Complete genome sequence of Actinosynnema mirum type strain (101T)

Actinosynnema mirum Hasegawa et al. 1978 is the type species of the genus, and is of phylogenetic interest because of its central phylogenetic location in the Actino-synnemataceae, a rapidly growing family within the actinobacterial suborder Pseudo-nocardineae. A. mirum is characterized by its motile spores borne on synnemata and as a producer of nocardicin antibiotics. It is capable of growing aerobically and under a moderate CO2 atmosphere. The strain is a Gram-positive, aerial and substrate mycelium producing bacterium, originally isolated from a grass blade collected from the Raritan River, New Jersey. Here we describe the features of this organism, together with the complete genome sequence and annotation. This is the first complete genome sequence of a member of the family Actinosynnemataceae, and only the second sequence from the actinobacterial suborder Pseudonocardineae. The 8,248,144 bp long single replicon genome with its 7100 protein-coding and 77 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain 101 T (DSM 43827 = ATCC 29888 = NBRC 14064, and other culture collections) is the type strain of Actinosynnema mirum, which is the type species of the genus Actinosynnema [1] (Figure 1). A. mirum was described by Hasegawa et al. in 1978 [1] as an aerobic actinobacterium which forms synnemata (compacted groups of erect hyphae which bear conidia) with zoospores [1]. The organism is of interest due to its position in the tree of life where the small genus Actino-synnema, currently comprising only two species, is located on a rather long branch within the rapidly growing actinobacterial suborder Pseudonocardineae [2]. We here present a summary classification and a set of features for A. mirum strain 101 T (Table 1), together with the description of the complete genomic sequencing and annotation.

Classification and features
No closely related cultivated strains are known from the literature that can be linked to the species A. mirum. Curiously, the 16S rRNA gene sequences of the type strains from the two subspecies within the second species of the genus Actinosynnema, A. pretiosum subsp. auranticum (AB303364) and A. pretiosum subsp. pretiosum (AB303365) [3], seem to have an equally or even higher degree of similarity to the 16S rRNA gene sequence derived from the genome sequence reported here than the previously reported gene sequences of strain 101 T (see Figure 1). None of the phylotypes reported from environmental screenings or genomic surveys could be linked to A. mirum with a convincing degree of sequence similarity (maximal observed degree of similarity 92%; status June 2009).
A. mirum strain 101 T cells are non-motile with fine hyphae which form aerial and substrate mycelia. Both the aerial and substrate mycelia are about 0.5 to 1.0 µm in diameter. Aerial mycelia are long branching hyphae, white to pale yellow in color ( Figure 2). The substrate mycelia are also long, branching hyphae, white to yellowish orange, and penetrate into the agar medium and form synnemata [1]. Cells stain Gram-positive and are non-acid fast [1].
A. mirum is capable of producing a yellowishbrown soluble pigment on tyrosine agar and a pale greenish pigment on oatmeal agar [1]. Capable of hydrolyzing starch, casein, tyrosine and gelatin, but not xanthine, hypoxanthine, adenine and urea [1]; produces nitrate reductase and phosphatase. Positive for utilization of tartrate, pyruvate, lactate and malate, but negative for benzoate, acetate, citrate and succinate [1]. Acid is produced aerobically from fructose, lactose, maltose, D-mannitol, L-arabinose, D-melibiose, Dmannose, L-rhamnose, xylose, dextrin, galactose, glucose, trehalose, raffinose, starch, sucrose, cellobiose, glycogen and adonitol, but not from inositol, sorbitol, D-ribose, salicin, inulin, glycerol, dulcitol, erythritol, α-methyl-D-glucoside and αmethyl-D-mannoside. A. mirum is a producer of nocardicin antibiotics [4] and inhibits the growth of several Gram-positive bacteria including: Bacillus megaterium, Sarcina lutea, Mycobacterium smegmatis; as well as the filamentous fungi, Aspergillus niger, Penicillium notatum and the yeasts, Saccharomyces cerevisiae and Candida tropicalis. Figure 1 shows the phylogenetic neighborhood of A. mirum strain 101 T in a 16S rRNA based tree. The sequences of the five 16S rRNA genes in the A. mirum genome differ by no more than one nucleotide (nt) from each other, and by up to six nts from the previously reported reference sequences derived from NBRC 14064 (AF328679) and from DSM 43827 (X84447). The differences between the genome data and the previously reported 16S rRNA gene sequence are probably due to sequencing errors in the previously reported sequence data.

Figure 1.
Phylogenetic tree highlighting the position of A. mirum 101 T relative to all type strains of the genus and to the type strains of the type species of all other genera within the family. The tree was inferred from 1,491 aligned characters [5,6] of the 16S rRNA gene sequence under the maximum likelihood criterion [7] and rooted in accordance with current actinobacterial taxonomy. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 1,000 bootstrap replicates if larger than 60%. Lineages with a type strain genome-sequencing project registered in GOLD [8] are printed in blue; published genomes in bold.

Genome sequencing and annotation
Genome project history 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 Genomes OnLine Database [8] and the complete genome sequence in GenBank (CP001630). Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.

Growth conditions and DNA isolation
A. mirum strain 101 T , DSM 44827, was grown in DSMZ medium 535 (GYM Streptomyces Medium at 28°C. DNA was isolated from 1-1.5 g of cell paste using Qiagen Genomic 500 DNA Kit (Qiagen, Hilden, Germany) with a modified lysis buffer (1 ml achromopeptidase and 0.5 ml lysostaphin added) and one hour incubation at 37°C.

Genome sequencing and assembly
The genome was sequenced using a combination of Sanger and 454 sequencing platforms. All general aspects of library construction and sequencing performed at the JGI can be found on the JGI website. 454 Pyrosequencing reads were assembled using the Newbler assembler version 1.1.02.15 (Roche). Large Newbler contigs were broken into 10,493 overlapping fragments of 1,000 bp and entered into assembly as pseudoreads. The sequences were assigned quality scores based on Newbler consensus q-scores with modifications to account for overlap redundancy and to adjust inflated q-scores. A hybrid 454/Sanger assembly was made using the phrap assembler (High Performance Software, LLC). Possible mis-assemblies were corrected with Dupfinisher or transposon bombing of bridging clones [16]. Gaps between contigs were closed by editing in Consed, custom primer walk or PCR amplification. 1,564 Sanger finishing reads were produced to close gaps and to raise the quality of the finished sequence. The error rate of the completed genome sequence is less than 1 in 100,000. Together all sequence types provided 28.9x coverage of the genome. The final assembly contains 105,508 Sanger reads in addition to the 454 based pseudo reads.

Genome annotation
Genes were identified using Prodigal [17] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [18]. 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. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes (IMG-ER) platform [19].

Genome properties
The genome is 8,248,144 bp long and comprises one circular chromosome with a 73.7% GC content (Table 3 and Figure 3). Of the 7,174 genes predicted, 7100 were protein coding genes, and 74 RNAs., One hundred and eight four pseudogenes were also identified. The majority of genes (67.3%) of the genes were assigned a putative function while the remaining ones were annotated as hypothetical proteins. 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.