Transcription of two adjacent carbohydrate utilization gene clusters in Bifidobacterium breve UCC2003 is controlled by LacI- and repressor open reading frame kinase (ROK)-type regulators

Abstract

Members of the genus Bifidobacterium are commonly found in the gastrointestinal tracts of mammals, including humans, where their growth is presumed to be dependent on various diet- and/or host-derived carbohydrates. To understand transcriptional control of bifidobacterial carbohydrate metabolism, we investigated two genetic carbohydrate utilization clusters dedicated to the metabolism of raffinose-type sugars and melezitose. Transcriptomic and gene inactivation approaches revealed that the raffinose utilization system is positively regulated by an activator protein, designated RafR. The gene cluster associated with melezitose metabolism was shown to be subject to direct negative control by a LacI-type transcriptional regulator, designated MelR1, in addition to apparent indirect negative control by means of a second LacI-type regulator, MelR2. In silico analysis, DNA-protein interaction, and primer extension studies revealed the MelR1 and MelR2 operator sequences, each of which is positioned just upstream of or overlapping the correspondingly regulated promoter sequences. Similar analyses identified the RafR binding operator sequence located upstream of the rafB promoter. This study indicates that transcriptional control of gene clusters involved in carbohydrate metabolism in bifidobacteria is subject to conserved regulatory systems, representing either positive or negative control.

FIG 1

Representation of the raffinose and melezitose utilization operons of B. breve UCC2003.

FIG 2

(A) Final OD₆₀₀ values after 16 h of wild-type strain B. breve UCC2003 and insertion mutants B. breve UCC2003-melR1 and B. breve UCC2003-melR2 growth on 1% melezitose or 1% lactose. The results are mean values obtained from three separate experiments. The final optical densities reached by B. breve UCC2003 and insertion mutants B. breve UCC2003-melR1 and B. breve UCC2003-melR2 during growth on lactose or melezitose were not statistically significantly different (P < 0.1). (B) Final OD₆₀₀ values after 16 h of growth of wild-type strain B. breve UCC2003 and insertion mutant B. breve UCC2003-rafR on 1% stachyose, 1% raffinose, 1% melibiose, or 1% lactose. The final optical densities attained by B. breve UCC2003 compared to insertion mutant B. breve UCC2003-rafR during growth on 1% raffinose, 1% stachyose, or 1% melibiose were statistically significantly different (*, P < 0.001; **, P < 0.01). No significant difference (P < 0.1) was observed in the final optical densities achieved by B. breve UCC2003 and B. breve UCC2003-rafR during growth on 1% lactose. The error bars indicate standard deviations.

FIG 3

(A) Representation of the melezitose utilization cluster of B. breve UCC2003 and DNA fragments used in EMSAs for the melA and Bbr_1862 promoter regions. The plus and minus signs indicate ability or inability of MelR1 or MelR2 to bind to the melA or Bbr_1862 DNA promoter-encompassing fragments, respectively. (B) EMSAs showing MelR1 interaction with DNA fragments encompassing fragment M1 (i) and MelR2 interaction with DNA fragments encompassing fragment K1 (ii). (C) EMSAs illustrating MelR1 interaction with melAIR (i) and mutated derivative mu-melAIR (ii) and MelR2 interaction with Bbr1862IR (iii) and mutated derivative mu-Bbr1862IR (iv). The minus signs indicate lanes with binding reactions to which no protein was added, while the remaining lanes represent binding reactions with the respective DNA probes incubated with increasing amounts of protein at concentrations ranging from 0.04 nM to 0.01 μM. Each successive lane, from right to left, corresponds to a doubling in the amount of protein used for the EMSA. (D) EMSAs showing MelR1 interaction with the DNA fragment M1 with the addition of melezitose at concentrations ranging from 2.5 to 20 mM.

FIG 4

(A) Representation of the raffinose utilization cluster of B. breve UCC2003 and DNA fragments used in EMSAs for the rafB and rafA promoter regions. The plus and minus signs indicate the ability or inability, respectively, of RafA to bind to the rafB or rafA DNA fragments. (B) EMSAs showing RafR interaction with DNA fragments encompassing fragments R1 (i), R2 (ii), and R3 (iii) and the annealed oligonucleotides representing rafBIR (iv). The minus signs indicate lanes with binding reactions to which no protein was added, while the remaining lanes represent binding reactions with the respective DNA probes incubated with increasing amounts of protein at concentrations ranging from 0.04 nM to 0.01 μM. Each successive lane, from right to left, corresponds to a doubling in the amount of protein used for the EMSA. (C) EMSAs showing RafR interaction with the mutated operator motif of the rafB promoter region. DNA fragments were obtained by PCR using IRD700-labeled primers. The operator sequence and incorporated mutations are shown above the image. wt and +, original promoter sequence; −, no added protein. The conserved ROK motif is highlighted in yellow. The arrows indicate the inverted-repeat structure of the motif.

FIG 5

Schematic representation of the melA (A), Bbr_1862 (B), rafB (C), and rafA (D) promoter regions. Boldface type and underlining indicate the −10 and −35 hexamers deduced from the primer extension results; deduced ribosomal binding sites (RBS) and experimentally determined transcriptional start sites (TSS) are indicated by asterisks. The arrows under sequences with names in boldface indicate the inverted-repeat MelR1, MelR2, and RafR binding sequences displayed above the arrows. The arrows in the right panels indicate the primer extension products.