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Int J Syst Evol Microbiol 55 (2005), 1129-1131; DOI  10.1099/ijs.0.63420-0
© 2005 International Union of Microbiological Societies

Corynebacterium mooreparkense, a later heterotypic synonym of Corynebacterium variabile

Roberto Gelsomino1, Marc Vancanneyt1, Cindy Snauwaert1, Katrien Vandemeulebroecke1, Bart Hoste1, Timothy M. Cogan2 and Jean Swings1

1 BCCM/LMG Bacteria Collection, Laboratorium voor Microbiologie, Universiteit Gent, Ledeganckstraat 35, Gent, Belgium
2 Dairy Products Research Centre, Teagasc, Fermoy, Ireland

Correspondence
Roberto Gelsomino
Roberto.Gelsomino{at}Ugent.be


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Strains of a Gram-positive bacterium were isolated from the Irish smear-ripened cheese Gubbeen, and assigned to a new species, Corynebacterium mooreparkense, in 2001. During a further study on the same cheese, no additional isolates from this species could be found. Instead, multiple isolates of its nearest phylogenetic neighbour, Corynebacterium variabile, were found. A first screening with rep-PCR and SDS-PAGE pointed to a similarity between C. mooreparkense and C. variabile. Following this peculiar result, attempts were made to collect all type strains deposited at different culture collections and all strains described by Brennan et al. [Int J Syst Evol Microbiol (2001) 51, 843–852]Go. Subsequently, 16S rRNA gene sequencing and DNA–DNA hybridizations were performed. All C. mooreparkense strains had a 16S rRNA gene sequence similarity of at least 99·5 % with C. variabile and the DNA–DNA relatedness was 95 %. On the basis of these results, it is concluded that C. mooreparkense is a later heterotypic synonym of C. variabile.

Published online ahead of print on 23 December 2004 as DOI 10.1099/ijs.0.63420-0.

The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of LMG 19265T and LMG 22560T are AJ783438 and AJ767054, respectively.


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Corynebacterium mooreparkense (LMG S-19265T=LMG 19265T=NCIMB 30131T=DPC 5310T) is a species whose strains were isolated from an Irish smear-ripened cheese (Gubbeen) and was described by Brennan et al. (2001)Go. Ten isolates were assigned to the species by these authors: DPC 5305, DPC 5306, DPC 5307, DPC 5308, DPC 5309, DPC 5312, DPC 5313, DPC 5314, DPC 5315 and DPC 5310T. The type strain was originally deposited in the BCCM/LMG Bacteria Collection as LMG S-19265T and at the National Collection of Industrial and Marine Bacteria as NCIMB 30131T.

During a recent research project, the surface microflora of Gubbeen was again thoroughly studied. A first screening method consisted of comparing the band patterns obtained with rep-PCR by using primer BOXA1R (Versalovic et al., 1994Go). Twenty-five cheese-surface isolates showed a similar band pattern with the type strain of C. mooreparkense. However, all band patterns were also very similar to those of strains of Corynebacterium variabile (LMG 22560T, LMG 22561 and LMG 22562). This peculiar result persuaded us to ask the author of the C. mooreparkense description to send us all ten above-mentioned DPC isolates and we requested from NCIMB the strain deposited in their culture collection (NCIMB 30131T). rep-PCR was repeated with all these strains. Fig. 1Go shows that the rep-PCR band patterns obtained with primer BOXA1R of C. mooreparkense strains DPC 5305 to DPC 5310T (LMG 19265T) and NCIMB 30131T are very similar to those of C. variabile strains and some recent Gubbeen isolates (numerical analysis was performed by using the Pearson coefficient and the UPGMA dendrogram type with the Bionumerics software; Applied Maths).



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  		Fig. 1. rep-PCR patterns obtained with primer BOXA1R of the ten DPC strains described by Brennan et al. (2001)Go, NCIMB 30131T, representative cheese isolates and C. variabile strains [cheese isolates with *, strains isolated recently (2003) from Gubbeen cheese].

 
Representatives of these isolates were compared by SDS-PAGE of whole-cell proteins, a method that has been proven to differentiate on the species level (Pot et al., 1994Go; Kersters & De Ley, 1975Go). Fig. 2Go shows that there is no significant difference between both the type strains and other isolates of C. mooreparkense and C. variabile. Moreover, two recent representative isolates from Gubbeen cheese (LMG 22338 and LMG 22363) also show high similarity with C. variabile and C. mooreparkense (numerical analysis with Pearson coefficient and UPGMA dendrogram type using the GelCompar software; Applied Maths).



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  		Fig. 2. SDS-PAGE band patterns and corresponding dendrogram of representative isolates of C. mooreparkense and C. variabile [cheese isolates with *, strains isolated recently (2003) from Gubbeen cheese].

 
Next, we tried to collect all type strains of C. mooreparkense from the different institutes involved in the original description or available in other culture collections. The University of Newcastle, where the 16S rRNA gene sequencing was performed, and the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ), where the DNA–DNA hybridization was performed, were contacted. However, these research groups disposed of the strain after analysis. The ten strains from the DPC collection and NCIMB 30131T were therefore used for partial (~570 bp) 16S rRNA gene sequences. All strains showed a similarity of at least 99·5 % among each other (data not shown) and with the newly determined complete sequence of C. mooreparkense LMG 19265T (GenBank/EMBL/DDBJ accession no. AJ783438), the type strain of C. variabile LMG 22560T sequenced in our laboratory (AJ767054) and the deposited sequence of C. variabile NCDO 2097T (X53185). However, a similarity of only 96·6 % was obtained with the original sequence of C. mooreparkense LMG 19265T (AF267148) of Brennan et al. (2001)Go (Fig. 3Go). This could be explained by mistakes during the sequencing procedure, which resulted in many inserts that inexplicably can be found in the 16S rRNA gene sequences of DPC 5305, DPC 5307, DPC 5315 and DPC 5310T (GenBank/EMBL/DDBJ accession nos AF267149, AF267151, AF267150 and AF267148, respectively) but cannot be found in any other strain of C. mooreparkense when re-sequenced in our laboratory.



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  		Fig. 3. Phylogenetic relationships of C. variabile, C. mooreparkense and their closest neighbours based on neighbour-joining analysis of the 16S rRNA gene sequences. Bootstrap values (expressed as percentages of 1000 replications) are shown at branch points. C., Corynebacterium; R., Rhodococcus.

 
In a following step, DNA–DNA hybridizations between C. variabile LMG 22560T and C. mooreparkense LMG 19265T were performed. The G+C content of the DNA was determined by HPLC (Mesbah et al., 1989Go) using the further specifications given by Logan et al. (2000)Go. DNA–DNA hybridization was performed by using a modification of the microplate method of Ezaki et al. (1989)Go, as described by Willems et al. (2001)Go. A hybridization temperature of 47 °C (calculated with correction for the presence of 50 % formamide) was used. A relatedness of 95 % between LMG 22560T and LMG 19265T was obtained (reciprocal values of 91·4 and 98·9 %), thus assigning the two strains to a single species, according to the recommendations for species designation (Wayne et al., 1987Go; Stackebrandt et al., 2002Go). This value is in contradiction with the value obtained by Brennan et al. (2001)Go, who state that the level of DNA re-association is 26 %.

We conclude that the strains described as C. mooreparkense match the genomic characteristics of C. variabile and that C. mooreparkense must therefore be seen as a later heterotypic synonym of C. variabile.


   ACKNOWLEDGEMENTS
 
The authors wish to acknowledge the European Community's project Smear Cheese Microflora ‘QLK1-CT-2001-02228’.


   REFERENCES
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 ABSTRACT
 MAIN TEXT
 REFERENCES
 
Brennan, N. M., Brown, R., Goodfellow, M., Ward, A. C., Beresford, T. P., Simpson, P. J., Fox, P. F. & Cogan, T. M. (2001). Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov., isolated from the surface of a smear-ripened cheese. Int J Syst Evol Microbiol 51, 843–852.[Abstract]

Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric deoxyribonucleic acid-deoxyribonucleic acid hybridization in microdilution wells as an alternative to membrane filter hybridization in which radioisotopes are used to determine genetic relatedness among bacterial strains. Int J Syst Bacteriol 39, 224–229.[Abstract/Free Full Text]

Kersters, K. & De Ley, K. (1975). Identification and grouping of bacteria by numerical analysis of their electrophoretic protein patters. J Gen Microbiol 87, 333–342.[Abstract/Free Full Text]

Logan, N. A., Lebbe, L., Hoste, B. & 7 other authors (2000). Aerobic endospore-forming bacteria from geothermal environments in northern Victoria Land, Antarctica, and Candlemas Island, South Sandwich archipelago, with the proposal of Bacillus fumarioli sp. nov. Int J Syst Evol Microbiol 50, 1741–1753.

Mesbah, M., Premachandran, U. & Whitman, W. B. (1989). Precise measurement of the G+C content of deoxyribonucleic acid by high-performance liquid chromatography. Int J Syst Bacteriol 39, 159–167.

Pot, B., Vandamme, P. & Kersters, K. (1994). Analysis of electrophoretic whole-organism protein fingerprints. In Chemical Methods in Prokaryotic Systematics, pp. 493–521. Edited by M. Goodfellow & A. G. O'Donnell. Chichester, UK: Wiley.

Stackebrandt, E., Frederiksen, W., Garrity, G. M. & 10 other authors (2002). Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Evol Microbiol 52, 1043–1047.[Abstract]

Versalovic, J., Schneider, M., de Bruijn, F. J. & Lupski, J. R. (1994). Genomic fingerprinting of bacteria using repetitive sequence-based polymerase chain reaction. Methods Mol Cell Biol 5, 25–40.

Wayne, L. G., Brenner, D. J., Colwell, R. R. & 9 other authors (1987). International Committee on Systematic Bacteriology. Report of the ad hoc committee on reconciliation of approaches to bacterial systematics. Int J Syst Bacteriol 37, 463–464.[Free Full Text]

Willems, A., Doignon-Bourcier, F., Goris, J., Coopman, R., de Lajudie, P., De Vos, P. & Gillis, M. (2001). DNA–DNA hybridization study of Bradyrhizobium strains. Int J Syst Evol Microbiol 51, 1315–1322.[Abstract]




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