Structural and Functional Analysis of a Putative Gene Cluster for Palatinose Transport on the Linear Chromosome of Agrobacterium tumefaciens MAFF301001

Abstract

We identified a putative pal gene cluster (palR, palE, palF, palG, palK, palA, and palB) in the plant-tumorigenic bacterium Agrobacterium tumefaciens MAFF301001; by sequencing analyses, this cluster was found to be involved in palatinose transport, and its functional importance was revealed by mutational analyses. The pal gene products were highly homologous to those of putative trehalose/maltose ABC-type transport systems but were not essential to bacterial growth on trehalose. Insertion mutations in the palK and palE genes showed the necessity of these genes for bacterial growth and chemotaxis with palatinose as the carbon source, but no inhibition of tumorigenesis was observed. Growth on trehalose and maltose was not influenced by the mutations.

The importance of sugars as a source of energy (11) and as signal compounds in chemotaxis, biofilm formation, and virulence gene induction has been reported in a broad spectrum of bacteria (4, 12, 13). Moreover, disaccharides such as trehalose provide a protective role to many microbes by enabling them to withstand environmental stresses, such as desiccation (14) and high osmolarity (9, 14). Palatinose or isomaltulose (6-O-α-d-glucopyranosyl-d-fructofuranose) has been reported as a unique energy source in some bacteria (16). As one of the functional isomers of sucrose, palatinose is produced from sucrose by some bacteria as a reserve material under excess carbon conditions (3). However, the genes for uptake and metabolism of palatinose are induced only when other carbon sources are limited (3).

It is believed that these biologically converted forms of sucrose are available to the organism itself but not to competitors such as host plants and other microorganisms (3). In addition, inhibition of yeast invertase by palationose was investigated, and its potential ability to exclude competitors has been suggested as a pathogenicity strategy (3). Moreover, palatinose has been reported to be important as an osmoprotectant under hyperosmotic conditions in the soil bacteria Sinorhizobium meliloti and Rhizobium spp. (9), conferring a selective advantage over other bacteria in adverse environmental conditions.

Agrobacterium tumefaciens is an α-proteobacterium of the family Rhizobiaceae and the causal agent of crown gall disease of dicotyledonous plants (1). As a soil microorganism, A. tumefaciens frequently encounters various environmental conditions and nutritional stresses in the heterogeneous rhizosphere (11). Also, during infection, it has to overcome adverse microenvironmental conditions such as low pH and phenolic compounds that exist at the infection site. Under such critical conditions, sugars can play a key role in the survival of the bacterium; therefore, sugar transport systems are critical to bacteria.

Our previous study analyzing the genome for the left region of the linear chromosome of A. tumefaciens MAFF301001 identified a region highly homologous to the gene components of the putative trehalose/maltose (thu) transport system of S. meliloti (6). Nevertheless, the mutational and growth analyses described below revealed the relevance of the gene cluster in palatinose uptake and transport. Therefore, we tentatively designated it the pal gene cluster.

Localizing the putative pal gene cluster in the A. tumefaciens genome.

Pulsed-field gel electrophoresis and Southern hybridization of total DNA revealed the presence of the putative pal gene cluster only on the linear chromosome in the genome of A. tumefaciens MAFF301001. According to the Southern hybridization signals, the putative pal gene cluster was located in the SwaI-#2/PmeI-#4 macrorestriction fragments of the linear chromosomal physical map of A. tumefaciens MAFF301001 constructed by Suzuki et al. in 2001 (15). Furthermore, signals were shown on two overlapping fosmid clones (SE2 and LL13) of the linkage map of this region (6) when the linkage library was used as a hybridization template, which further confirmed the presence of the putative pal gene cluster in the left region of the linear chromosome. For comparison, total genomic DNA from another strain (A. tumefaciens C58) separated by pulsed-field gel electrophoresis was hybridized with the same probe containing all the open reading frames (ORFs) of the pal gene cluster. The putative pal gene cluster was also present only on the linear chromosome of the A. tumefaciens C58 strain. On the basis of Southern hybridization signals, this gene cluster was located in the SwaI-D macrorestriction fragment of the linear chromosome in the physical map of the C58 strain constructed by Goodner et al. in 1999 (8). Similarly, with respect to the PacI macrorestriction map of the linear chromosome of A. tumefaciens C58, the putative pal cluster was located between the PacI-B and PacI-F fragments.

Organization and genetic features of the putative pal gene cluster.

A 8.2-kb region was completely sequenced by the primer walking method (10). The entire putative pal gene cluster was contained in the overlapping fosmid clones SE2 and LL13 of the linkage map of the linear chromosome (Fig. 1a). Moreover, we cloned the putative pal gene cluster in a suicide vector, pK19mob (kindly provided by the National Institute of Genetics, Mishima, Japan); this resulted in two clones, pK19mob-palGKAB and pK19mob-palREFG. Sequence analysis and a subsequent homology search revealed seven ORFs belonging to the putative pal gene cluster and named palR, palE, palF, palG, palK, palA, and palB (Fig. 1b). The direction of transcription of the putative regulatory gene palR was opposite to that of the other pal genes, and palR was upstream of palE. Table 1 gives the proposed function and sequence similarity of each gene compared to the best-matching genes of S. meliloti, Erwinia rhapontici, and A. tumefaciens C58 strain. The gene organization and transcriptional direction of this region are highly similar to those of the putative thu gene cluster in S. meliloti, which is the best match identified by amino acid homology search. Despite the significant size variation in palG, palA, and palB, all the other ORFs show very slight size variations compared to those of S. meliloti.

FIG. 1.

Gene organization and genetic features of the putative pal gene cluster in A. tumefaciens MAFF301001. (a) Fosmid clones harboring the pal gene cluster. (b) Gene organization of the pal gene cluster. The direction of transcription is indicated by the arrowheads. (c) Physical maps for restriction sites of seven restriction enzymes in the pal region.

TABLE 1.

Characteristics of the ORFs in the pal gene cluster and comparison with the best-matching genes of S. meliloti and E. rhapontici and A. tumefaciens C58

A. tumefaciens MAFF301001			S. meliloti		E. rhapontici		A. tumefaciens C58
Gene or ORF	Gene or ORF size (no. of aaa)	Proposed function (best-matched gene/organism/accession no.b)	% aa identity	ORF size (no. of aa)	% aa identity	ORF size (no. of aa)	% aa identity	ORF size (no. of aa)
palR	275	Transcriptional regulator of the LacI-Ga1R family (putative thuR/ S. meliloti/dadAF175299-1)	56.4	338		298	98	354
palE	443	Palatinose binding protein (putative thuE/S. meliloti/dadAF175299-2)	76.6	424	39.7	423	94.6	452
palF	325	Inner membrane permease (putative thuF/S. meliloti/dadAF175299-3)	79.8	328	45.1	328	96.9	324
palG	189	Inner membrane permease (putative thuG/S. meliloti/dadAF175299-4)	78.4	276	49.6	289	95.5	277
palK	327	ATP binding protein (putative thuK/S. meliloti/dadAF175299-5)	75.8	342	61.7	370	94.1	342
palA	169	Utilization of palatinose (putative thuA/S. meliloti/dadAF175299-6)	86.3	270			98.8	263
palB	208	Utilization of palatinose (putative thuB/S. meliloti/dadAF175299-7)	65.1	365			98.1	349

^aaa, amino acids.

^bAccession numbers for DDBJ amino acid sequence data bank.

However, the palH, palQ, and palZ genes which exist downstream of the palG gene in the pal gene cluster of E. rhapontici (3) were not found in A. tumefaciens MAFF301001, and no such components were found in the DNA databases of related species such as A. tumefaciens C58 and S. meliloti.

The palR gene in E. rhapontici showed homology to the transcriptional regulator of the LysR family. However, PalR in A. tumefaciens MAFF301001 was homologous to the LacI-GalR family of transcriptional regulators and showed no homology with the regulatory protein of E. rhapontici. Figure 1(c) shows the restriction map for seven major restriction enzymes in the sequenced region. PalE, PalF, PalG, and PalK are probably components of the ABC transporter for palatinose, as shown in Table 1.

Growth on different carbon sources.

Because little information is available on palatinose transport and metabolism in plant-pathogenic bacteria, except for the recently identified pal gene cluster in E. rhapontici (3), the following physiological experiments were conducted using pal mutants.

A. tumefaciens mutant strains with palE::Gm^r and palK::Gm^r mutations were constructed by insertion of the Gm^r cassette. The palK mutation was placed upstream of the linker peptide and Walker B motif of the palK ORF according to the consensus sequence for the ABC transporter (2, 5). The mutants were tested for growth on six different sugars as the sole carbon source in AB minimal medium. There was no difference in the growth patterns of all the palK mutants grown with the sugars tested, except for palatinose, from that of the wild type (Fig. 2). The growth patterns of the palE mutants were also similar to that of the wild type under these growth conditions (data not shown). Both palK and palE mutants had almost no growth (98 and 95% growth reduction from the wild-type level, respectively) in medium containing palatinose. This growth pattern was quite similar to the growth of the wild type and palE and palK mutants when they were cultured in AB minimal medium without sugar. These observations prove that the gene cluster is critical for palatinose uptake and transport and that it affects bacterial growth. Furthermore, the growth patterns of palK mutants of A. tumefaciens C58 were identical to those of palK mutants of A. tumefaciens MAFF301001 (data not shown) and confirmed the importance of the gene cluster in the C58 strain as well.

FIG. 2.

Growth of palK mutants in six different sugars. The bacteria were grown on AB minimal medium with the indicated sugar as the sole carbon source. The number of days after inoculation is shown on the x axis, and optical density (OD) at 600 nm is shown on the y axis. The wild-type strain and the palK mutant were grown in minimal medium alone (□ and ▪, respectively) or in minimal medium supplemented with a sugar (▴ and ○, respectively). Each treatment was repeated at least three times. Means ± standard errors are shown.

Growth under high osmotic conditions.

Potential osmoprotectant properties of palatinose have been reported elsewhere (9). Considering the adverse microenvironmental conditions in the plant wound site during Agrobacterium infection (high pH and phenolic compounds), defects in tumorigenesis by the mutants were anticipated. Although the putative pal gene cluster was not essential for growth on trehalose, we investigated whether the gene cluster has an effect on trehalose uptake. The wild type and mutants were grown in AB minimal medium containing 0.5 M NaCl as the osmoticum and 1 mM trehalose as the osmoprotectant (9). The pal mutations had no effect on the growth of wild-type and mutant bacteria in medium containing the osmoticum plus trehalose or in medium lacking the osmoticum (data not shown), as shown by measurements of the optical density at 600 nm. These results further confirmed that the gene cluster has no effect on trehalose uptake.

Chemotaxis and virulence assay.

The sugar binding protein functions not only in substrate translocation but also in signal recognition in chemotaxis (7). Chemotaxis in a swarm agar plate (4) by the wild type and palE and palK mutants towards palatinose and other sugars is shown in Fig. 3. The absence of a growth zone towards palatinose by palE and palK mutants showed the inability of the bacteria to move toward sugar and to grow. These results offer additional support that the identified gene cluster is responsible for uptake and transport of palatinose.

FIG. 3.

Insertion mutation sites and chemotaxis characteristics of palE and palK mutants. (a) Restriction map of the pal gene cluster with restriction enzymes EcoRV, SphI, SacII, PstI, and BamHI. (b and c) Insertion sites of the Gm^r cassette on a palE mutant (b) and palK mutant (c). (d) Chemotaxis of the wild type and palE and palK mutants towards filter papers soaked in sugar. Panel A shows a typical growth pattern towards glucose, trehalose, maltose, galactose, or sucrose by the wild type (Wt) and palE and palK mutants. Panel B shows a typical growth pattern towards palatinose; a growth zone can be seen only for the wild type.

Inoculations with wild-type bacteria produced galls on inoculation sites including leaves and stems. Although only a slight decrease in tumorigenesis was observed when leaves were treated with palK and palE mutants and when the stems were treated with the palK mutant, mutants did not inhibit tumorigenesis (Table 2).

TABLE 2.

Tumorigenesis of A. tumefaciens pal mutants on kalanchoe plants

Strain (relevant genotype)	Tumorigenesis (%)a
	Stem	Leaf
MAFF301001 (wild type)	100	94
palK mutant (palK::Gmr)	88	88
palE mutant (palE::Gmr)	100	75

^aEight and 16 inoculations were done at stems and leaves, respectively. The number of tumors was counted after 4 weeks.

Palatinose as a substance advantageous to pathogens due to the inhibition of invertase activity has been reported elsewhere (3). Although inoculations of palK and palE mutants on kalanchoe plants did not inhibit tumorigenesis, the above data strongly support the effect of palatinose on growth of bacteria and therefore, the importance of the pal gene cluster in microbial survival with a range of energy sources.

Nucleotide sequence accession number.

The nucleotide sequence of the putative pal gene cluster has been deposited in the DDBJ/EMBL/GenBank databases under the accession no. AB074877.