Complete genome sequence of Kosakonia sacchari type strain SP1T

Kosakonia sacchari sp. nov. is a new species within the new genus Kosakonia, which was included in the genus Enterobacter. K sacchari is a nitrogen-fixing bacterium named for its association with sugarcane (Saccharum officinarum L.). K sacchari bacteria are Gram-negative, aerobic, non-spore-forming, motile rods. Strain SP1T (=CGMCC1.12102T=LMG 26783T) is the type strain of the K sacchari sp. nov and is able to colonize and fix N2 in association with sugarcane plants, thus promoting plant growth. Here we summarize the features of strain SP1T and describe its complete genome sequence. The genome contains a single chromosome and no plasmids, 4,902,024 nucleotides with 53.7% GC content, 4,460 protein-coding genes and 105 RNA genes including 22 rRNA genes, 82 tRNA genes, and 1 ncRNA gene.


Organism information Classification and general features
K. sacchari type strain SP1T is a Gram-negative, non-spore-forming, motile rod with peritrichous flagella (Figure 1., Table 1. [2]). It grows aerobically but reduces N2 to NH3 at a low pO2. It is able to grow and fix N2 on media containing 10% (w/v) cane sugar or sucrose and forms circular, convex, smooth colonies with entire margins on solid media. It grows best around 30°C and pH 7. Phylogenetic analysis of the 16S rRNA gene sequences from SP1T, the type strains of species of the genus Enterobacter and the type strains of type species of other genera in the family Enterobacteriaceae showed that SP1T formed a monophyletic group with the type strain of E. cloacae (the type species of the genus Standards in Genomic Sciences Enterobacter) [2]. However, phylogenetic analysis of the rpoB gene sequences showed that SP1T diverged from E. cloacae [2]. Here, phylogenetic analysis of the 16S rRNA gene sequences from SP1T, other type strains in the genus Kosakonia, and the type strain of E. cloacae showed that K. sacchari formed a monophyletic group with K. radicincitans, K. oryzae, and K. arachidis and diverged from K. cowanii (the type species of the genus Kosakonia) and E. cloacae (Figure 2.). Like typical members in the genera Enterobacter and Kosakonia, K. sacchari SP1T utilizes L-alanine, D-cellobiose, citrate, D-fructose, D-galactose, Dglucose, glycerol, maltose, D-mannitol and Dmannose [2,6,7]. K. sacchari differentiates from E. cloacae by utilization of D-arabitol and L-fucose, differentiates from K. radicincitans by utilization of putrescine, D-arabitol, L-fucose and α-methyl-Dglucoside, and differentiates from K. oryzae by utilization of putrescine, D-arabitol and Lrhamnose [2].  aligned with the CLUSTAL W program and were constructed with the neighbor-joining algorithm integrated in the MEGA 5.0 program [5]. The phylogenetic tree was tested with 1,000 bootstrap replicates. Bootstrap values are shown at the nodes. The GenBank accession numbers of the sequences are indicated in parentheses. The scale bar represents a 0.1% nucleotide sequence divergence.

Genome sequencing information
Genome project history K. sacchari SP1 T was selected for sequencing because it is the type strain of K. sacchari, and on the basis of its scientific interest as an endophyte that has the potential to promote the growth of agriculturally important crops by nitrogen fixation [8].

Genome annotation
). A 4 -10 Kb insert library was constructed. Sequencing was run on a single SMRT Cell. The sequencing data were assembled using the Hierarchical Genome Assembly Process (HGAP) with smrtanalysis-2.1.1. The final assembly of the chromosome produced 63-fold coverage of the genome.
Automated genome annotation was completed using the NCBI Prokaryotic Genome Annotation Pipeline. Product description annotations were obtained using searches against the KEGG, InterPro, and COG databases. Genes with signal peptides were predicted using SignalP [9]. Genes with transmembrane helices were predicted using TMHMM [10]. Genes for tRNA were found by tRNAScanSE [11]. Ribosomal RNAs were found by using BLASTN vs. ribosomal RNA databases, and 5S rRNA hits were further refined using Cmsearch (http://manpages.ubuntu.com/manpages/rari ng/man1/cmsearch.1.html
Two hundred twenty seven disrupted genes were replaced by the complete gene sequences obtained from the first Illumina HiSeq 2000 sequencing.
The genome of K. sacchari SP1 T contains a single chromosome of 4,902,024 nucleotides with 53.7% GC content and no plasmids (Table 3, Figure 3.). The genome contains 4,585 predicted genes, 4,460 protein-coding genes and 105 RNA genes including 22 rRNA genes, 82 tRNA genes and 1 ncRNA gene. A total of 3,752 genes (81.8%) were assigned a putative function. The remaining genes were annotated as hypothetical or unknown proteins ( Table 3). The distribution of genes into COGs functional categories is presented in Table 4 .   Insights from the genome.

Comparison with the genome of Enterobacter sp. strain R4-368
The chromosome of K. sacchari SP1 T shows the highest sequence similarities ranging from 69.5% to 100% to the chromosome of Enterobacter sp.
strain R4-368, which is an endophytic nitrogenfixing bacterium isolated from the biofuel plant Jatropha curcas [13]. The genome of the strain R4-368 comprises a single circular chromosome of 5,039,027 bp with 54.0% GC content (deposited in GenBank under the accession number CP005991) and one plasmid pENT01 of 116,007 bp with 52.8% GC content (deposited in GenBank under the accession number CP005992) [13].