Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray.
The genus Brucella contains bacteria producing a zoonosis of large sanitary and economical impact. The complete nucleotide sequence of eight Brucella isolates is currently available. This information can be used for high throughput approaches to the biology of this genus such as the construction of comprehensive collections of ORF clones or ORFeomes. The ORFeome of Brucella melitensis was a first contribution to this goal. Using the Brucella ORFeome as starting material we have amplified each ORF and printed them in duplicate onto coated glass slides along with the appropriate positive and negative controls. Quality control of the microarray was performed by image analysis after ethidium bromide staining. This Brucella DNA microarray was used to determine the global transcriptional profile of Brucella abortus grown under laboratory conditions. Two sets of genes representing strongly and poorly expressed genes have been defined. The occurrence of several genes of the same operon in the same data set has been taken as additional proof of the significance of the results. The two sets have been validated by RT-PCR of retrotranscribed RNA. Among the more abundant transcripts we found ribosomal proteins, Krebs cycle and oxidative phosphorylation enzymes. virB, flagellar components and other genes related with virulence and intracellular growth were in the poorly transcribed set. This report demonstrated the usefulness of the ORFeome for the construction of a PCR product microarray for the analysis of global gene expression in Brucella and also applicable to other microorganisms. The results provided here represent a comprehensive description of the global transcriptional profile of B. abortus grown under laboratory conditions and, at the same time, validate the use of this Brucella microarray for the study of the biology and pathogenesis of Brucella through the analysis of gene expression under any experimental conditions.
Viadas
C
C
Departamento de Microbiología y Parasitología, Universidad de Navarra, Irunlarrea N1, 31008 Pamplona, Navarra, Spain. cviadas@gmail.com
Rodríguez
M C
MC
García-Lobo
J M
JM
Sangari
F J
FJ
López-Goñi
I
I
D002003:Brucella abortus / Q000235:genetics; D017347:Brucella melitensis / Q000235:genetics*; D020869:Gene Expression Profiling; D016680:Genome, Bacterial; D008967:Molecular Biology / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis; D016366:Open Reading Frames