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Plasmids of Ketogulonicigenium robustum - strains NRRL B-21627 and SPU_B003

Evidence presented here suggests that Ketogulonicigenium robustum strain SPU_B003, reportedly a novel isolate from soil, is a subculture of the species type strain K. robustum NRRL B-21627 (ADM X6L).

Bacteria of the genus Ketogulonicigenium (Ketogulonigenium) are known for their efficient oxidation of L‑sorbose to 2‑keto‑L‑gulonic acid (2‑KLG, 2‑KGA), an important intermediate in the manufacture of ascorbic acid (vitamin C).  Ketogulonicigenium robustum ADM X6L, the species type strain, was isolated from cotton field soil in the USA in the mid-1990s (1). ADM X6L is attractive as a superior 2‑KLG producer (2) and is available from culture collections in the USA, South Korea and Europe under accessions NRRL B‑21627, KCTC 0858BP and LMG 21292.

The DNA sequences of four plasmids carried by ADM X6L appeared in US patent 7053197 (3) and were assigned GenBank accessions AR884692.1, AR884693.1, AR884694.1 and AR884695.1 for pADMX6L1 through pADMX6L4, respectively. The ADM X6L plasmids are present in the NCBI BLAST search database 'patnt'.

A second K. robustum was introduced in 2018 in the form of strain SPU_B003, claimed to have been isolated from soil in Yunnan province of the PRC (4). Strain SPU_B003 also carries four plasmids which were labeled "unnamed2" through "unnamed5", plus a megaplasmid or chromid that was labeled "unnamed1". The complete genome assembly of strain SPU_B003 was published in 2017 under accession GCF_002117445.1.

This report shows that the four plasmids of SPU_B003 "unnamed2" through "unnamed5" are virtually identical to the four plasmids of ADM X6L, suggesting that SPU_B003 is a subculture of ADM X6L.

Are the ADM X6L and SPU_B003 plasmids similar?

BLAST searches (5) of the patnt database (6) were performed using each of the four SPU_B003 plasmid sequences as the query. One of the SPU_B003 plasmids showed full-length 100% identity with a counterpart plasmid in strain ADM X6L. The other three SPU_B003 plasmids also had counterparts in ADM X6L, in each case differing by only one base pair.   Click here to access the BLAST alignments and some comments.

The pairing of equivilent plasmids is as follows:

SPU_B003 unnamed2  =  pADMX6L3
Plasmid unnamed2 (19,694 bp) is one base pair shorter than pADMX6L3, but the sequences are otherwise identical. The two versions are reverse complements and are linearized at different coordinates.

SPU_B003 unnamed3 = pADMX6L1
Plasmid unnamed3 (7,028 bp) is one base pair shorter than pADMX6L1, but the sequences are otherwise identical. The two versions represent the same DNA strand but are linearized at different coordinates.

SPU_B003 unnamed4 = pADMX6L2
Plasmid unnamed4 (4,005 bp) is identical to pADMX6L2. The sequences are linearized at different coordinates and are reverse complements.

SPU_B003 unnamed5 = pADMX6L4
Plasmid unnamed5 (4,211 bp) differs from pADMX6L4 at one base pair, but the sequences are otherwise identical. They are reverse complements and are linearized at different coordinates.

Have the ADM X6L plasmids been found in any other bacteria?

BLAST nucleotide searches against the NCBI comprehensive nucleotide database 'nt' (7) were performed using the ADM X6L plasmid sequences as the queries. The highest-scoring alignments were of course to the SPU_B003 plasmids. Beyond that the highest-scoring alignments were to a Paracoccus sp. plasmid (NZ_CP070370.1, 4983 bp) sharing 93‑96% identity over 63% of pADMX6L1 in two segments, and a Paracoccus marcusii plasmid (NZ_CP065896.1, 96460 bp) sharing 82‑84% identity over 34% of pADMX6L3 in three segments. Hence, segments of plasmids pADMX6L1 and pADMX6L3 show some sequence relatedness to plasmids from bacteria of the order Rhodobacterales...more so for pADMX6L1.

Plasmids pADMX6L2 and pADMX6L4 appear to be wholly unique at the DNA level, as meaningful alignments in the 'nt' database could not be found except to their counterpart plasmids in strain SPU_B003. In amino acid sequence the predicted Rep protein of pADMX6L2 shows relatedness to replication proteins of the RepW type (9).

pADMX6L1 and RepL-type plasmids

Removing either base‑1 or base‑7029 from the pADMX6L1 sequence (both are a 'G') makes it identical to plasmid unnamed3. Doing this lengthens the predicted MobA/MobL ORF of pADMX6L1 by 9 codons and improves its alignment with similarly-annotated ORFs from other Rhodobacterales. Whether the 1‑bp difference between pADMX6L1 and unnamed3 is real or is a sequencing artifact is unknown.

In 2019 Jörn Petersen et al. published a study of the RepL‑type plasmid pLA6_12 (GenBank CP031597 7053 bp) from Marinibacterium anthonyi strain La 6 (8). Their work included a similarity analysis of 36 other RepL‑type plasmids based on predicted protein sequences from the conserved backbone region of pLA6_12. Plasmid "unnamed3" (pADMX6L1) of K. robustum was in the study and was considered to be a RepL‑type plasmid.


  1. Urbance JW, Bratina BJ, Stoddard SF, Schmidt TM. Taxonomic characterization of Ketogulonigenium vulgare gen. nov., sp. nov. and Ketogulonigenium robustum sp. nov., which oxidize L-sorbose to 2-keto-L-gulonic acid. Int J Syst Evol Microbiol. 2001 May;51(Pt 3):1059-1070. doi: 10.1099/00207713-51-3-1059. PMID: 11411674.
  2. Liaw HJ, Edington J, Yang Y. Bacterial strains and use thereof in fermentation processes for 2-keto-L-gulonic acid production. US Patent 5989891 (PDF download), filed July 1997.
  3. D'Elia J, Stoddard SF. Ketogulonigenium endogenous plasmids. US Patent 7053197 (PDF download), filed October 2002.
  4. Wang CY, Li Y, Gao ZW, Liu LC, Zhang MY, Zhang TY, Wu CF, Zhang YX. Establishing an innovative carbohydrate metabolic pathway for efficient production of 2-keto-L-gulonic acid in Ketogulonicigenium robustum initiated by intronic promoters. Microb Cell Fact. 2018 May 19;17(1):81. doi: 10.1186/s12934-018-0932-9. PMID: 29778095; PMCID: PMC5960096.
  5. Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, Madden TL. BLAST+: architecture and applications. BMC Bioinformatics. 2009 Dec 15;10:421. doi: 10.1186/1471-2105-10-421. PMID: 20003500; PMCID: PMC2803857.
  6. Database: Nucleotide sequences derived from the Patent division of GenBank (patnt); 45,782,177 sequences; 25,558,976,770 total bases; Date: Nov 5, 2021 8:54 PM Longest sequence: 33,862,096 bases; BLASTDB Version: 5
  7. Database: Nucleotide collection (nt); 79,893,514 sequences; 674,287,044,366 total bases; Date: Feb 20, 2022 9:10 AM; Longest sequence: 99,791,824 bases; BLASTDB Version: 5.
  8. Petersen J, Vollmers J, Ringel V, Brinkmann H, Ellebrandt-Sperling C, Spröer C, Howat AM, Murrell JC, Kaster AK. A marine plasmid hitchhiking vast phylogenetic and geographic distances. Proc Natl Acad Sci U S A. 2019 Oct 8;116(41):20568-20573. doi: 10.1073/pnas.1905878116. Epub 2019 Sep 23. PMID: 31548387; PMCID: PMC6789725.
  9. Freese HM, Ringel V, Overmann J, Petersen J. Beyond the ABCs-Discovery of Three New Plasmid Types in Rhodobacterales (RepQ, RepY, RepW). Microorganisms. 2022 Mar 29;10(4):738. doi: 10.3390/microorganisms10040738. PMID: 35456790; PMCID: PMC9025767.