Two unusual chlorocatechol catabolic gene clusters in <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. TFD44

The genes responsible for the degradation of 2,4-dichlorophenoxyacetate (2,4-D) by -Proteobacteria have previously been difficult to detect by using gene probes or polymerase chain reaction (PCR) primers. PCR products of the chlorocatechol 1,2-dioxygenase gene, tfdC, now allowed cloning of two chlorocatechol gene clusters from the Sphingomonas sp. strain TFD44. Sequence characterization showed that the first cluster, tfdD,RFCE, comprises all the genes necessary for the conversion of 3,5-dichlorocatechol to 3-oxoadipate, including a presumed regulatory gene, tfdR, of the LysR-type family. The second gene cluster, tfdC2E2F2, is incomplete and appears to lack a chloromuconate cycloisomerase gene and a regulatory gene. Purification and N-terminal sequencing of selected enzymes suggests that at least representatives of both gene clusters (TfdD of cluster 1 and TfdC2 of cluster 2) are induced during the growth of strain TFD44 with 2,4-D. A mutant constructed to contain an insertion in the chloromuconate cycloisomerase gene tfdD still was able to grow with 2,4-D, but more slowly and with a longer lag phase. This, and the detection of additional activity peaks during protein purification suggest that strain TFD44 harbors at least another chloromuconate cycloisomerase gene. The sequence of the tfdCE region was almost identical to that of a partially characterized chlorocatechol catabolic gene cluster of Sphingomonas herbicidovorans MH, whereas the sequence of the tfdC2E2F2 cluster was different. The similarity of the predicted proteins of the tfdD,RFCE and tfdC2E2F2 clusters to known sequences of other Proteobacteria in the database ranged from 42 to 61% identical positions for the first cluster and from 45.5 to 58% identical positions for the second cluster. Between both clusters, the similarities of their predicted proteins ranged from 44.5 to 64% identical positions. Thus, both clusters (together with those of S. herbicidovorans MH) represent deep-branching lines in the respective dendrograms, and the sequence information will help future primer design for the detection of corresponding genes in the environment.     

Peripheral sequences of the Serratia entomophila pADAP virulence-associated region

Some strains of the Enterobacteriaceae Serratia entomophila and Serratia proteamaculans cause amber disease in the grass grub, Costelytra zealandica (Coleoptera: Scarabaeidae), an important pasture pest in New Zealand. The genes responsible for this disease reside on a large, 155-kb plasmid designated amber disease-associated plasmid (pADAP). Herein, we report the DNA sequencing of approximately 50 kb upstream and 10 kb downstream of the virulence-encoding region. Based on similarity with proteins in the current databases, and potential ribosome-binding sites, 63 potential ORFs were determined. Eleven of these ORFs belong to a type IV pilus cluster (pilL-V) and a further eight have similarities to the translated products of the plasmid transfer traH-N genes of the plasmid R64. In addition, a degenerate 785-nt direct repeat flanks a 44.7-kb region with the potential to encode three Bacillus subtilis Yee-type proteins, a fimbrial gene cluster, the sep virulence-associated genes and several remnant IS elements.     

Xylose metabolism in the anaerobic fungus<Emphasis Type="Italic"> Piromyces</Emphasis> sp. strain E2 follows the bacterial pathway

The anaerobic fungus Piromyces sp. strain E2 metabolizes xylose via xylose isomerase and d-xylulokinase as was shown by enzymatic and molecular analyses. This resembles the situation in bacteria. The clones encoding the two enzymes were obtained from a cDNA library. The xylose isomerase gene sequence is the first gene of this type reported for a fungus. Northern blot analysis revealed a correlation between mRNA and enzyme activity levels on different growth substrates. Furthermore, the molecular mass calculated from the gene sequence was confirmed by gel permeation chromatography of crude extracts followed by activity measurements. Deduced amino acid sequences of both genes were used for phylogenetic analysis. The xylose isomerases can be divided into two distinct clusters. The Piromyces sp. strain E2 enzyme falls into the cluster comprising plant enzymes and enzymes from bacteria with a low G+C content in their DNA. The d-xylulokinase of Piromyces sp. strain E2 clusters with the bacterial d-xylulokinases. The xylose isomerase gene was expressed in the yeast Saccharomyces cerevisiae, resulting in a low activity (25±13 nmol min^–1mg protein^-1). These two fungal genes may be applicable to metabolic engineering of Saccharomyces cerevisiae for the alcoholic fermentation of hemicellulosic materials.     

The organization of the keratin I and II gene clusters in placental mammals and marsupials show a striking similarity

The genomic database for a marsupial, the opossum Monodelphis domestica, is highly advanced. This allowed a complete analysis of the keratin I and keratin II gene cluster with some 30 genes in each cluster as well as a comparison with the human keratin clusters. Human and marsupial keratin gene clusters have an astonishingly similar organization. As placental mammals and marsupials are sister groups a corresponding organization is also expected for the archetype mammal. Since hair is a mammalian acquisition the following features of the cluster refer to its origin. In both clusters hair keratin genes arose at an interior position. While we do not know from which epithelial keratin genes the first hair keratins type-I and -II genes evolved, subsequent gene duplications gave rise to a subdomain of the clusters with many neighboring hair keratin genes. A second subdomain accounts in both clusters for 4 neighboring genes encoding the keratins of the inner root sheath (irs) keratins. Finally the hair keratin gene subdomain in the type-I gene cluster is interrupted after the second gene by a region encoding numerous genes for the high/ultrahigh sulfur hair keratin-associated proteins (KAPs). We also propose a tentative synteny relation of opossum and human genes based on maximal sequence conservation of the encoded keratins. The keratin gene clusters of the opossum seem to lack pseudogenes and display a slightly increased number of genes. Opossum keratin genes are usually longer than their human counterparts and also show longer intergenic distances.     

Efficient authentic fine mapping of the rice blast resistance gene <Emphasis Type="Italic">Pik-h</Emphasis> in the <Emphasis Type="Italic">Pik</Emphasis> cluster,using new <Emphasis Type="Italic">Pik-h</Emphasis>-differentiating isolates

The Pik-h gene in rice confers resistance to several races of rice blast fungus (Magnaporthe oryzae), and has been classified as a member of the Pik cluster, one of the most resistance (R) gene-dense regions in the rice genome. However, the loss of a key mutant isolate has long made it difficult to differentiate Pik-h from other Pik group genes especially from Pik-m. We identified new natural isolates enabling the differentiation between Pik-h and Pik-m genes, and first confirmed the authenticity of the International Rice Research Institute (IRRI) “monogenic” line IRBLkh-K3, and then fine-mapped the Pik-h gene in the Pik cluster. Using 701 susceptible individuals among 3,060 siblings from a cross of IRBLkh-K3×CO39, the Pik-h region was delimited to 270 kb, the narrowest interval among the Pik group genes reported to date, in the cv. Nipponbare genome. Annotation of this genome region first revealed 6 NBS-LRR type R-gene analogs (RGAs), clustered within the central 120 kb, as possible counterparts of Pik-h and 6 other Pik group R genes. Interestingly, the Pik-h region and the cluster of RGAs were shown to be located 130 kb and 230 kb apart from Xa4 and Xa2 bacterial blight resistance genes, respectively, once classified as belonging to the Pik cluster. The closest recombination events were limited to the margins of the Pik-h region, and recombination was suppressed in the core interval with the RGA cluster. This fine-mapping, performed in a short time using an HEGS system, will facilitate utilization of the cluster’s genetic resources and help to elucidate the mechanism of evolution of R-genes. The presence of natural isolates also confirmed that evolution of Pik-h corresponds to pathogen evolution.     

Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

The tomato Orion locus comprises a unique class of <Emphasis Type="Italic">Hcr9</Emphasis> genes

Resistance against the tomato fungal pathogen Cladosporium fulvum is often conferred by Hcr9 genes (Homologues of the C. fulvum resistance gene Cf-9) that are located in the Milky Way cluster on the short arm of chromosome 1. These Hcr9 genes mediate recognition of fungal avirulence gene products. In contrast, the resistance gene Cf-Ecp2 mediates recognition of the virulence factor Ecp2 and is located in the Orion (OR) cluster on the short arm of chromosome 1. Here, we report the map- and homology-based cloning of the OR Hcr9 cluster. A method was optimised to generate clone-specific fingerprint data that were subsequently used in the efficient calculation of genomic DNA contigs. Three Hcr9s were identified as candidate Cf-Ecp2 genes. By PCR-based cloning using specific OR sequences, orthologous Hcr9 genes were identified from different Lycopersicon species and haplotypes. The OR Hcr9s are very homologous. However, based on the relative low sequence homology to other Hcr9s, the OR Hcr9s are classified as a new subgroup.Data deposition: The sequence of the Cf-Ecp2 Hcr9 gene cluster and the orthologous Hcr9 sequences have been deposited in the GenBank database (accession No. AY639600..AY639604)     

一株稀有海洋真菌Stachybotrys longispora FG216的De Novo测序及基因组学分析

【背景】长孢葡萄穗霉菌(Stachybotrys longispora) FG216是一株稀有海洋真菌,其次生代谢产物FGFC1具有纤溶活性。进行S. longispora FG216的基因组序列分析,将充实和促进海洋微生物功能基因和次生代谢产物合成生物学的基础研究和应用研究。【目的】解析S. longispora FG216的基因组序列,分析基因组生物功能和同源相似性关系,分析次生代谢产物纤溶活性化合物FGFC1的相关基因。【方法】基于Illumina HiSeq高通量测序平台对S. longispora FG216菌株进行De Novo测序,使用SSPACE、Augustus等软件进行组装、编码基因预测、基因功能注释、物种共线性分析以及预测FGFC1次生代谢产物合成基因簇。【结果】S. longispora FG216的基因组测序总长度为45622830bp,共得到605个Scaffold,GC含量为51.31%,注释预测得到13329个编码基因和169个非编码RNA。基因组测序数据提交至国家微生物科学数据中心(编号为NMDC60016264),其中13 053、8 422、8 460、7 714和2 847个基因分别能够在NR、KEGG、KOG、GO和CAZy数据库匹配到注释信息。比较基因组学分析发现,Stachybotrys具有保守性,核心基因占基因家族总数目的71.44%,S. longispora FG216与S. chlorohalonata IBT 40285的相似性最高;同时,预测得到101个次生代谢产物合成基因簇,其中18个基因簇与已知的化合物相匹配。通过antiSMASH预测,Cluster57是编码合成FGFC1母核结构异吲哚啉酮的基因簇,与S.chlorohalonataIBT40285中的基因簇相似度为40%。【结论】海洋稀有真菌S.longisporaFG216的基因组信息已上传至国家微生物科学数据中心公开使用,为Stachybotrys种属的研究提供了重要的参考意义,同时发现了S. longispora FG216次生代谢产物纤溶活性化合物FGFC1母核部分编码基因是Cluster 57。     

Organization of<Emphasis Type="Italic"> Iroquois</Emphasis> genes in fish

In mammals, a total of six iroquois (Irx) genes exist, which are organized into two clusters. Here we report on the organization of all iroquois genes present in fish, using zebrafish (Danio rerio) and pufferfish (Fugu rubripes and Tetraodon nigroviridis) as examples. A total of 10 Irx genes were found in pufferfish, and 11 in zebrafish; all but one of these genes are organized into clusters (four clusters plus one isolated gene locus). The extra fish clusters result from chromosome duplication in the fish lineage, after its divergence from tetrapod vertebrates. Two of the four fish clusters are highly conserved to the ones in mammals, with regard to similarity of genes and cluster architecture. Irx genes within the other two clusters have diverged in sequence and cluster organization, suggesting functional divergence. These results will allow us to use the zebrafish system for functional and comparative studies of iroquois genes in vertebrate development.Electronic Supplementary Material Supplementary material is available in the online version of this article at Edited by D. Tautz     

来自南极洲类诺卡氏菌(Nocardioides sp.) InS609-2的全基因组序列分析

【背景】类诺卡氏菌（Nocardioides sp.） InS609-2是一株分离自南极洲罗斯海特拉诺瓦湾的恩克斯堡岛土壤中潜在的极地放线菌新种。尚无研究报道Nocardioides sp.InS609-2的全基因组序列，缺少对其功能基因、代谢产物合成途径及比较基因组学等的研究。【目的】解析Nocardioides sp.InS609-2的基因组序列信息，以深入挖掘次级代谢产物基因资源。【方法】利用Illumina HiSeq高通量测序平台对菌株InS609-2进行全基因组完成图测序，使用相关软件对测序数据进行基因组组装、基因预测和功能注释、预测次级代谢产物合成基因簇和共线性分析等。【结果】基因组最后得到的总长度为4 524 052 bp，G+C含量为69.42%，预测到4 656个基因、56个tRNA和6个rRNA。根据Nocardioides sp.InS609-2的全基因组测序结果，分别有3 761、3 052、1 767、4 134和2 725个基因在COG、GO、KEGG、NR和Swiss-Prot数据库中提取到注释信息。同时，还预测得到19个次级代谢产物合成基因簇。基因组测序数据提交至NCBI获得GenBank登录号为CP060034。Nocardioides sp.InS609-2与N. dokdonensis CP015079、N. yefusunii CP034929、N. euryhalodurans CP038267、N. seonyuensis CP038436、N. daphniae CP038462和N. okcheonensis CP087710这6株基因组同源性比较高的类诺卡氏菌进行共线性分析和蛋白聚类分类分析，得到的结果是7个基因组间既有保守性又各自有独特性。七个基因组共有44个蛋白聚类簇。最后进行16S rRNA基因系统发育树、泛基因组、core基因组和基因组进化树分析，进一步挖掘了Nocardioides sp.InS609-2的基因组信息。【结论】从基因组层面上预测了Nocardioides sp.InS609-2的次级代谢产物的生物合成基因簇，为InS609-2的后续相关研究提供了参考信息，具有重要意义。     

The lipopeptide antibiotic A54145 biosynthetic gene cluster from Streptomyces fradiae

Ca(2+)-dependent cyclic lipodepsipeptides are an emerging class of antibiotics for the treatment of infections caused by Gram-positive pathogens. These compounds are synthesized by nonribosomal peptide synthetase (NRPS) complexes encoded by large gene clusters. The gene cluster encoding biosynthetic pathway enzymes for the Streptomyces fradiae A54145 NRP was cloned from a cosmid library and characterized. Four NRPS-encoding genes, responsible for subunits of the synthetase, as well as genes for accessory functions such as acylation, methylation and hydroxylation, were identified by sequence analysis in a 127 kb region of DNA that appears to be located subterminally in the bacterial chromosome. Deduced epimerase domain-encoding sequences within the NRPS genes indicated a D: -stereochemistry for Glu, Lys and Asn residues, as observed for positionally analogous residues in two related compounds, daptomycin, and the calcium-dependent antibiotic (CDA) produced by Streptomyces roseosporus and Streptomyces coelicolor, respectively. A comparison of the structure and the biosynthetic gene cluster of A54145 with those of the related peptides showed many similarities. This information may contribute to the design of experiments to address both fundamental and applied questions in lipopeptide biosynthesis, engineering and drug development.     

Molecular cloning, genetic organization of gene cluster encoding phenol hydroxylase and catechol 2,3-dioxygenase in Alcaligenes faecalis IS-46

Alcaligenes faecalis IS-46 can utilize phenol as the sole carbon and energy source at concentration up to 1000 mg/l. In this report we created a cosmid library of this strain and the two clones specifying the whole L-46d type of phenol hydroxylase gene cluster were identified and characterized. Sequence analysis revealed that although the overall gene organization of the clusters was quite similar, few coding sequences differed or were found to have two copies compared with other source organisms. One of these coding sequences showed a good protein sequence similarity to a hypothetical protein and one matched with a regulatory protein of the LysR system. Their putative role in phenol degradation was discussed. Bioinformatic analysis suggested tentative phylogenetic assignments of the retrieved clusters. This work described for first time the complete nucleotide sequence and genetic organization of the whole phenol hydroxylase gene cluster in A. faecalis species.     

Comparative and functional analysis of the rRNA-operons and their tRNA gene complement in different lactic acid bacteria

The complete genome sequences of the lactic acid bacteria (LAB), Lactobacillus plantarum, Lactococcus lactis, and Lactobacillus johnsonii were used to compare location, sequence, organisation, and regulation of the ribosomal RNA (rrn) operons. All rrn operons of the examined LAB diverge from the origin of replication, which is compatible with their efficient expression. All operons show a common organisation of 5'-16S-23S-5S-3' structure, but differ in the number, location and specificity of the tRNA genes. In the 16S-23S intergenic spacer region, two of the five rrn operons of Lb. plantarum and three of the six of Lb. johnsonii contain tRNA-ala and tRNA-ile genes, while L. lactis has a tRNA-ala gene in all six operons. The number of tRNA genes following the 5S rRNA gene ranges up to 14, 16, and 21 for L. lactis, Lb. johnsonii and Lb. plantarum, respectively. The tRNA gene complements are similar to each other and to those of other bacteria. Micro-heterogeneity was found within the rRNA structural genes and spacer regions of each strain. In the rrn operon promoter regions of Lb. plantarum and L. lactis marked differences were found, while the promoter regions of Lb. johnsonii showed a similar tandem promoter structure in all operons. The rrn promoters of L. lactis show either a single or a tandem promoter structure. All promoters of Lb. plantarum contain two or three -10 and -35 regions, of which either zero to two were followed by an UP-element. The Lb. plantarum rrnA, rrnB, and rrnC promoter regions display similarity to the rrn promoter structure of Esherichia coli. Differences in regulation between the five Lb. plantarum promoters were studied using a low copy promoter-probe plasmid. Taking copy number and growth rate into account, a differential expression over time was shown. Although all five Lb. plantarum rrn promoters are significantly different, this study shows that their activity was very similar under the circumstances tested. An active promoter was also identified within the Lb. plantarum rrnC operon preceding a cluster of 17 tRNA genes.     

Characterisation of the <Emphasis Type="Italic">Aspergillus niger dapB</Emphasis> gene,which encodes a novel fungal type IV dipeptidyl aminopeptidase

We have cloned the Aspergillus niger dapB gene. Analysis of its nucleotide sequence and the corresponding protein sequence indicates that the gene encodes a type IV dipeptidyl aminopeptidase (DPP IV). Based upon its deduced sequence we predict the presence of a transmembrane domain in the protein. Furthermore, dapB-overexpressing transformants display an increase in intracellular DPP IV activity. This is the first reported characterisation of a dipeptidyl aminopeptidase with a transmembrane domain from a filamentous fungus. Using the dapB sequence as a query, we were able to identify 14 DPP IV-encoding genes, and 12 additional DPPIV proteases in public genomic databases. Phylogenetic analysis reveals that in yeasts there are two clades of genes that encode DPP IV proteases with a transmembrane domain. In this study we demonstrate that, as in yeasts, two classes of DPP IV-encoding genes exist in filamentous fungi. However, only one of these codes for DPP IV proteases with a transmembrane domain. The second type present in filamentous fungi encodes extracellular DPP IV proteases. The dapB gene belongs to the first cluster. We propose that DapB plays a role in the proteolytic maturation of enzymes produced by A. niger.Electronic Supplementary Material Supplementary material is available for this article at