The Brucella abortus two-component system response regulator BvrR binds to three DNA regulatory boxes in the upstream region of omp25

Abstract

Brucella abortus is a facultative extracellular-intracellular bacterial zoonotic pathogen worldwide. It is also a major cause of abortion in bovines, generating economic losses. The two-component regulatory system BvrR/BvrS modulates the expression of genes required to transition from extracellular to intracellular lifestyles. However, few regulatory regions of BvrR direct target genes have been studied. In this study, we characterized the regulatory region of omp25, a gene encoding an outer membrane protein that is positively regulated by TCS BvrR/BvrS. By omp25-lacZ reporter fusions and β-galactosidase activity assays, we found that the region between-262 and + 127 is necessary for transcriptional activity, particularly a 111-bp long fragment located from-262 to −152. In addition, we demonstrated the binding of P-BvrR to three sites within the −140 to +1 region. Two of these sites were delimited between −18 to +1 and − 99 to −76 by DNase I footprinting and called DNA regulatory boxes 1 and 2, respectively. The third binding site (box 3) was delimited from −140 to −122 by combining EMSA and fluorescence anisotropy results. A molecular docking analysis with HDOCK predicted BvrR-DNA interactions between 11, 13, and 12 amino acid residue-nucleotide pairs in boxes 1, 2, and 3, respectively. A manual sequence alignment of the three regulatory boxes revealed the presence of inverted and non-inverted repeats of five to eight nucleotides, partially matching DNA binding motifs previously described for BvrR. We propose that P-BvrR binds directly to up to three regulatory boxes and probably interacts with other transcription factors to regulate omp25 expression. This gene regulation model could apply to other BvrR target genes and to orthologs of the TCS BvrR/BvrS and Omp25 in phylogenetically closed Rhizobiales.Brucella abortus es un patógeno zoonótico bacteriano extracelular-intracelular facultativo en todo el mundo. También es una de las principales causas de aborto en bovinos, lo que genera pérdidas económicas. El sistema regulador de dos componentes BvrR/BvrS modula la expresión de los genes necesarios para la transición del modo de vida extracelular al intracelular. Sin embargo, se han estudiado pocas regiones reguladoras de los genes diana directos de BvrR. En este estudio, hemos caracterizado la región reguladora de omp25, un gen que codifica una proteína de membrana externa que está regulada positivamente por TCS BvrR/BvrS. Mediante fusiones reporteras omp25-lacZ y ensayos de actividad β-galactosidasa, descubrimos que la región comprendida entre-262 y + 127 es necesaria para la actividad transcripcional, en particular un fragmento de 111 pb de longitud situado entre-262 y -152. Además, demostramos la unión de P-BvrR a tres sitios dentro de la región -140 a +1. Dos de estos sitios se delimitaron entre -18 y +1 y -99 y -76 mediante la huella de DNasa I y se denominaron cajas reguladoras de ADN 1 y 2, respectivamente. El tercer sitio de unión (caja 3) se delimitó de -140 a -122 combinando los resultados de EMSA y anisotropía de fluorescencia. Un análisis de acoplamiento molecular con HDOCK predijo interacciones BvrR-ADN entre 11, 13 y 12 pares de aminoácidos residuo-nucleótido en las cajas 1, 2 y 3, respectivamente. Una alineación manual de la secuencia de las tres cajas reguladoras reveló la presencia de repeticiones invertidas y no invertidas de cinco a ocho nucleótidos, que coinciden parcialmente con los motivos de unión al ADN previamente descritos.