Genome Sequence of the Ethanol-Producing Zymomonas mobilis subsp. mobilis Lectotype Strain ATCC 10988

Abstract

Zymomonas mobilis ATCC 10988 is the type strain of the Z. mobilis subsp. mobilis taxon, members of which are some of the most rigorous ethanol-producing bacteria. Isolated from Agave cactus fermentations in Mexico, ATCC 10988 is one of the first Z. mobilis strains to be described and studied. Its robustness in sucrose-substrate fermentations, physiological characteristics, large number of plasmids, and overall genomic plasticity render this strain important to the study of the species. Here we report the finishing and annotation of the ATCC 10988 chromosomal and plasmid genome.

GENOME ANNOUNCEMENT

Zymomonas mobilis is a natural fermentative bacterium that has been studied for decades due to its remarkable ethanologenic properties. Among isolates deriving from various parts of the world, ATCC 10988, identified in the 1920s in Mexican pulque fermentations (initially called “Thermobacterium mobile”) (17), is one of the longest-studied Zymomonas strains and has served for most biochemical and physiological works on the organism. ATCC 10988 exhibits fermenting performances that equal that of industrial strain CP4 in sucrose-rich media (15) and has also attracted attention due to its split genome: it bears multiple different plasmids, six of which are reported in this work, while the smallest have been cloned and sequenced in earlier works (2, 10, 12). Most species-specific vectors constructed and used for Z. mobilis, indeed, derive from the small multicopy plasmids of ATCC 10988 (16, 19, 21).

Total and plasmid DNA from ATCC 10988 was prepared (11) and used for whole-genome shotgun sequencing at the US DOE Joint Genome Institute after a 8.9-kb DNA library was constructed. Draft assemblies were based on 8,950 reads, providing 5.4× coverage. In addition to Sanger sequencing, 454 pyrosequencing was done to 20× coverage. The Phred/Phrap/Consed software package was used for sequence assembly and quality assessment, while after the shotgun stage, misassemblies and gaps were corrected as previously reported (7). The completed genome of ATCC 10988 contains 10,920 reads, achieving an average of 6-fold sequence coverage per base and an error rate of less than 1 in 100,000.

Coding gene prediction and annotation were performed using Prodigal (http://compbio.ornl.gov/prodigal/) and BLAST (1); tRNA and rRNA genes were identified with tRNAscan-SE and RNAmmer (8, 9). Functional gene assignment was based on the SPTR (TrEMBL) (3), Pfam (5), TIGRFAMs (13), COG (18), and KEGG (6) databases. Genome structure comparisons relied on ACT (4), BLASTN (1), and MegaBLAST (22).

ATCC 10988 contains a single, circular chromosome of 2,021,773 bp and six plasmids reported in this work, p10988_1 to p10988_6, of 32,479 bp, 32,283 bp, 31,692 bp, 18,461 bp, 4,023 bp, and 2,750 bp, respectively. The chromosome and plasmids have GC contents of 46.22% and 43.46%, 45.41%, 43.23%, 41.79%, 37.63% and 41.31%, correspondingly. The entire genome contains 1,875 protein-encoding genes and 48 tRNA and 6 rRNA genes (two rRNA clusters) in the chromosome.

The chromosome is 34,590 bp smaller than that of ATCC 31821 (ZM4) (20), with an average of 98% sequence identity. Four large syntenic units, shuffled in order and direction compared to ZM4, are discerned (ranging from 732 kb to 179 kb). In their junctions identical transposase genes are met. In total, 36 transposase genes are found in this organism, a number far exceeding those for the Z. mobilis strains sequenced so far. Additionally, 54 indels are found scattered, the largest of which is 25.3 kb in size and corroborates previous microarray findings comparing ZM4 to ATCC 10988 (14). The plasmids harbor genes implicated in replicon sustenance and in transport, regulation, transposition, DNA restriction/modification, and metabolism. They share variable regions of homology, albeit no full-length identity, with plasmids from previously sequenced Z. mobilis strains.

Nucleotide sequence accession numbers.

The ATCC 10988 genome received GenBank accession numbers CP002850 for the chromosome and CP002851 to CP002856 for the six plasmids.