TABLE 1.
Straina | Small-subunit rRNA patternsb | R. tropici plasmidsc | nifH gened | Acetylene reduction activity with P. vulgaris nodules (nmol of ethylene/h/plant)e
|
|
---|---|---|---|---|---|
14 days postinoculation | 18 days postinoculation | ||||
E. adhaerens transconjugants | |||||
CFNEA40 | ACEGIK | a, cf | + | 102.7 | NDg |
CFNEA41 | ACEGIK | a, b, c | + | 104.8 | ND |
CFNEA50 | ACEGIK | a, c | + | 108.0 | 84.9 |
CFNEA51 | ACEGIK | a, c | + | 106.9 | 85.9 |
CFNEA52 | ACEGIK | a, b, cf | + | 103.8 | 84.9 |
CFNEA53 | ACEGIK | a, b, c | + | 115.3 | 96.4 |
CFNEA54 | ACEGIK | b, c | + | 113.2 | 94.3 |
CFNEA55 | ACEGIK | b, c | + | 126.9 | 104.8 |
CFNEA56 | ACEGIK | b, c | + | 114.3 | 94.3 |
E. adhaerens ATCC 33499 | ACEGIK | ||||
R. tropici strains | |||||
CFN299 | BDFHJL | + | |||
CFN299 Tn5-mob-6 | 166.8 | ||||
CFN299 Tn5-mob-7 | 182.4 |
Only transconjugant CFNEA41 was derived from CFN299 Tn5-mob-7; all other transconjugants were derived from CFN299 Tn5-mob-6. R. etli CFN42 was also used in this study.
The different letters represent distinct patterns obtained with restriction enzymes HinfI, MspI, RsaI, HhaI, Sau3A1, and DdeI; each letter position refers to a different restriction enzyme used for PCR-synthesized 16S rRNA genes (15) obtained with primers rD1 and fD1 (29).
Eckhardt gel analysis was performed as modified (23) with liquid early-exponential-phase cultures in horizontal gels with sodium dodecyl sulfate in the agarose. The plasmid sizes were as follows: plasmid a, 185 kb; plasmid b, 220 kb; and plasmid c, 500 kb (nod and nif plasmid).
The presence of nifH was determined by Southern blot hybridization or by PCR synthesis with nif primers as described previously (6).
Acetylene reduction activity was determined 14 and 18 days postinoculation. The data are averages for five plants from one experiment. The plant-to-plant variation was not more than 10% for each transconjugant. The levels of nitrogen fixation with the E. adhaerens transconjugants were around 40% of the levels with the parental CFN299 strains in the four other experiments. Plants were maintained in a growth chamber at 28°C.
Plasmid cointegration or rearrangement occurred, which led to an approximately 700-kb plasmid or to an additional 300-kb plasmid.
ND, not determined.