Characterization of Pentapeptide Insertion Mutants

1SUPPLEMENTAL MATERIAL

4Characterization of pentapeptide insertion mutants

5 Cgs-region (1-418). The functional role of the extreme N-terminus of Cgs is not

6known although in silico analysis of the Cgs-region (1-418) predicted a possible protein-

7protein interaction module (2). In agreement with the motility phenotype, all insertions in

8this region were active or partially active for the synthesis of CG (Fig. 1B and S2A)

9indicating that this region can tolerate pentapeptide insertions without significant loss of

10activity. The inactive mutant Cgs-344 (Fig. 1B) did not produce a protein readily detected

11by Coomassie Blue-staining SDS-PAGE (Fig. S2B) or immunobloting (data not shown);

12therefore, it was not considered in the analysis. Interestingly, Cgs-160 and Cgs-302

13insertions produced CG with a DP higher than that of the wild type; this phenotype is

14similar to that previously observed with truncated or some pentapeptide insertion mutants

15in the C-terminal region of Cgs, which is involved in the control of CG DP (1). In these

16mutants an important reduction of the incorporation of [14C]-glucose in the protein

17intermediate was observed; probably do to instability of the proteins (Fig. S2).

18Furthermore, in Cgs-313 and Cgs-315 mutants the synthesis of CG was significantly

19reduced but the formation of the intermediate was less affected indicating that in these

20mutants glucose residues are incorporated in the linear -1-2-glucooligosaccharide

21protein-linked intermediate but are not efficiently released through the cyclization

22reaction (Fig. S2). Taken together these results suggest a role of this region in protein-

23protein interaction. We hypothesize that the N-terminal domain may be interacting with

1 1 24the cyclization and phosphorylase domains at the C-terminal region allowing the protein

25to reach the spatial conformation required for the catalysis of these reactions.

26 Cgs-region (475-818). Cgs pentapeptide insertion mutants Cgs-638, Cgs-681,

27Cgs-686, Cgs-728, Cgs-751, Cgs-778 and Cgs-785 were inactive while mutant Cgs-696

28was partially active for the synthesis of CG (Fig. S3A); these results are in agreement

29with the motility phenotype (Fig. 1B). Furthermore, in these mutants no incorporation of

30[14C]-glucose in the -1,2-glucooligosaccharide protein-linked intermediate was detected

31(mutants 638, 728, 751, 778 and 785) or it was significantly reduced (mutants 681, 686

32and 696) (Fig. S3B). These results are in agreement with those previously obtained after

33site-directed disruption of the GT-84 domain (2), which result in protein failure to

34catalyze the initiation and/or elongation reaction/s required for the synthesis of the linear

35-1,2-glucoologosaccharide protein-linked intermediate.

36 Cgs-region (870-938). In this small cytoplasmic loop only five pentapeptide

37insertion mutants were isolated (Fig. 1B). Four of them were inactive for the synthesis of

38CG in vivo; while surprisingly Cgs-918 was active in vivo but not in vitro (Fig. S4). In

39addition, Cgs-907 mutant did not produce a protein readily detected by Coomassie Blue-

40staining SDS-PAGE but it was still active for the incorporation of [14C]-glucose in the -

411,2-glucooligosaccharide protein-linked intermediate (Fig. S4B). These results indicate

42that this region may be structurally and/or functionally important for the synthesis of

43CG.

44 Cgs-region (1545-2867). Characterization of pentapeptide insertion mutants that

45map in this region was previously described (1). In agreement with the motility

46phenotype (Fig. 1B), all these mutants were active for the synthesis of CG.

2 2 47Legends to figures

49Figure S1: Screening: motility assay. Individual clones were inoculated in 96 wells

50plates and grown at 28°C during 48 h. These cultures were used as inoculums for the

51motility assay which was carried out in yeast extract-mannitol medium (3) with 0.3%

52agar using a 96 well replica plater. Plus sign, motile; minus sign, non-motile. A1045, A.

53tumefaciens A1045 mutant strain (chvB::Tn5). A1045 (pBA24), A1045 strain harboring

54the plasmid pBA24 which expresses wild type Cgs. A1045 strain overproduces

55exopolysaccharide (4) that accumulates on the surface of the plate.

57Figure S2: In vivo and in vitro characterization of Cgs-region (1-418) pentapeptide

58insertion mutants. (A) TLC analysis of CG produced by A1045 strain harboring the

59indicated plasmid. * and **, migration of anionic and neutral CG, respectively. In this

60system, slower migration of neutral-CG is higher DP. (B) Incorporation of [14C]-glucose

61into Cgs. Permeabilized cells of A. tumefaciens A1045 strain harboring the indicated

62plasmid were incubated with UDP-[14C]glucose during 20 min at 28C. TCA-insoluble

63fractions were analyzed by Coomassie Blue-staining 10% SDS-PAGE (upper panel) and

64the radioactivity was detected by autoradiography (lower panel). The position of

65molecular mass standard (in kDa) is indicated on the left. The arrows on the right indicate

66the position of wild type Cgs. pBA24, plasmid expressing wild type Cgs.

68Figure S3: In vivo and in vitro characterization of Cgs-region (475-818) pentapeptide

69insertion mutants. See legend of Fig. S2.

3 3 70Figure S4: In vivo and in vitro characterization of Cgs-region (870-938) pentapeptide

71insertion mutants. See legend of Fig. S2.

4 4 72References

741. Ciocchini, A. E., L. S. Guidolin, A. C. Casabuono, A. S. Couto, N. Iñón de

75 Iannino, and R. A. Ugalde. 2007. A glycosyltransferase with a length-

76 controlling activity as a mechanism to regulate the size of polysaccharides. Proc

77 Natl Acad Sci U S A 104:16492-7.

782. Ciocchini, A. E., M. S. Roset, G. Briones, N. Iñón de Iannino, and R. A.

79 Ugalde. 2006. Identification of active site residues of the inverting

80 glycosyltransferase Cgs required for the synthesis of cyclic beta-1,2-glucan, a

81 Brucella abortus virulence factor. Glycobiology 16:679-91.

823. de Iannino, N. I., and R. A. Ugalde. 1993. Biosynthesis of cyclic beta-(1-

83 3),beta-(1-6) glucan in Bradyrhizobium spp. Arch Microbiol 159:30-8.

844. Geremia, R. A., S. Cavaignac, A. Zorreguieta, N. Toro, J. Olivares, and R. A.

85 Ugalde. 1987. A Rhizobium meliloti mutant that forms ineffective pseudonodules

86 in alfalfa produces exopolysaccharide but fails to form beta-(1----2) glucan. J

87 Bacteriol 169:880-4.