Figure 5. Consequences of disrupting CdnL interaction with RNAP in vivo.

(A) Scheme of the strategy employed to check for cdnL complementation in M. xanthus. A pMR2873 construct with the required cdnL allele (cdnL*) under P_cdnL control and DNA segments flanking cdnL upstream (grey) and downstream (black) in the genome, was introduced into strain MR1467, which bears the ΔcdnL allele and a copy of cdnL at a heterologous site under the control of the photoinducible P_B promoter (expressed in the light but repressed in the presence of B₁₂ in the dark). Merodiploids resulting from plasmid integration by recombination at either “1” or “2” would constitutively express cdnL* from P_cdnL and conditionally express the wild-type allele from P_B at a heterologous site. (B) Complementation analyses in M. xanthus with the F36A, M49A and P51A CdnL mutants. CTT/B₁₂ plates that were spotted with 5 µl of liquid cultures (OD₅₅₀ ∼1) grown under permissive conditions and diluted as indicated, and then incubated for 2 days at 33°C under restrictive (dark and B₁₂) or permissive (light, hence the red color) conditions. “WT” is the positive control derived from using pMR2873 with wild-type cdnL, and “ΔcdnL” is the recipient strain MR1467. Western blots (using polyclonal anti-CdnL antibodies) of M. xanthus cell extracts from strains expressing each CdnL variant and in which CdnL-eGFP supplied the essential CdnL function, as described in the text. (C) BACTH analysis of the interaction of CdnL homologs (BbCdnL, ScCdnL, and CgCdnL) with Bbβ_31–538, Scβ_29–426, and Cgβ_32–428 fragments, respectively. “+” below the open bars indicates that both members of the pair are present, while “−” is the negative control (without the indicated CdnL homolog). We have shown the interaction of TtCdnL with its cognate β-fragment elsewhere [18]. (D) BACTH analysis of the interaction of BbCdnL, ScCdnL, CgCdnL or TtCdnL with M. xanthus Mxβ_19–537.