Non-contiguous finished genome sequence of Aminomonas paucivorans type strain (GLU-3)

Aminomonas paucivorans Baena et al. 1999 is the type species of the genus Aminomonas, which belongs to the family Synergistaceae. The species is of interest because it is an asaccharolytic chemoorganotrophic bacterium which ferments quite a number of amino acids. This is the first finished genome sequence (with one gap in a rDNA region) of a member of the genus Aminomonas and the third sequence from the family Synergistaceae. The 2,630,120 bp long genome with its 2,433 protein-coding and 61 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.


Introduction
Strain GLU-3 T (= DSM 12260 = ATCC BAA-6) is the type strain of the species Aminomonas paucivorans, which in turn is the type and only species of the genus Aminomonas [1,2]. The generic name derives from the Latin word 'aminum' meaning 'amine' and the Greek word 'monas' meaning 'a unit or monad', referring to amine-degrading monads [2]. The species epithet is derived from the Latin word 'paucus' meaning 'few or little' and the Latin word 'vorans' meaning 'digesting', referring to digesting little [2]. Strain GLU-3 T was isolated from anaerobic sludge of a dairy wastewater treatment plant in Santafé de Bogota, Colombia [2]. So far, no further isolates have been obtained for A. paucivorans. Here we present a summary classification and a set of features for A. paucivorans GLU-3 T , together with the description of the non-contiguous finished genomic sequencing and annotation.

Classification and features
The 16S rRNA gene of A. paucivorans GLU-3 T shares 96% sequence identity with that of the type strain of Thermanaerovibrio acidaminovorans, which was isolated from an upflow anaerobic sludge bed reactor of a sugar refinery, Breda, the Netherlands [3] (Figure 1), and 82.2-96.4% sequence identity with the type strains from the other members of the family Synergistaceae [11].
The sequences of four marine metagenomic clones in the env_nt database, 1096626071844 (AACY020063505), 1096626840052 (AA-CY020539193), 1096626748225 (AACY020105546) and 1096626774924 (AACY020274567) share 96% sequence identity with A. paucivorans GLU-3 T (as of October 2010). A representative genomic 16S rRNA sequence of A. paucivorans was compared using NCBI BLAST under default values with the most recent release of the Greengenes database [12] and the relative frequencies of taxa and keywords, weighted by BLAST scores, were determined. The four most frequent genera were Thermanaerovibrio (65.5%), Aminomonas (18.0%), Anaerobaculum (9.0%) and Aminiphilus (7.6%). The species yielding the highest score was T. acidaminovorans. The five most frequent keywords within the labels of environmental samples which yielded hits were 'anaerobic' (7.2%), 'sludge' (6.9%), 'wastewater' (6.8%), 'municipal' (6.8%) and 'digester' (6.7%). These keywords corroborate the physiological and ecological features on strain GLU-3 T as depicted in the original description [2]. The single most frequent keyword within the labels of environmental samples which yielded hits of a higher score than the highest scoring species was 'harbor/sediment' (50.0%). Figure 1 shows the phylogenetic neighborhood of A. paucivorans GLU-3 T in a 16S rRNA based tree. The sequences of the three 16S rRNA gene copies in the genome of A. paucivorans differ from each other by up to one nucleotide, and differ by up to eleven nucleotides from the previously published 16S rRNA sequence (AF072581), which contains 59 ambiguous base calls (ambiguous bases not count as differences).

Genome project history
This organism was selected for sequencing on the basis of its phylogenetic position [18], and is part of the Genomic Encyclopedia of Bacteria and Archaea project [19]. The genome project is deposited in the Genome OnLine Database [8,20] and the non-contiguous finished genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession AEIV00000000. The version described in this paper is the first version, AEIV01000000. Sequencing, finishing and annotation were performed by the DOE Joint Genome Institute (JGI). A summary of the project information is shown in Table 2.  [4,5] of the 16S rRNA gene sequence under the maximum likelihood criterion [6] and rooted in accordance with the current 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 [7] if larger than 60%. Lineages with type strain genome sequencing projects registered in GOLD [8] are shown in blue, published genomes in bold [3,9,10].
A. paucivorans GLU-3 T is described as Gramnegative, slightly curved, rod-shaped bacterium (0.3 × 4.0-6.0 µm), which occurs singly or in pairs ( Figure 2 and Table 1). Colonies of strain GLU-3 T are round, smooth and white, with a diameter up to 1 mm [2]. Strain GLU-3 T does not produce endospores [2]. The organism does not have flagella and motility is not observed [2], although plenty of motility genes are present in the genome. Strain GLU-3 T is a strictly anaerobic, mesophilic, chemoorganotrophic and asaccharolytic bacterium [2]. The temperature range for growth is 20-40°C, with an optimum at 35°C [2]. The pH range for growth is 6.7-8.3, with an optimum at 7.5 [2]. The organism does not require NaCl for growth but tolerates up to 2.0% [2]. The optimum growth occurs in media with 0.05-0.5% of NaCl [2]. The species requires yeast extract for growth [2]. The organism is able to ferment arginine, histidine, glutamine, threonine, and glycine [2]. Arginine is fermented to acetate, formate and ornithine [2]. Histidine is fermented to acetate and formate [2]. Glutamate is fermented to acetate, formate and trace amounts of propionate [2]. Threonine and glycine are fermented to acetate [2]. Casamino acid, peptone and cysteine are only poorly used by the strain GLU-3 T , and acetate is the end-product of the amino acid metabolism [2]. A mixed culture of strain GLU-3 T and Methanobacterium formicicum does not extend the range of substrate utiliza-tion [2], as is observed for, e.g., Aminobacterium colombiense [9]. Methane is not detectable in mixed cultures, when grown in glycine and threonine [2], however, the end-product profiles are the same as in pure culture [2]. The major endproduct is shifted from acetate to propionate, when strain GLU-3 T was grown together with M. formicicum on arginine, histidine and glutamate [2]. Ornithine is not accumulated during arginine degradation in mixed culture [2]. Strain GLU-3 T does not degrade alanine and branched-chain amino acids, valine, leucine and isoleucine either in pure culture or in syntrophic growth with M. formicicum [2]. Also, the range of amino acid utilization is not increased in co-culture with M. formicicum [2]. Strain GLU-3 T does not grow on carbohydrates, gelatin, casein, pyruvate, succinate, malate, fumarate, α-ketoglutarate, mesaconate, βmethylaspartate, oxaloacetate, glycerol, ethanol, acetate, propionate, butyrate, lactate, citrate, leucine, lysine, alanine, valine, proline, serine, methionine, asparagines, phenylalanine and aspartate [2]. The organism does not utilize sulfate, thiosulfate, elemental sulfur, sulfite, nitrate and fumarate as electron acceptors [2].

Chemotaxonomy
No chemotaxonomic data are currently available for A. paucivorans or for other members of the genus Aminomonas.  , not directly observed for the living, isolated sample, but based on a generally accepted property for the species, or anecdotal evidence). These evidence codes are from of the Gene Ontology project [17]. If the evidence code is IDA, then the property was directly observed by one of the authors or an expert mentioned in the acknowledgements.

Growth conditions and DNA isolation
A. paucivorans GLU-3 T , DSM 12260, was grown anaerobically in DSMZ medium 846 (Anaerobic Serine/Arginine medium) [21] at 37°C. DNA was isolated from 0.5-1 g of cell paste using the Mas-terPure Gram-positive DNA purification kit (Epi-centre MGP04100) following the standard protocol as recommended by the manufacturer, with modification st/LALM for cell lysis as described in Wu et al. [19].

Genome sequencing and assembly
The genome was sequenced using a combination of Illumina and 454 sequencing platforms. All general aspects of library construction and sequencing can be found at the JGI website. Pyrosequencing reads were assembled using the Newbler assembler version 2.0.00.20-PostRelease-11-05-2008-gcc-3.4.6 (Roche). The initial Newbler assembly consisted of 126 contigs in 103 scaffolds and was converted into a phrap assembly by making fake reads from the consensus for collecting the read pairs in the 454 paired end library. Illumina GAii sequencing data (525.3 Mb) was assembled with Velvet [22] and the consensus sequences were shredded into 1.5 kb overlapped fake reads and assembled together with the 454 data. The 454 draft assembly was based on 190.7 Mb 454 draft data and all of the 454 paired end data. Newbler parameters were -consed -a 50 -l 350 -g -m -ml 20. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment in the subsequent finishing process. After the shotgun stage, reads were assembled with parallel phrap (High Performance Software, LLC). Possible mis-assemblies were corrected with ga-pResolution, Dupfinisher, or sequencing cloned bridging PCR fragments with subcloning or transposon bombing (Epicentre Biotechnologies, Madison, WI) [23]. Gaps between contigs were closed by editing in Consed, by PCR and by Bubble PCR primer walks (J.-F.Chang, unpublished). A total of 259 additional reactions were necessary to close gaps and to raise the quality of the finished sequence. Illumina reads were also used to correct potential base errors and increase consensus quality using a software (Polisher) developed at JGI [24]. The error rate of the completed genome sequence is less than 1 in 100,000. Together, the combination of the Illumina and 454 sequencing platforms provided 274.4× coverage of the genome. The final assembly contained 535,052 pyrosequences and 15,007,632 Illumina reads.

Genome annotation
Genes were identified using Prodigal [25] as part of the Oak Ridge National Laboratory genome annotation pipeline, followed by a round of manual curation using the JGI GenePRIMP pipeline [26]. The predicted CDSs were translated and used to search the National Center for Biotechnology Information (NCBI) nonredundant database, Uni-Prot, TIGR-Fam, Pfam, PRIAM, KEGG, COG, and In-terPro databases. Additional gene prediction analysis and functional annotation was performed within the Integrated Microbial Genomes -Expert Review (IMG-ER) platform [27].

Genome properties
The genome consists of a 2,630,120 bp long chromosome with an overall GC content of 67.6% (Table 3 and Figure 3). Of the 2,494 genes predicted, 2,433 were protein-coding genes, and 61 RNAs; 34 pseudogenes were also identified. The majority of the protein-coding genes (77.2%) 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. Standards in Genomic Sciences