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Saccharomonospora viridis (Schuurmans et al. 1956) Nonomurea and Ohara 1971 is the type species of the genus Saccharomonospora which belongs to the family Pseudonocardiaceae. S. viridis is of interest because it is a Gram-negative organism classified among the usually Gram-positive actinomycetes. Members of the species are frequently found in hot compost and hay, and its spores can cause farmer’s lung disease, bagassosis, and humidifier fever. Strains of the species S. viridis have been found to metabolize the xenobiotic pentachlorophenol (PCP). The strain described in this study has been isolated from peat-bog in Ireland. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first complete genome sequence of the family Pseudonocardiaceae, and the 4,308,349 bp long single replicon genome with its 3906 protein-coding and 64 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.
Strain P101T (= DSM 43017 = ATCC 15386 = JCM 3036 = NCIMB 9602) is the type strain of Saccharomonospora viridis, and the type species of the genus Saccharomonospora [1,2], which currently contains eight species . Although phylogenetically a member of the Gram-positive actinomycetes, already the initial report on S. viridis strain P101T noticed the astonishing feature of the organism to be Gram-negative, despite showing the typical mycelium morphology of Saccharomonospora . Like in other actinomycetes, spores of S. viridis are readily dispersed in air, and the prolonged exposure to spores can apparently result in acute respiratory distress (farmer’s lung disease) which may lead to irreversible lung damage [4,5]. Here we present a summary classification and a set of features for S. viridis P101T, together with the description of the complete genomic sequencing and annotation.
Members of the species S. viridis have been isolated or molecularly identified on several occasions from hot composts in Europe and USA [12-14,17], and also from soil in Japan . One novel, yet unpublished, cultivated member of the species has been reported by Lu and Liu from Chinese soil (AF127525). Uncultured clone sequences with significant (99%) sequence similarity were observed from composting mass in China (AM930281 and AM930338). Screening of environmental genomic samples and surveys reported at the NCBI BLAST server indicated no closely related phylotypes that can be linked to the species or genus, with the closest matches (about 90% sequence similarity) to strain P101T 16S rRNA identified in a marine metagenome from the Sargasso Sea .
Figure 1 shows the phylogenetic neighborhood of S. viridis strain P101T in a 16S rRNA based tree. The sequences of all three copies of the 16S rRNA gene are identical and perfectly match the previously published 16S rRNA sequence generated from NCIMB 9602 (Z38007).
The hyphae of the vegetative mycelium of strain P101T are branched and sometimes show curved endings . Single spores are observed only on the aerial mycelium either directly on the hyphae or on short sporophores (Table 1 and Figure 2). The spores are oval, 0.9-1.1 µm × 1.2-1.4 µm in size. Only very occasionally two spores are observed. The aerial mycelium is either grayish green in color, or turns from white to greenish as on Czapek Agar. The optimal temperature for growth is 55°C, but 45°C for aerial mycelium formation and pigment production. At 37°C and 60°C the growth is very limited and without aerial mycelia. No growth occurs at 27°C and 70°C .
Strain P101T has been observed to be sensitive to a variety of phages . Members of S. viridis are apparently able to metabolize pentachlorophenol but not other chlorophenols . It was suggested that S. viridis metabolizes PCP by conjugation to form a more polar transformation product, but, unlike other PCP-degrading bacteria, the organism is incapable of effecting total degradation of the xenobiotic . Microorganisms such as S. viridis may therefore contribute to PCP removal by microbial communities in situ, despite being unable to completely mineralize chlorophenols in pure culture . S. viridis produces a thermostable α-amylase which forms 63% (w/w) maltose on hydrolysis of starch . Maltotriose and maltotetraose are the only intermediate products observed during this reaction, with maltotriose accumulating to 40% (w/w). Both unimolecular and multimolecular mechanisms (transfers and condensation) have been shown to occur during the concentration-dependent degradation of maltotriose and maltotetraose. Such reactions result in the almost exclusive formation of maltose from maltotriose at high initial concentration . S. viridis produces thermoviridin, an antibiotic that is primarily active against the Gram-positive bacteria (growth inhibition) [2,11]. At higher concentrations, also Gram-negative bacteria were growth-inhibited .
The murein of P101T is of cell wall type IV. It contains meso-diaminopimelic acid in the peptidoglycan and arabinose and galactose in whole-cell hydrolysates (sugar type A). Mycolic acids and teichonic acids were not reported. Strain P101T contains menaquinones MK-9(H4) (60%) and MK-8(H4) (20 to 30%). The combination of the tetrahydromultiprenyl menaquinones MK-9(H4) and MK-8(H4) is characteristic for the genus Saccharomonospora . The major cellular fatty acids are saturated, iso-branched acids with 16 and 18 carbon atoms, and 2-hydroxydodecanoic acids. Details are described in the Compendium of Actinobacteria . Phosphatidylethanolamine, hydroxy-phosphatidyl-ethanolamine, and lyso-phosphatidyl-ethanolamine were identified as the main phospholipids.
This organism was selected for sequencing on the basis of its phylogenetic position, and is part of the Genomic Encyclopedia of Bacteria and Archaea project. The genome project is deposited in the Genome OnLine Database  and the complete genome sequence in GenBank. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.
S. viridis strain P101T, DSM 43017, was grown in DSMZ medium 535 (Trypticase soy broth, ) at 45°C. DNA was isolated from 1-1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with a modified protocol, st/FT, for cell lysis, as described in Wu et al. .
The genome was sequenced using Sanger sequencing platform only. All general aspects of library construction and sequencing can be found at the JGI website (http://www.jgi.doe.gov). The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment. After the shotgun stage reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher  or transposon bombing of bridging clones (Epicentre Biotechnologies, Madison, WI). Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification (Roche Applied Science, Indianapolis, IN). A total of 354 finishing reactions were produced to close gaps and to raise the quality of the finished sequence. The completed genome sequences of S. viridis contains 66,210 Sanger reads, achieving an average of 12.9 sequence coverage per base, with an error rate less than 1 in 100,000.
Genes were identified using GeneMark  as part of the genome annotation pipeline in the Integrated Microbial Genomes Expert Review (IMG-ER) system , followed by a round of manual curation using the JGI GenePRIMP pipeline (http://geneprimp.jgi-psf.org) . The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, UniProt, TIGRFam, Pfam, PRIAM, KEGG, COG, and InterPro databases. The tRNAScanSE tool  was used to find tRNA genes, whereas ribosomal RNAs were found by using the tool RNAmmer . Other non coding RNAs were identified by searching the genome for the Rfam profiles using INFERNAL (v0.81) . Additional gene prediction analysis and manual functional annotation was performed within the Integrated Microbial Genomes (IMG) platform (http://img.jgi.doe.ogv/er) .
The metabolic Pathway/Genome Database (PGDB) was computationally generated using Pathway Tools software version 12.5  and MetaCyc version 12.5 , based on annotated EC numbers and a customized enzyme name mapping file. It has undergone no subsequent manual curation and may contain errors, similar to a Tier 3 BioCyc PGDB .
The genome is 4,308,349 bp long and comprises one main circular chromosome with a 67.3% GC content (Table 3 and Figure 3). Of the 3,970 genes predicted, 3,906 were protein coding genes, and 64 RNAs; 78 pseudogenes were also identified. The majority of the protein-coding genes (71.2%) were assigned with a putative function, while the remaining ones were annotated as having hypothetical function. The properties and the statistics of the genome are summarized in Table 3. The distribution of genes into COGs functional categories is presented in Table 4 and a cellular overview diagram is presented in Figure 4, followed by a summary of metabolic network statistics shown in Table 5.
We would like to gratefully acknowledge the help of Marlen Jando for growing S. viridis cultures and Susanne Schneider for DNA extraction and quality analysis (both at DSMZ). This work was performed under the auspices of the US Department of Energy's Office of Science, Biological and Environmental Research Program, and by the University of California, Lawrence Berkeley National Laboratory under contract No. DE-AC02-05CH11231, Lawrence Livermore National Laboratory under Contract No. DE-AC52-07NA27344, and Los Alamos National Laboratory under contract No. DE-AC02-06NA25396, as well as German Research Foundation (DFG) INST 599/1-1 and SI 1352/1-1.