Dissection of exopolysaccharide biosynthesis in Kozakia baliensis

Abstract

BackgroundAcetic acid bacteria (AAB) are well known producers of commercially used exopolysaccharides, such as cellulose and levan. Kozakia (K.) baliensis is a relatively new member of AAB, which produces ultra-high molecular weight levan from sucrose. Throughout cultivation of two K. baliensis strains (DSM 14400, NBRC 16680) on sucrose-deficient media, we found that both strains still produce high amounts of mucous, water-soluble substances from mannitol and glycerol as (main) carbon sources. This indicated that both Kozakia strains additionally produce new classes of so far not characterized EPS.ResultsBy whole genome sequencing of both strains, circularized genomes could be established and typical EPS forming clusters were identified. As expected, complete ORFs coding for levansucrases could be detected in both Kozakia strains. In K. baliensis DSM 14400 plasmid encoded cellulose synthase genes and fragments of truncated levansucrase operons could be assigned in contrast to K. baliensis NBRC 16680. Additionally, both K. baliensis strains harbor identical gum-like clusters, which are related to the well characterized gum cluster coding for xanthan synthesis in Xanthomanas campestris and show highest similarity with gum-like heteropolysaccharide (HePS) clusters from other acetic acid bacteria such as Gluconacetobacter diazotrophicus and Komagataeibacter xylinus. A mutant strain of K. baliensis NBRC 16680 lacking EPS production on sucrose-deficient media exhibited a transposon insertion in front of the gumD gene of its gum-like cluster in contrast to the wildtype strain, which indicated the essential role of gumD and of the associated gum genes for production of these new EPS. The EPS secreted by K. baliensis are composed of glucose, galactose and mannose, respectively, which is in agreement with the predicted sugar monomer composition derived from in silico genome analysis of the respective gum-like clusters.ConclusionsBy comparative sugar monomer and genome analysis, the polymeric substances secreted by K. baliensis can be considered as unique HePS. Via genome sequencing of K. baliensis DSM 14400 + NBRC 16680 we got first insights into the biosynthesis of these novel HePS, which is related to xanthan and acetan biosynthesis. Consequently, the present study provides the basis for establishment of K. baliensis strains as novel microbial cell factories for biotechnologically relevant, unique polysaccharides.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0572-x) contains supplementary material, which is available to authorized users.

Comparative genomics of the Komagataeibacter strains—Efficient bionanocellulose producers

“…Komagataeibacter species harbor the acetan biosynthesis cluster. These genes are homologous to gum ‐like heteropolysaccharide genes of Xanthomonas campestris responsible for xanthan synthesis, which has been recently discovered in Kozakia baliensis (Brandt, Jakob, Behr, Geissler, & Vogel, ). In K. xylinus E25 strain, this cluster consists of seventeen genes (Figure a).…”

“…It is completely absent in K. europaeus LMG 18494, whereas the majority of these genes is missing in the K. hansenii strains, which was additionally verified using tblastn (Figure b). The most important seems to be the absence of aceA ( gumD) , which is thought to initiate acetan biosynthesis by transferring a glucosyl‐1‐phosphate residue from UDP‐glucose to an undecaprenyl‐phosphate lipid carrier anchored in the inner membrane (Brandt et al., ; Ishida, Sugano, & Shoda, ). This would implicate that the K. hansenii strains and K. europaeus LMG 18494 should not produce acetan.…”

“…The gene symbols relate to putative functions: polD (rmlD) — dTDP ‐4‐dehydrorhamnose reductase; polC (rmlC) — dTDP ‐4‐dehydrorhamnose 3,5‐epimerase; e.g.,—endoglucanase; gumE —polymerization or export protein; gumF —acyltransferase; gumK —glucuronosyltransferase; aceR —rhamnosyl transferase; aceQ —glucosyl transferase; aceP —glucosyl transferase; aceM ( ugd )— UDP ‐glucose 6‐dehydrogenase; aceF ( mpg )—mannose‐phosphate‐guanyl transferase; aceA ( gumD )— UDP ‐glucose:undecaprenyl‐phosphate glucose‐1‐phosphate transferase; aceB ( gumM )—glycosyltransferase; aceD ( gumC )—putative tyrosine‐protein kinase; aceC ( gumH )—glycosyltransferase; aceE —polysaccharide transporter or flippase; aceG —putative beta‐barrel porin 2 family protein; aceH ( gumB )—polymerization and export protein; aceI —acyltransferase; polA ( aceJ; rmlB )— dTDP ‐glucose 4,6‐dehydratase; polB ( rmlA )—glucose‐1‐phosphate thymidylyltransferase. Annotation is based on homology with gum ‐like gene cluster in Kozakia baliensis and acetan biosynthesis cluster from K. sucrofermentans DSM 15973 (Brandt et al., ; Ishida, Sugano, & Shoda, ). (c) Organization and conservation of the levan operon in K. kakiaceti JCM 25156, K. xylinus NBRC 13693, and Ga. diazotrophicus PA l 5.…”

Gluconacin fromGluconacetobacter diazotrophicusPAL5 is an active bacteriocin against phytopathogenic and beneficial sugarcane bacteria

“…Similar phylogenetic relationships were also demonstrated between K. baliensis and G. diazotrophicus for the gene cluster of exopolysacharides biosynthesis (Brandt et al . ). Therefore, these phylogenetic analyses suggest that the Linocin M18‐like bacteriocin is a highly conserved molecule showing correlation with 16S rRNA sequences, so that it could be used to cluster species in the Acetobacteraceae family.…”

Multiple Genome Sequences of Heteropolysaccharide-Forming Acetic Acid Bacteria

“…High-molecular-weight DNA was purified from modified sodium-gluconate (NaG) medium liquid cultures using the Genomic-tip 100/G kit (Qiagen, Hilden, Germany), as described previously (9). Single-molecule real-time (SMRT) sequencing (PacBio RS II) was carried out at GATC (Konstanz, Germany) (10).…”