1887

Abstract

Three Gram-negative, rod-shaped, catalase- and oxidase-positive, facultatively anaerobic and motile bacteria, strains WS 4538, WS 4539 and WS 4540, were isolated from the surfaces of two fully ripened French red smear soft cheeses. Based on 16S rRNA gene sequence similarity, all three strains were shown to belong to the genus . They are most closely related to S-1 (96.3 % similarity) and MANO22D (95.9 %). DNA–DNA hybridization confirmed that all three isolates belong to the same species and clearly separated strain WS 4539 from DSM 19141 (38–42 % relatedness) and DSM 17657 (28–37 %). In contrast to their nearest relatives, the strains exhibited -galactosidase and aesculin hydrolase activities. A 14 bp insertion in the 16S rRNA gene sequence forms an elongated structure at helix 10 in the rRNA molecule and provides a tool for PCR-based identification of the novel species. Partial sequences of the housekeeping genes , , and supported the conclusion that the three isolates constitute a separate species within the genus . The name sp. nov. is proposed for the novel taxon. Strain WS 4539 (=DSM 22364 =LMG 25240; DNA G+C content 41.8 mol%) is the type strain and WS 4540 (=DSM 22378 =LMG 25241) is a reference strain.

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2010-08-01
2019-10-18
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vol. , part 8, pp. 1745 - 1749

DNA–DNA hybridization results.

Members of the that carry an insertional fragment in their 16S rRNA gene sequences and the habitats from which they have been isolated.

Neighbour-joining trees based on comparison of the sequences of selected housekeeping genes.

Models of secondary structure of the mature 16S rRNA molecule, showing a short helix 10 as calculated for and an elongated helix 10 as calculated for sp. nov.

[PDF file of Supplementary Figures and Tables](229 KB)

Supplementary Methods.

Phylogenetic trees based on all five loci were calculated using representative type strains of species. Sequences were retrieved either from the NCBI database ( http://www.ncbi.nlm.nih.gov/sites/entrez?db=nucleotide) or from the Taxonomy of the Vibrios website ( http://www.taxvibrio.lncc.br). The sequences were aligned using CLUSTAL_X version 2.09 (Larkin , 2007). Distance matrices of the resulting multiple sequence alignments were calculated according to Kimura's two-parameter model using TREECON version 1.3b (Van de Peer & De Wachter, 1997).

Total RNA of the novel strains was extracted using Trizol (Invitrogen) following the instructions of the manufacturer. Reverse transcription was performed on extracts standardized to 1 µg RNA by spectroscopic measurements at 280 nm (Nanodrop ND-1000; Peqlab). The reaction volume of 20 µl contained DNase-digested RNA (TURBO DNase; Ambion), dNTP mixture (500 µM), DDT (5 mM), RNase OUT (40 U; Invitrogen) and first-strand buffer. An aliquot containing 1 pmol insertion-specific primer 188r [5′-GCTTTRCTTCTTTCCTTTAA-3′, consisting of 10 nucleotides insertional region (underlined) and 10 nucleotides downstream of the insertion] was added. Samples were completed by the addition of Superscript III (200 U; Invitrogen); for blanks, ultrapure water was added instead. As a positive control, reverse transcription was conducted using the internal primer 198r (1 pmol; 5′-AAGGTCCCCCGCTTTRCTTC-3′), which covers only the region downstream of the insertion. The incubation conditions for the reverse transcription reaction were 25 °C for 5 min, 50 °C for 90 min and 70 °C for 15 min. PCR amplification of cDNA was carried out in a 50 µl reaction mixture containing 1 pmol primers 27f and 188r, 200 µM dNTP mixture, 1 U Thermoprime Plus Polymerase (Thermo Scientific) and 20 ng template DNA. PCR conditions were 95 °C for 2 min followed by 30 cycles of 95 °C for 20 s, 45 °C for 30 s and 72 °C for 15 s. The size of the resulting fragment was in accordance with the position of the 14 bp insertion in the 16S rRNA gene and could be further proven by sequencing. No PCR amplification could be achieved when RNA samples did not undergo reverse transcription (blank samples) or when water was added instead of template (blind samples).

References

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