Effect of truncation and mutation of C-terminal extension of
RpoE10 on the activation of abm-rpoE10 promoter:
In A. brasilense , the gene encoding RpoE10 is preceded by a gene
encoding an antibiotic biosynthesis monooxygenase (abm ) in a
dicistronic operon (abm-rpoE10 ). To compare the regulation of theabm-rpoE10 promoter with that of the other ECF41 σ dependent
promoters of R. sphaeroides and B. licheniformis13, we constructed an abm:gfp fusion (pAPD5) and
mobilized it into A. brasilense Sp245. Since ECF41 σ factors have
been shown to activate their own promoters, the full lengthrpoE10 was also cloned downstream of an IPTG inducible promoter
in a broad host range expression vector, pMMB206 (designated as pAPD7)
and mobilized in A. brasilense Sp245 harbouring pAPD5. We did not
find any significant increase in the fluorescence from abmpromoter even after inducing it with IPTG. Because of the suggested
negative role of the C-terminal extension of ECF41 σ factors8, we constructed two recombinants by cloning genes
encoding two different truncated derivatives of RpoE10 in pMMB206. While
construct RpoE10(Del1) had a deletion up to the DGGGR motif (pAPD8), the
other construct RpoE10(Del2) had deletion excluding DGGGR residues
(Figure 1B). When we mobilized these recombinant plasmids in A.
brasilense Sp245 harboringabm:gfp fusion, we found that
RpoE10(Del1), lacking 277NPDKV281motif with deletion up to200DGGGR204 motif, increased GFP
fluorescence by almost 15-fold after IPTG induction (Figure 1C). But,
RpoE10(Del2), which lacked the entire C-terminal region,including200DGGGR204and277NPDKV281 motifs of SnoaL, did
not show any promoter activity. To analyze the role of the conserved
motifs NPDKV and DGGGR, we also constructed two site-directed mutants,
RpoE10(Mut1) and RpoE10(Mut2), and mobilized them in A.
brasilense Sp245 harboringabm:gfp fusion. In the RpoE10(Mut1),
the 277NPDKV281 motif was replaced
with the NAAAV motif, and in RpoE10(Mut2), the200DGGGR204 motif was replaced with
the AAAGR motif. The effect of RpoE10(Mut1) and RpoE10(Mut2) on the
expression of abm:gfp was similar to that observed with
RpoE10(Del1) and RpoE10(Del2), respectively (Figure 1C).