Identification of Brevibacteriaceae by Multilocus Sequence Typing and Comparative Genomic Hybridization Analyses

Abstract

Multilocus sequence typing with nine selected genes is shown to be a promising new tool for accurate identifications of Brevibacteriaceae at the species level. A developed microarray also allows intraspecific diversity investigations of Brevibacterium aurantiacum showing that 13% to 15% of the genes of strain ATCC 9174 were absent or divergent in strain BL2 or ATCC 9175.Brevibacteriaceae play a major part in the cheese smear community (6, 11). The classification and typing of cheese-related Brevibacteriaceae have been based mainly on molecular methods such as amplified ribosomal DNA restriction enzyme analysis, pulsed-field gel electrophoresis, and ribotyping (8,10,12). Recently, the original Brevibacterium linens group was split into two species on the basis of their physiological and biochemical characteristics, the sugar and polyol composition of their teichoic acids, and their 16S rRNA sequence and DNA-DNA hybridization levels. One species remains B. linens and is represented by type strain ATCC 9172. The other, represented by type strain ATCC 9175, has been renamed Brevibacterium aurantiacum. Regarding this new classification, the taxonomic position of cheese-related isolates has to be revisited and potential relationships between phylogenetic affiliation and the potential occurrence of given metabolic characteristics redefined (7). The unfinished genome sequence of B. aurantiacum ATCC 9174 has recently been released by the Joint Genome Institute (http://genome.jgi-psf.org/draft_microbes/breli /breli.home.html). The development of focused phylogenetic approaches using multiple markers in conjunction with whole-genome screening techniques such as comparative genomic hybridization (CGH) has proven to be useful for the detailed characterization of pathogenic species, including food pathogens (3, 5, 9). However, only a few technological species have been investigated at an intraspecies level (2). Our intention was thus to develop modern tools to facilitate the typing of strains of technological interest, for which Brevibacteriaceae could be used as a case study.Phylogenetic analysis of cheese-related Brevibacteriaceae shows an organization in three main branches. Three cheese Brevibacterium sp. strains, BL2, CNRZ918, and ATCC 9174, and B. aurantiacum and B. linens type strains ATCC 9175 and ATCC 9172, respectively, (7), were analyzed both by 16S rRNA sequencing with the universal primers and by multilocus sequence typing (MLST). The 16S rRNA analysis showed the phylogenic relationship between these strains (Table 1). B. linens ATCC 9172 is an independent lineage. BL2 and ATCC 9174 were related to B. aurantiacum type strain ATCC 9175. Interestingly, strain CNRZ918 presents similarities to the B. aurantiacum lineage, but this strain appeared to be closely related to Brevibacterium antiquum, with 99% identity between their 16S rRNA sequences.To extend the 16S rRNA phylogenetic organization of technological Brevibacterieceae, an MLST approach was used. Nine genes (cysN, glnA, gyrA, metY, metX, mgl, pheS, sahH, and ...

Global Regulation of the Response to Sulfur Availability in the Cheese-Related Bacterium Brevibacterium aurantiacum

“…2). Most of the genes of ATCC 9174 involved in sulfur metabolism, with two exceptions, are present in ATCC 9175 (20). Both strains were able to grow on sulfate as the sole sulfur source, an observation in agreement with the presence of a sulfate assimilation pathway (CysIHDN-BL2146-BL2147-CysJ) in the B. aurantiacum genome.…”

“…2B and data not shown). This result may be correlated with the deletion of the BL615-to-BL621 region containing genes similar to ssuD and ssuE in the genome of ATCC 9175, as observed by CGH analysis (20). B. aurantiacum further utilized sulfide for cysteine synthesis using the OAS thiol-lyase, CysK, and the serine acetyltransferase, CysE (Fig.…”

“…The expression profiles of B. aurantiacum ATCC 9175 grown in the presence of various sulfur sources were analyzed by using DNA microarrays (Agilent Technologies). The DNA microarray was described in a previous study (20). RNAs were labeled with either Cy3 or Cy5 fluorescent dye (GE Healthcare, Little Chalfont, United Kingdom) by using a SuperScript Direct cDNA labeling system kit (Invitrogen) according to the manufacturer's recommendations.…”

“…The unfinished genome sequence of the B. aurantiacum ATCC 9174 strain (20) has been recently released by the Joint Genome Institute (http://genome.jgi-psf.org/). In the present study, we performed a reconstruction of sulfur metabolism in FIG.…”

Phylogenetic, Microbiological, and Glycoside Hydrolase Diversities within the Extremely Thermophilic, Plant Biomass-Degrading Genus Caldicellulosiruptor

“…As log 2 hybridization ratios approach zero, those open reading frames, and by extension the organism, are considered to be more genetically similar to C. saccharolyticus. The more genetically divergent the species tested are from C. saccharolyticus (based on M-CGH analysis), the more the plots of log 2 ratios will deviate from the horizontal axis (17). In addition, pair-wise scatter plots of hybridization intensities from unknown versus reference strains have previously been used to assess levels of diversity between strains (11).…”