The thuEFGKAB operon of rhizobia and agrobacterium tumefaciens codes for transport of trehalose, maltitol, and isomers of sucrose and their assimilation through the formation of their 3-keto derivatives

Abstract

The thu operon (thuEFGKAB) in Sinorhizobium meliloti codes for transport and utilization functions of the disaccharide trehalose. Sequenced genomes of members of the Rhizobiaceae reveal that some rhizobia and Agrobacterium possess the entire thu operon in similar organizations and that Mesorhizobium loti MAFF303099 lacks the transport (thuEFGK) genes. In this study, we show that this operon is dedicated to the transport and assimilation of maltitol and isomers of sucrose (leucrose, palatinose, and trehalulose) in addition to trehalulose, not only in S. meliloti but also in Agrobacterium tumefaciens. By using genetic complementation, we show that the thuAB genes of S. meliloti, M. loti, and A. tumefaciens are functionally equivalent. Further, we provide both genetic and biochemical evidence to show that these bacteria assimilate these disaccharides by converting them to their respective 3-keto derivatives and that the thuAB genes code for this ketodisaccharide-forming enzyme(s). Formation of 3-ketotrehalose in real time in live S. meliloti is shown through Raman spectroscopy. The presence of an additional ketodisaccharide-forming pathway(s) in A. tumefaciens is also indicated. To our knowledge, this is the first report to identify the genes that code for the conversion of disaccharides to their 3-ketodisaccharide derivatives in any organism.

Fig 1

The trehalose transport and utilization operon, thuEFGKAB, in members of Rhizobiaceae. The proposed biochemical functions encoded by the genes are indicated at the top. Numbers in bold above each gene indicate the percentages of identity with S. meliloti 1021 at the amino acid level (or at nucleotide level [*]). Where available, locus tags are indicated directly below clusters: for S. meliloti 1021, the prefix is SM_b20; for S. medicae WSM 419, it is Smed_3; for M. loti MAFF303099, it is mll3; for R. etli CFN42, it is RHE_PF00; for R. leguminosarum bv. viciae 3841, it is pRL120; for R. leguminosarum bv. trifolii WSM2304, it is Rleg2_5; for A. tumefaciens C58, it is Atu3; and for B. suis ATCC 23445, it is BSUIS_B0.

Fig 2

Growth of wild-type S. meliloti Rm1021 (●), the trehalose utilization mutants Sm7023 (▲) and Sm7024 (○), the trehalose transport mutant Sm7019 (△), the sucrose transport mutant Rm9628 (■), and the trehalose and sucrose transport mutant Sm7025 (□) on M9 minimal medium containing palatinose or maltitol at 0.4% (wt/vol).

Fig 3

Complementation analysis for utilization of leucrose, maltitol, palatinose, and trehalose in S. meliloti Sm7023. Shown is the growth of Sm7023 (○) and of Sm7023 containing plasmid pRK7813::MthuAB (●) expressing the thuAB genes of M. loti from the lac promoter in pRK7813. The cells were grown in M9 medium containing various carbon sources at 0.4% (wt/vol) as indicated.

Fig 4

Absorption spectra of ketosugars formed by A. tumefaciens MAFF301001 (□) and mutant At7023 (■) grown overnight with various disaccharides (1%, wt/vol) as indicated. An asterisk indicates that no growth of At7023 was observed. The presence of ketosugars in the cell-free supernatant was determined by the alkaline method described by Fukui and Hayano (33). Each value represents the mean from at least three different experiments ± standard error.

Fig 5

(A) Complementation analysis for utilization of leucrose, maltitol, palatinose, and trehalulose in A. tumefaciens At7023 containing cosmid pRK7813 expressing thuA (□), thuB (■), or thuAB (●) of S. meliloti from the lac promoter. (○), At7023 without cosmid. The cells were grown in M9 medium containing various carbon sources at 0.4% (wt/vol) as indicated. (B) Absorption spectra of 3-ketotrehalose formed by A. tumefaciens At7023 containing cosmid pRK7813 expressing thuA (□), thuB (■), or thuAB (●) of S. meliloti from the lac promoter. (○), At7023 without cosmid. The cells were grown overnight in M9 medium containing trehalose (1%, wt/vol), and the presence of ketosugars in the cell-free supernatant was determined by the alkaline method described by Fukui and Hayano (33). Each value represents the mean from three different experiments ± standard error.

Fig 6

(A) Raman spectra of 5% (wt/vol) aqueous solutions of trehalose (upper spectrum) and 3-ketotrehalose (lower spectrum). The spectra were acquired within a 2-min acquisition time with an excitation power of 30 mW at a 785-nm wavelength. (B) Plot of the difference of normalized intensities of the 1,734-cm⁻¹ peak upon trehalose application in the S. meliloti wild type (Rm1021) grown in the presence of trehalose (■), S. meliloti Rm1021 grown without trehalose (□), and the trehalose utilization mutant Sm7023 grown in the presence of trehalose (▲) versus time. Prior to actual measurements, the safety of the method was evaluated by monitoring the bacterial cells in the optical trap for up to 5 min under experimental conditions (data not shown). Each value represents the mean from three different experiments ± standard error.