Comparative genomics of Streptomyces avermitilis

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2 Antonie van Leeuwenhoek 2008 93 1 25, 9 025 608 bp 7 577 protein coding genes for http www sanger ac uk Projects S coelicolor sche. S avermitilis Secondly that the genomes of these me shtml A microarray analysis of the genomes of. two species are linear and both ends contain unique these Streptomyces using the S coelicolor microarray. terminal inverted repeats that probably covalently is able to provide a wide ranging comparative. bind a terminal protein Terminal inverted repeats and analysis of the conserved genome content of these. covalently bound terminal proteins are not found in Streptomyces This type of approach where a heter. the limited number of other bacteria that have linear ologous microarray is used to analyze the genome. chromosomes such as Borrelia burgdorferi and content of a range of strains or species has been. Agrobacterium tumefaciens and up to the present successfully used in a wide range of organisms. seem to be unique to the Streptomyces and perhaps Akman and Aksoy 2001 Akman et al 2001 Behr. other Actinobacteria Lin et al 1993 Chen et al et al 1999 Chan et al 2003 Cho and Tiedje 2001. 2002 Goodner et al 1999 Huang et al 2004 Over Dorrell et al 2001 Dziejman et al 2002 Fitzgerald. 2 500 Streptomyces strains are present in the Ribo et al 2001 Gill et al 2002 Leonard et al 2003. somal Database Project http www rdp cme msu e Murray et al 2001 Porwollik et al 2002 Salama. du over 1 500 are available at the American Type et al 2000 Israel et al 2001 Rajashekara et al. Culture Collection http www atcc org and many 2004 The strains analyzed using this approach range. more are held in both public and private culture from intraspecies comparisons such as Campylobac. collections throughout the world Analysis of the ter jejuni Vibrio cholerae and Staphylococcus aureus. small subunit ribosomal RNA gene sequences of Dorrell et al 2001 Dziejman et al 2002 Fitzgerald. Streptomyces confirms that they form a monophyletic et al 2001 to interspecies comparisons such as. clade but one with considerable diversity In addi Sodalis glossinidiusversus an Escherichia coli array. tion there is significant gene diversity at the inter Salmonella bongori versus a Salmonella enterica. species level across the genomes of both completely array Shewanella species versus Shewanella oneid. sequenced Streptomyces with 2 291 gene unique to ensis and E coli arrays and Brucella species versus a. S avermitilis and 2 307 genes unique to S coelicol Brucella melitensis array Akman et al 2001 Chan. or This makes them particularly interesting targets et al 2003 Murray et al 2001 Rajashekara et al. for comparative genomic studies In this study we 2004. chose four species to begin an analysis of the In this study we used both versions of the. genomic diversity of the Streptomyces S avermitilis S coelicolor genome microarrays to compare the. was chosen because of the availability of the gene complements of the three Streptomyces species. complete genome sequence of this species while and one Kitasatospora species The genus Kitasatos. Streptomyces maritimus was chosen because of its pora is closely related to the genus Streptomyces in. intermediate position in terms of phylogeny within terms of morphology chemical taxonomy and small. the Streptomyces Streptomyces cattleya was chosen subunit ribosomal RNA sequence analysis Thus the. because based on small subunit ribosomal RNA choice of a species from this genus acts as potential. sequence this species is phylogenetically quite outgroup in terms of overall genome structure In. divergent from S coelicolor and branches near the terms of genes that are conserved the types of genes. root of the Streptomyces clade Streptomyces cattleya of particular interest include genes involved in. is a b lactam producing species Finally Kitasatos secondary metabolism genes involved in chromo. pora aureofaciens was chosen as this genus is very some replication genes in the terminal regions of the. closely related to the Streptomyces chromosome sigma factors genes involved in dif. The availability of two microarrays for S coeli ferentiation and hypothetical genes In terms of gene. color Lum et al 2004 Huang et al 2001 Vinciotti absence the distribution of such genes along the. et al 2005 http www surrey ac uk SBMS Fgenom chromosome and the apparent absence of any major. ics Microarrays index html makes possible a com housekeeping genes in a specific species are of. parative genomic analysis of Streptomyces species interest This information provides insights into genes. The genes that make up the genome of S coelicolor that make up the core complement for a member of. have been classified based on scheme of Riley and the Streptomyces and into which genes are central to. colleagues for E coli and modified for S coelicolor defining a Streptomyces species. Antonie van Leeuwenhoek 2008 93 1 25 3, Materials and methods mycelium cultured in TSB liquid medium with 0 5. glycine at 308C overnight,16S phylogeny,Preparation of labeled DNA. This was carried out on selected small subunit 16S. ribosomal RNA gene sequences obtained from Ribo Genomic DNA from a stationary phase culture was. somal Database Project II Release 9 http purified by the salting out procedure Pospiech and. www rdp cme msu edu index jsp and aligned using Neumann 1995 and had been sonicated to 2 Kb. CLUSTALX Thompson et al 1997 The analysis Four to six micrograms of sonicated genomic DNA. was carried out using Neighbor Joining algorithm were used as template and this was denatured in. from the same program In the case of S maritimus the presence of 12 mg of 72 GC content random. the taxonomy of the strain was confirmed by DNA hexamers in a total volume of 25 ml at 1008C for. sequencing of the 16S ribosomal RNA gene 10 min The mixture was then snap cooled on ice. before adding the remaining reaction components,Arrays 1 5 ml of Cy3 dCTP or Cy5 dCTP Amersham. Pharmacia Biotech 4ml Klenow fragment NEB, Two series of arrays that cover about 97 of the 212 5ml Klenow buffer 0 5 ml dNTP 4 mM.
complete genome of Streptomyces coelicolor A3 2 dATP 4 mM dTTP 10 mM dGTP and 0 2 mM. Lum et al 2004 http www surrey ac uk SBMS dCTP and 14 ml ddH2O The random primed. Fgenomics Microarrays index html were used in this labeling reaction was carried out for 2 3 h at 378C. study Both arrays are PCR arrays but from different Buffer exchange purification and concentration of. sources namely Stanford University USA and the the DNA products was accomplished by three. University of Surrey UK and made up of different cycles of diluting the reaction mixture in 0 5 ml TE. PCR products The Stanford array as used in this buffer 10 mM Tris and 1 mM EDTA pH 8 0 and. study contained sequences covering 7603 open filtering though a Microcon 30 microconcentrators. reading frames The Surrey microarray is made up Millipore. of 7 758 unique PCR amplified sequences 7 563, from the chromosome and 195 from SCP1 There are Microarray hybridization and data analysis. an additional 376 non unique alternative and cross. hybridizing sequences that are also spotted on to the The two DNA pools to be compared were mixed. array together with no probe spots and control spots and applied to an array in a hybridization mixture. The two types of arrays were used to improve that contained 3 68 SSC 0 18 SDS and 1 mg. validation with a system using heterologous hybrid yeast tRNA total 16 3 ml which had been heated. ization however only the University of Surrey array at 1008C for 5 min before being applied to array. was hybridized and analyzed in duplicate The major Hybridization took place under a glass coverslip. difference between the two arrays was that the Surrey sealed by glue in a humidified Omnislide Thermo. array did not include a number of transposition Hybaid at 608C for 12 14 h The slides were. element related genes although there were other washed dried and scanned for fluorescence using a. overlap differences The sequences of the PCR GenePix TM 4000B scanner Axon instruments. products are not available for either array due to Average signal intensity and local background. intellectual property protection requirements measurements were obtained for each spot on each. array using GenePixPro software The dataset was, Strains and growth conditions screened for aberrant spots and these were elimi. nated from the analysis after manual checking, S coelicolor A3 2 SCP1 104 S avermitilis Most genes are present in duplicate on the two. ATCC 31267 S cattleya ATCC 35852 S maritimus arrays and the signal from each pair of spots was. Yang Ming and K aureofaciens ATCC 10762 were inputted into the computer program available from. used in these studies Fresh spores were collected and ScanAlyze Eisen et al 1998 Gollub et al 2003. 4 Antonie van Leeuwenhoek 2008 93 1 25, The data was then processed into a mean log2 Cy3 Results and discussion. Cy5 ratio format The dataset was normalized for, each array separately and outputted to Excel where Comparison of S avermitilis S cattleya.
after checking the alignment of the datasets from S maritimus and K aureofaciens with. each array a mean signal for each common gene the S coelicolor genome. was calculated Genes that were absent from either, array mostly transposon related genes in the In total after spot and data validation a total of 7 083. University of Surrey array were not included in open reading frames were included in this analysis as. the analysis Based on Bentley et al 2002 the presence on both types of array and giving analyzable. mean signal and standard deviation for the core signal on all three arrays Validity in this study was. region of genes from SCO2050 to SCO5800 was initially obtained by using microarrays from two. calculated The standard deviation was used to set a sources that presumably use different PCR products. cut off for gene absence at 2SD below the core to create the arrays In addition the University of. mean The microarray data is presented relative to Surrey array was hybridized and analyzed in dupli. the S coelicolor standard in two ways This is cate In terms of gene absence based on two standard. either as a color plot of the genes where green deviations as described in the Materials and meth. presents a negative hybridization signal black ods section the agreement between the Stanford. represents an equal hybridization signal and red array and the duplicated University of Surrey array. indicates a positive hybridization signal using the was about 95 while the agreement between the two. program Treeview Eisen et al 1998 or as numeric University of Surrey arrays was about 98 In order. values for the signal from each gene The micro to minimize the effect of divergent individual array. array data for the four species described here and spots the signal mean for each gene from the three. additional unpublished species can be accessed via arrays was used throughout this study. rkirby ym edu tw In this study the genomic content of three Strepto. myces species and one Kitasatospora species with,divergent taxonomy antibiotic production and SSU. Comparison of the microarray dataset for,rRNA sequence are compared using two different S. S avermitilis with the complete genome sequence,coelicolor microarrays It is clear that there are. inherent limitations to this approach Firstly only gene. The nucleotide sequences for all the identified open. absence or divergence rather than the presence of new. reading frame from the S avermitilis genome, genes can be identified Secondly it is not possible to.
sequence Ikeda et al 2003 were compared with,clearly separate the absence of a gene from the. the genome sequence of S coelicolor using blastn, presence of a divergent homologue of the same gene. limiting the output to the best match This E value. Finally although the order of the genes in S coelicolor. dataset for the genes was then aligned with the,and S avermitilis are known from their complete. S avermitilis microarray dataset and a comparison,genome sequences and are well conserved this does. plotted as a scatterplot Genes showing disagree, not mean that the synteny of most of them is conserved.
ment between the two datasets were identified, in other Streptomyces species However the detection. based on a 2 Standard Deviation SD cutoff for, of synteny across Actinobacteria including Mycobac. the microarray dataset and a E 10 cutoff for the, terium tuberculosis Corynebacteriun glutamicum and. blast value,other species Bentley et al 2002 and unpublished. data supports a conserved central core structure to the. Analysis of gene presence across the chromosome genomes of the Actinomycetes and a priori most. Streptomyces Thus although major chromosomal, A graphical display was created by counting the reorganizations in the central core region cannot be.
number of gene detected as present from the signal detected by microarray data a basic chromosomal. based on the 2SD cutoff from each normalized structure can be assumed as a first approximation. microarray dataset using a moving window of 10 namely a linear chromosome with variable terminal. genes in steps of one regions and a relatively well conserved core region. Antonie van Leeuwenhoek 2008 93 1 25 5, When the pooled data from the two arrays for Fig 1 Gene differences were present in the order. the four species was analyzed using Cy 3 labeled S cattleya K aureofaciens S avermitilis. used to compare the genome content of Streptomyces avermitilis Streptomyces cattleya Streptomyces maritimus and Kitasatospora aureofaciens with that of Streptomyces coelicolor A3 2 The array data showed an about 93 agreement with the genome sequence data available for S avermitilis and also showed a number of trends in the genome structure

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