Study of mural painting isolates, leading to the transfer of 'Bacillus maroccanus' and 'Bacillus carotarum' to Bacillus simplex, emended description of Bacillus simplex, re-examination of the strains previously attributed to 'Bacillus macroides' and description of Bacillus muralis sp. nov

1 Vakgroep BFM WE10V, Laboratorium voor Microbiologie, Universiteit Gent, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium 2 Department of Biological and Biomedical Sciences, Glasgow Caledonian University, Cowcaddens Road, Glasgow G4 0BA, UK 3 Department of Animal Product Quality, Brusselsesteenweg 370, B-9090 Melle, Belgium 4 BCCM/LMG Bacteria Collection, K. L. Ledeganckstraat 35, B-9000 Gent, Belgium Correspondence Jeroen Heyrman Jeroen.Heyrman{at}UGent.be A group of 24 strains was isolated from deteriorated mural paintings situated in Spain (necropolis of Carmona) and Germany (church of Greene-Kreiensen). (GTG) 5 -PCR genomic fingerprinting was performed on these strains to assess their genomic variability and the strains were delineated into four groups. Representatives were studied by 16S rRNA gene sequencing and were found to be closely related to Bacillus simplex and the species ‘ Bacillus macroides ’ (strain NCIMB 8796) and ‘ Bacillus maroccanus ’ (names not validly published) according to a FASTA search. The close similarity between B. simplex , ‘ B. macroides ’ NCIMB 8796, ‘ B. maroccanus ’ and the mural painting isolates was confirmed by additional (GTG) 5 -PCR, ARDRA, FAME and SDS-PAGE analyses. Furthermore, these techniques revealed that strains of ‘ Bacillus carotarum ’, another name that has not been validly published, also showed high similarity to this group of organisms. On the other hand, it was shown that the strains labelled ‘ B. macroides ’ in different collections do not all belong to the same species. Strain NCIMB 8796 can be allocated to B. simplex , while strain DSM 54 (=ATCC 12905) shares the highest 16S rRNA gene sequence similarity with Bacillus sphaericus and Bacillus fusiformis (both around 98·6 %). On the basis of further DNA–DNA hybridization data and the study of phenotypic characteristics, one group of five mural painting strains was attributed to a novel species in the genus Bacillus , for which the name Bacillus muralis sp. nov. is proposed. Finally, the remaining mural painting strains, one (LMG 18508=NCIMB 8796) of two strains belonging to ‘ B. macroides ’ and strains belonging to ‘ B. maroccanus ’ and ‘ B. carotarum ’ are allocated to the species B. simplex and an emended description of B. simplex is given. Published online ahead of print on 22 October 2004 as DOI 10.1099/ijs.0.63221-0. The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of Bacillus muralis sp. nov. LMG 20238 T is AJ628748 . An ARDRA-