用的80碱基对延长到684碱基对;并通过提高诱导筛选基因表达的四环素的浓度,由500 μg/ml提高到1000 μg/ml后,使无痕敲除效率高达90%以上。生长曲线研究表明,缺失nanKETA基因后的菌株生长状态与原菌株基本一致。结论：通过延长与基因组同源的双链核苷酸的长度和诱导筛选基因表达的四环素的浓度可显著提高无痕敲除的效率。     

Genetic variation at the O-antigen biosynthetic locus in Pseudomonas aeruginosa

The outer carbohydrate layer, or O antigen, of Pseudomonas aeruginosa varies markedly in different isolates of these bacteria, and at least 20 distinct O-antigen serotypes have been described. Previous studies have indicated that the major enzymes responsible for O-antigen synthesis are encoded in a cluster of genes that occupy a common genetic locus. We used targeted yeast recombinational cloning to isolate this locus from the 20 internationally recognized serotype strains. DNA sequencing of these isolated segments revealed that at least 11 highly divergent gene clusters occupy this region. Homology searches of the encoded protein products indicated that these gene clusters are likely to direct O-antigen biosynthesis. The O15 serotype strains lack functional gene clusters in the region analyzed, suggesting that O-antigen biosynthesis genes for this serotype are harbored in a different portion of the genome. The overall pattern underscores the plasticity of the P. aeruginosa genome, in which a specific site in a well-conserved genomic region can be occupied by any of numerous islands of functionally related DNA with diverse sequences.     

Genome mining demonstrates the widespread occurrence of gene clusters encoding bacteriocins in cyanobacteria

Cyanobacteria are a rich source of natural products with interesting biological activities. Many of these are peptides and the end products of a non-ribosomal pathway. However, several cyanobacterial peptide classes were recently shown to be produced through the proteolytic cleavage and post-translational modification of short precursor peptides. A new class of bacteriocins produced through the proteolytic cleavage and heterocyclization of precursor proteins was recently identified from marine cyanobacteria. Here we show the widespread occurrence of bacteriocin gene clusters in cyanobacteria through comparative analysis of 58 cyanobacterial genomes. A total of 145 bacteriocin gene clusters were discovered through genome mining. These clusters encoded 290 putative bacteriocin precursors. They ranged in length from 28 to 164 amino acids with very little sequence conservation of the core peptide. The gene clusters could be classified into seven groups according to their gene organization and domain composition. This classification is supported by phylogenetic analysis, which further indicated independent evolutionary trajectories of gene clusters in different groups. Our data suggests that cyanobacteria are a prolific source of low-molecular weight post-translationally modified peptides.     

Identification and characterization of nucleotide-binding site-leucine-rich repeat genes in the model plant Medicago truncatula

The nucleotide-binding site (NBS)-Leucine-rich repeat (LRR) gene family accounts for the largest number of known disease resistance genes, and is one of the largest gene families in plant genomes. We have identified 333 nonredundant NBS-LRRs in the current Medicago truncatula draft genome (Mt1.0), likely representing 400 to 500 NBS-LRRs in the full genome, or roughly 3 times the number present in Arabidopsis (Arabidopsis thaliana). Although many characteristics of the gene family are similar to those described on other plant genomes, several evolutionary features are particularly pronounced in M. truncatula, including a high degree of clustering, evidence of significant numbers of ectopic translocations from clusters to other parts of the genome, a small number of more evolutionarily stable NBS-LRRs, and numerous truncations and fusions leading to novel domain compositions. The gene family clearly has had a large impact on the structure of the genome, both through ectopic translocations (potentially, a means of seeding new NBS-LRR clusters), and through two extraordinarily large superclusters. Chromosome 6 encodes approximately 34% of all TIR-NBS-LRRs, while chromosome 3 encodes approximately 40% of all coiled-coil-NBS-LRRs. Almost all atypical domain combinations are in the TIR-NBS-LRR subfamily, with many occurring within one genomic cluster. This analysis shows the gene family not only is important functionally and agronomically, but also plays a structural role in the genome.