Article Figures & Data
Figures
- Fig. 1.
SDS-PAGE comparison of relative amounts of LPS I and LPS II in strains CE166, CE166α, and wild-type CE3. SDS extracts of washed cell pellets were loaded to give roughly equal amounts of total LPS. The band that migrates faster than LPS II comigrates with purified lipid A. Lanes: 1, CE3; 2, CE166; 3, CE166α.
- Fig. 2.
The R. etli CE3 lps α andlps-166 genetic regions. The lps α locus is a contiguous stretch of DNA shown on the left. The dotted line indicates that lps-166 is separated from the lps α locus by an undetermined distance along the chromosome. Ticks on the upper line indicate the positions of EcoRI sites. The relative locations of the Tn5 insertions of four mutants in this study are indicated by arrows above this line. The lines below indicate stretches of DNA from lps α that are contained in the specified plasmids. The lps α DNA responsible for the suppression of lps-166 is located in the region indicated by the striped box.
- Fig. 3.
SDS-PAGE of SDS extracts of washed cell pellets of strains showing variation in LPS I content and the same strains after complementation with lps region α. Lanes: 1, wild-type CE3; 2, CE395; 3, CE395α; 4, CE394; 5, CE394α.
- Fig. 4.
Proposed structure of LPS I of R. etli CE3 (16, 17). Features affected in mutants CE166 and CE395 are noted. Designation as O antigen is arbitrarily assigned to all contiguous residues released with the Kdo most distal to lipid A after mild hydrolysis that cleaves at Kdo residues (16, 23). Chemical analysis indicates that Fuc residues are only partially O-methylated at the positions shown (16). R, the various hydroxyfatty acyl groups that are ester- and amide-linked at the positions indicated in the lipid A portion; TOMFuc, tri-O-methylated Fuc; 3M6dTal, 3-O-methyl-6-deoxytalose; QuiNAc, N-acetylquinovosamine; GlcON, 2-aminogluconate. All other abbreviations are defined in Table2.
Tables
- Table 1.
Strains and plasmids used in this study
Straina or plasmidb Relevant characteristic(s) Reference(s) or source Strains CE3 R. etli wild-type LPS and symbiosis 33 CE166 lps-166::Tn5, QuiN− LPS, low I/IIc 10 CE166α CE166 carrying pCOS109.11 This work CE374 lps-233::Tn5 32, 45 CE394 lps-394::Tn5, altered LPS and I/II This work CE394α CE394 carrying pCOS109.11 This work CE395 lps-395::Tn5, altered LPS and I/II This work CE395α CE395 carrying pCOS109.11 This work CE408d mTn5SSgusA20in CE3 This work CE433d mTn5SSgusA20in CE109 (lacks LPS I) This work CE434d mTn5SSgusA20in CE166 This work CE436d mTn5SSgusA20in CE166α This work CE451 lps-166::Tn5 in CE3 by homogenotization This work Plasmids pCOS109.11 R. etli CE3lps region α in 31-kb insert 10, 29 pDEL2 22-kb DNA from pCOS109.11 insert 11 pDEL2-3 14-kb DNA from pDEL2 insert 11 pDEL14 16-kb DNA from pCOS109.11 insert 11 pDEL21 9-kb DNA from pCOS109.11 insert 11 pEQ166 pJQ200 carrying the 11-kb lps-166::Tn5 EcoRI fragment This work pJQ200 Vector for homogenotization; GmsacB 40 ↵a Strain CE3 is a streptomycin-resistant derivative of R. etli nodule isolate CFN42 (33). All other strains were derived from CE3.
↵b All R. etli DNA inserts, except in pEQ166, were carried in vector pLAFR1 (18).
↵c I/II, LPS I/LPS II ratio. The apparent ratios of LPS I/LPS II of the strains decrease in the following order: CE3 and CE374 (equal ratios), CE395, CE166, CE394.
↵d mTn5SSgusA20 was inserted at unknown locations after introduction into the respective parent strains on plasmid pCAM120 (47). These strains were used to compare extents of infection. Their LPS SDS-PAGE and symbiotic phenotypes were the same as those of respective parent strains (CE3, CE109, CE166, and CE166α) (data not shown).
- Table 2.
Sugar compositions of purified LPSs
Sugarb Molar ratioa found in strain: CE3 CE166 CE166α CE395 QuiN 0.6 ND ND 0.4 2OMe6Dhx 0.9 0.4 0.8 ND 3OMe6Dhx 2.9 1.3 2.5 1.6 Fuc 3.6 1.4 2.6 2.2 GlcA 3.0 1.7 2.4 1.9 Man 1.9 1.4 1.7 1.5 Gal 1.0 1.0 1.0 1.0 GalA 2.9 2.8 2.9 3.0 Kdo 2.1 1.9 2.2 — GlcN 0.8 0.9 0.9 0.9 ↵a Entries are molar ratios of the sugars normalized to 1.0 galactose. They are the mean values from five parallel analyses of CE3, CE166, and CE166α LPS and three additional parallel analyses of CE3 and CE166 LPS in two laboratories over the course of 10 years. In the data for these three strains, the average standard deviation of the mole ratios before normalization was 21% of the mean value. The CE395 data are mean values from two determinations. ND, not detected (<0.01); —, obscured by TMS methyl glycoside peaks due to residual EDTA in the CE395 LPS after Sepharose 4B chromatography.
↵b The sugars in boldface type are found in the portion of the R. etli CE3 designated as the O antigen (16, 17). Abbreviations: 2OMe6Dhx, 2-O-methyl-6-deoxyhexose; 3OMe6Dhx, 3-O-methyl-6-deoxy hexose; Fuc, fucose; Man, mannose; Gal, galactose; GalA, galacturonic acid; GlcN, glucosamine. Migration with methylated standards indicates that 2OMe6Dhx and 3OMe6Dhx are 2-O-methyl-fucose and 3-O-methyl-6-deoxytalose, respectively, in strain CE3 (16) (Fig. 4).
- Table 3.
Symbiotic performance of complemented and suppressed strains
Time and inoculant AR/planta Nodules/plantc Specific ARb No. wt 14 d.a.i. CE3 100 60 45 100 CE166α 1 12 7 10 CE166αNd 2 11 6 16 CE374 2 16 10 11 CE394α 42 45 29 67 CE395α 36 52 32 53 20 d.a.i. CE3 100 63 78 100 CE166e <0.1 25 2 <0.1 CE166α 29 17 39 59 26 d.a.i. CE3 100 68 120 100 CE166e <0.1 60 —f <0.1 CE166α 25 18 71 42 CE374 92 31 88 123 CE395α 110 48 123 112 CE394α 123 50 125 116 ↵a AR (acetylene reduction) normalized to values exhibited by strain CE3 (which is set to 100).
↵b AR per total nodule fresh weight, expressed as percent of the wild-type value.
↵c Number and total fresh weight (mg) of the nodules per plant.
↵d An isolate from nodules of CE166α.
↵e Representative of results with CE394 and CE395.
↵f —, small pseudonodules (not weighed).
