Genome Sequence of a Cellulose-Producing Bacterium, Gluconacetobacter hansenii ATCC 23769

Abstract

The Gram-negative bacterium Gluconacetobacter hansenii is considered a model organism for studying cellulose synthesis. We have determined the genome sequence of strain ATCC 23769.Plants produce cellulose, an unbranched chain of ␤-1,4-linked glucose units, as a structural polysaccharide. It is the most abundant polymer on earth, recently receiving much interest due to its potential use as a feedstock for bioethanol. Bacteria also produce cellulose. Among these, Gluconacetobacter hansenii (previously named Acetobacter xylinus) (4) has been extensively characterized and is a model system for cellulose biosynthesis (1, 2, 7). G. hansenii produces extracellular cellulose that is devoid of lignin or hemicellulose, making it an excellent source for pure cellulose. A lack of a completely sequenced genome for this organism has been a limiting factor in identifying other key proteins involved in cellulose synthesis.The whole-genome sequencing of G. hansenii ATCC 23769 was performed using the 454 FLX-Titanium pyrosequencing technology (5). A combinatorial sequencing approach using 489,201 reads obtained from the shotgun library and 195,088 reads from an 8-kb pair end library (3) produced a total of 221,294,116 bp. These reads were assembled using the Newbler assembler, producing 88 large contigs (Ͼ500 bp) and a chromosome-sized scaffold of 3,646,142 bp with an average coverage of ϫ50.5. This scaffold contained exclusively chromosomal DNA and no plasmid sequences. The gaps in the large scaffold were filled by primer walking and subsequent sequencing of the PCR products. The resulting high-quality draft assembly, consisting of a large scaffold with 71 contigs, was annotated using the Prokaryotic Genomes Automatic Annotation Pipeline (PGAAP) service of the National Institute of Biotechnology Information (NCBI).The chromosomal sequence of G. hansenii 23769 contains 3,547,122 bp, with a GϩC content of 59%. The genome contains 3,351 genes, of which 3,308 are protein-encoding genes, accounting for 84% of the genome. There are 43 genes for tRNAs and 2 rRNA loci. The genes encoding proteins involved in cellulose synthesis are in an operon consisting of acsAB (GXY_04277), acsC (GXY_04282), and acsD (GXY_04292), as previously shown by Saxena et al. (7). Interestingly, there are two additional copies of acsAB, GXY_08864 and GXY_14452, which share 69% and 72% sequence identity, respectively, with the acsAB genes in the operon; the deduced amino acid sequences are 40% and 46% identical, respectively, with that deduced from acsAB in the operon. There are also two additional copies of acsC, GXY_08869 and GXY_014472, which share 72% and 65% DNA sequence identity, respectively, with the acsC gene in the operon; the deduced amino acid sequences share 28% and 30% amino acid identity, respectively, with that deduced from acsC. acsAB (GXY_08864) and acsC (GXY_08869) are only 17 bp apart, less than the distance (66 bp) between the acsAB and acsC genes in the operon. acsAB (GXY_14452) and acsC (GXY_14472) are separated by 3,299 bp, with three ge...

Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain

“…2). Up to three acs operons have been reported in other bacterial cellulose-producing species [G. xylinus ATCC 23769 and G. hansenii ATCC 53582 (31,32)], indicating that the high cellulose synthase copy number may be a possible contributor to the high cellulose productivity observed here (Fig. 1B).…”

Identification and Characterization of Non-Cellulose-Producing Mutants of Gluconacetobacter hansenii Generated by Tn 5 Transposon Mutagenesis

“…dgc and pdeA are located in the cdg (cyclic diguanylate) operon, and pdeA lies upstream from dgc (36). The genome of G. hansenii ATCC 23769 encodes two cdg operons (21). The deduced amino acid sequence of dgc2 (GXY_01661) of the second cdg operon is 59.9% identical to that of dgc1.…”

“…It is not clear what the physiological significance is, if any, between the different organizations of the acs genes in G. hansenii and G. xylinus. It is interesting that the genome of G. hansenii ATCC 23769 has two additional acsAB-like genes (named acsAB1 and acsAB2) and acsC-like genes (named acsC1 and acsC2), but acsD is present in only one copy (21). The deduced amino acid sequences of acsAB1 (GXY_08864) and acsAB2 (GXY_14452) are 40 and 46% identical, respectively, to that of acsAB, and the deduced amino acid sequences of acsC1 (GXY_08869) and acsC2 (GXY_014472) are 28 and 30% identical, respectively, to that of acsC.…”

“…To gain a comprehensive understanding of this process, we set out to systematically identify the genes involved by using Tn5 transposon mutagenesis to isolate mutants that either fail to synthesize/secrete cellulose into the culture medium or produce cellulose with altered properties (e.g., with reduced crystallinity). G. hansenii ATCC 23769 offers an attractive system for this effort, because its complete genome sequence has recently been determined (21), making gene identification relatively straightforward.…”

Genome Sequences of the High-Acetic Acid-Resistant Bacteria Gluconacetobacter europaeus LMG 18890 T and G. europaeus LMG 18494 (Reference Strains), G. europaeus 5P3, and Gluconacetobacter oboediens 174Bp2 (Isolated from Vinegar)

“…The determination of the complete genome sequence for this microorganism is absolutely necessary for the identification of genes and proteins involved in such a high levels of acetic acid adaptation. The genome sequences of other industrially important AAB, such as Gluconobacter oxydans, Acetobacter pasteurianus, and Gluconacetobacter hansenii, have recently been published (1,6,8). The genome sequences of two reference strains of Gluconacetobacter europaeus, as well as those of G. europaeus and G. oboediens strains, isolated from submerged red wine and spirit vinegar, respectively, are herein presented.…”