Mutants in the nodFEL promoter of Rhizobium leguminosarum bv. viciae reveal a role of individual nucleotides in transcriptional activation and protein binding

Okker, RJH Spaink, HP Lugtenberg, BJJ Schlaman, HRM

Rjh. Okker et al., Mutants in the nodFEL promoter of Rhizobium leguminosarum bv. viciae reveal a role of individual nucleotides in transcriptional activation and protein binding, ARCH MICROB, 175(2), 2001, pp. 152-160

24

INGLESE

Article

Microbiology

ARCHIVES OF MICROBIOLOGY

0302-8933 → ACNP

175

2

2001

152 - 160

ISI

0302-8933(200102)175:2<152:MITNPO>2.0.ZU;2-L

The highly conserved nod box sequence in the promoters of the inducible nod ulation genes of rhizobia is required for transcription activation together with NodD, a LysR-type transcriptional regulator, and a flavonoid as a coi nducer. DNA fragments containing nod box sequences form two binding complex es when crude preparations of Rhizobium leguminosarum by. viciae are used: a NodD-dependent and an additional, NodD-independent complex. The role of i ndividual nucleotides in the conserved nod box sequence in complex formatio n and in nodulation gene expression was investigated by introducing 13 indi vidual base-pair substitutions in the nodF nod box of R. leguminosarum bv. viciae and studying their effect on promoter activity and protein-DNA compl ex formation. Two mutants showed decreased NodD binding and decreased promo ter activity. Five mutants showed a NodD-dependent complex as with the wild -type nodF nod box, whereas their promoter activity was severely reduced af ter induction. This result is in agreement with earlier observations that N odD DNA binding also occurs in the absence of inducer. Four mutants were im paired in the formation of the NodD-independent retardation complex. Three of them showed no alterations in promoter activity, meaning that no specifi c role for the protein forming the NodD-independent complex could be establ ished. The two mutants in the highly conserved LysR motif of the nod box we re unable to direct coinducer-dependent promoter activity but, unexpectedly , their retardation patterns were not altered. The remaining two mutants sh owed constitutive promoter activity. The results are discussed in terms of the relevance of conserved nucleotides and motifs identified in the nod box .