The Sugar Kinase That Is Necessary for the Catabolism of Rhamnose in Rhizobium leguminosarum Directly Interacts with the ABC Transporter Necessary for Rhamnose Transport

Abstract

Rhamnose catabolism in Rhizobium leguminosarum was found to be necessary for the ability of the organism to compete for nodule occupancy. Characterization of the locus necessary for the catabolism of rhamnose showed that the transport of rhamnose was dependent upon a carbohydrate uptake transporter 2 (CUT2) ABC transporter encoded by rhaSTPQ and on the presence of RhaK, a protein known to have sugar kinase activity. A linker-scanning mutagenesis analysis of rhaK showed that the kinase and transport activities of RhaK could be separated genetically. More specifically, two pentapeptide insertions defined by the alleles rhaK72 and rhaK73 were able to uncouple the transport and kinase activities of RhaK, such that the kinase activity was retained, but cells carrying these alleles did not have measurable rhamnose transport rates. These linker-scanning alleles were localized to the C terminus and N terminus of RhaK, respectively. Taken together, the data led to the hypothesis that RhaK might interact either directly or indirectly with the ABC transporter defined by rhaSTPQ. In this work, we show that both N-and C-terminal fragments of RhaK are capable of interacting with the N-terminal fragment of the ABC protein RhaT using a 2-hybrid system. Moreover, if RhaK fragments carrying either the rhaK72 or rhaK73 allele were used, this interaction was abolished. Phylogenetic and bioinformatic analysis of the RhaK fragments suggested that a conserved region in the N terminus of RhaK may represent a putative binding domain. Alanine-scanning mutagenesis of this region followed by 2-hybrid analysis revealed that a substitution of any of the conserved residues greatly affected the interaction between RhaT and RhaK fragments, suggesting that the sugar kinase RhaK and the ABC protein RhaT interact directly. IMPORTANCEABC transporters involved in the transport of carbohydrates help define the overall physiological fitness of bacteria. The two largest groups of transporters are the carbohydrate uptake transporter classes 1 and 2 (CUT1 and CUT2, respectively). This work provides the first evidence that a kinase that is necessary for the catabolism of a sugar can directly interact with a domain from the ABC protein that is necessary for its transport. C ells are separated from the external environment by a selectively permeable membrane. A means to transport physiologically relevant substrates across these membranes is required (1-3). The largest and most widely distributed family of transporters is the ATP binding cassette (ABC) transporters (1). ABC transporters are responsible for the transport of a very diverse set of substrates and can be broken into two main classes, importers and exporters (1, 3-5). Much of our understanding of the structure, mechanism, and kinetics of these systems has been derived from the study of relatively few model transporters (6).Gram-negative ABC importers typically consist of a permease component that is made up of two membrane-spanning proteins (encoded by either one or two genes), a periplasm...

Regulation of the rhaEWRBMA Operon Involved in l -Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis

“…In R. leguminosarum, rhamnose catabolism has an effect on its nodulation competitiveness, and the genes for rhamnose catabolism constitute two operons, i.e., the rhaDI and rhaRSTPQUK operons, which are oriented divergently so that their promoters are proximally situated (48). The member genes are predicted to encode a set of rhamnose catabolic enzymes (RhaU, RhaK, RhaD, and RhaI), an ABC-type rhamnose transport system (RhaS, RhaT, RhaP, and RhaQ), and a DeoR-type transcriptional regulator (RhaR), among which the functions of RhaK and RhaSTPQ have been characterized (48)(49)(50). The participation of a DeoR family member in the gene regulation for rhamnose catabolism was also suggested in Chloroflexus aurantiacus (7) and Thermotoga maritima (51).…”

The transport of mannitol in Sinorhizobium meliloti is carried out by a broad-substrate polyol transporter SmoEFGK and is affected by the ability to transport and metabolize fructose

“…It may be that FrcK affects the activity of the transporter(s) through a direct or indirect interaction. We note that kinases that affect transporter activity have been previously described in rhizobia [37, 52]. The substrate for the putative kinase activity of FrcK is not readily apparent from our work.…”

Identification of novel autoinducer-2 receptors in Clostridia reveals plasticity in the binding site of the LsrB receptor family

“…6C). As a negative control, we performed the ITC experiments with a substrate-binding protein from Rhizobium etli, which is homologous to LsrB and thought to bind rhamnose (13,39). Both the raw data and the heat of reaction curve show that, even with AI-2 at a concentration nearly 4 times higher than in the other ITC assays, there is no specific binding, although nonspecific binding seems to occur without apparent saturation of the binding site (Fig.…”