Strain 7 KIP^T (= DSM 43043 = ATCC 23552 = JCM 3097) is the type strain of the species Intrasporangium calvum, which is the type species of its genus Intrasporangium [1,2]. The generic name derived from the Latin word intra meaning within and the Greek word spora meaning a seed. The name Intrasporangium, was selected to emphasize the possibility of intercalary formation of sporangia in mycelial filaments [3]. Intrasporangium is the type genus of the family Intrasporangiaceae and one out of currently nineteen genera in the family Intrasporangiaceae [4-6]. Strain 7 KIP^T was first described in 1967 by Kalakoutskii et al. as an airborne organism, which was isolated under nonselective conditions on plates of meat-peptone agar exposed to the atmosphere of a school dining room [1,7,8]. I. calvum is of particular interest because the type of its menaquinones is different from all other representatives of the family Intrasporangiaceae [8]. Here we present a summary classification and a set of features for I. calvum 7 KIP^T, together with the description of the complete genomic sequencing and annotation.

The 16S rRNA gene of strain 7 KIP^T shares 92.6-98.7% sequence identity with the sequences of the type strains from the other members of the family Intrasporangiaceae [9], with Humihabitans oryzae as the closest relative. The 16S rRNA gene sequence of 7 KIP^T is 99% identical to the uncultured Intrasporangiaceae clone HT06Ba24, isolated from soil of a former coal gasification site in Gliwice, Poland [10,11] and AKAU4164, isolated from uranium contaminated soil in Oak Ridge, USA [10,12]. The environmental samples database (env_nt) contains the marine metagenome clone 1096626841081 (AACY020552144) from surface water (92% sequence identity with 7 KIP^T). The genomic survey sequences database (gss) contains the metagenomic clone 1061002660518 from Floreana island in Punta Cormorant, Ecuador [10], which shares 93% sequence identity with 7 KIP^T (as of July 2010). One of the 16S rRNA sequences of strain 7 KIP^T was compared using NCBI BLAST under default values (e.g., considering only the best 250 hits) with the most recent release of the Greengenes database [13] and the relative frequencies, weighted by BLAST scores, of taxa and keywords, weighted by BLAST scores, were determined. The five most frequent genera were Janibacter (29.6%), Terrabacter (19.8%), Sanguibacter (8.4%), Dermacoccus (7.7%) and Tetrasphaera (6.2%). The five most frequent keywords within the labels of environmental samples which yielded hits were 'skin' (9.1%), 'human' (4.7%), 'microbiome/temporal/topographical' (4.5%), 'sludge' (4.4%) and 'heel/plantar' (3.1%). The single most frequent keyword within the labels of environmental samples which yielded hits of a higher score than the highest scoring species was 'contaminated/soil/uranium' (33.3%).

Figure 1 shows the phylogenetic neighborhood of I. calvum 7 KIP^T in a 16S rRNA based tree. The sequences of the two 16S rRNA gene copies in the genome are differ by only one nucleotide from each other and by up to one nucleotide from the previously published sequence generated from DSM 43043 (AJ566282).

Figure 1 Phylogenetic tree highlighting the position of I. calvum 7 KIPT relative to the type strains of the genera within the family Intrasporangiaceae. The trees were inferred from 1,406 aligned characters [14,15] of the 16S rRNA gene sequence under the maximum likelihood criterion [16] and rooted with the type strains of the genera within the family Kineosporiaceae [17]. The branches are scaled in terms of the expected number of substitutions per site. Numbers above branches are support values from 650 bootstrap replicates [18] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [19] are shown in blue, published genomes in bold [20].

Strain 7 KIP^T forms a branching mycelium, which tends to break into irregular fragments, i.e., typically nocardioform [1,8]. The mycelium may bear intercalary vesicles that do not contain spores [Table 1, Figure 2, 7,26]. The vesicles of strain 7 KIP^T are ovoid and lemon-shaped (5-15 µm in diameter) [1,7]. Several round or oval bodies (1.2-1.5 µm in diameter) may be observed in the vesicles of older cultures [1,7]. The oval bodies in the vesicles of strain 7 KIP^T are nonmotile but may undergo a Brownian movement (in mature vesicles) [1,7]. There was no aerial mycelium observed from the strain 7 KIP^T [1,7,8]. The mycelial filaments penetrate the agar and form compact, small colonies (1-5 mm of diameter) [1]. These colonies are round, glistening and whitish (cream-whitish in old colonies) when the cells are grown on meat-extract peptone agar [1]. Strain 7 KIP^T is aerobic and Gram-positive (Gram-variable in old cultures) and not acid-fast [1]. Strain 7 KIP^T is rather fastidious in nutritional requirements [1]. Growth is seemingly dependent on some unidentified substances present in the peptone used in the growth medium [1]. The strain prefers complex media for growth, especially containing peptone and yeast extract [1,7]. As such, the growth characteristics on a variety of media such as meat-extract peptone, blood serum broth, oatmeal agar, Sauton medium agar and other media, also in combination of different atmospheric gases and their concentrations, have been studied in detail [1]. Strain 7 KIP^T is able to grow between 28°C and 37°C, however, the cells grow faster at 37°C than 28°C, but it does not at 45°C [1]. It grows slowly on meat-extract peptone medium and the first signs of macroscopic growth will appear after 3-5 days when incubated at 28°C [1]. Strain 7 KIP^T does not grow on the majority of synthetic mineral media that are routinely used for actinomycetes [1,7]. Strain 7 KIP^T is able to reduce nitrate to nitrite when KNO₃ is added to the growth medium (meat-extract peptone broth) [1]. The liquefaction of gelatin does not occur when the strain 7 KIP^T was grown on meat-extract peptone gelatine [1]. Strain 7 KIP^T has no antibiotic activity against Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Bacillus subtilis, Candida albicans and Mycobacterium sp. v-5 [1]

Figure 2 Scanning electron micrograph of I. calvum 7 KIPT

The genome consists of a 4,024,382 bp long chromosome with a 70.7% GC content (Table 3 and Figure 3). Of the 3,710 genes predicted, 3,653 were protein-coding genes, and 57 RNAs; ninety pseudogenes were also identified. The majority of the protein-coding genes (71.3%) were assigned with a putative function while the remaining ones were annotated as hypothetical proteins. The distribution of genes into COGs functional categories is presented in Table 4.

Figure 3 Graphical circular map of the genome. From outside to the center: Genes on forward strand (color by COG categories), Genes on reverse strand (color by COG categories), RNA genes (tRNAs green, rRNAs red, other RNAs black), GC content, GC skew.