Functional organization of MobB, a small protein required for efficient conjugal transfer of plasmid R1162

Abstract

MobB is a small (molecular weight = 15,097) protein encoded by the broad-host-range plasmid R1162 and is required for its efficient transfer by conjugation. The C-terminal half of the protein contains a membrane domain essential for transfer. This region can be replaced by a putative membrane domain from another, unrelated protein, and thus is likely to function independently from the rest of MobB. The other, functionally active region of MobB, identified by mutagenesis, is at the N-terminal end. One mutation affecting this region inhibits replication, suggesting that this part of the protein is contacting and sequestering the relaxase-linked primase. The overall organization reflects a multimeric and bipolar organization, with molecules of MobB anchored in the membrane at one end and engaging the relaxase at the other. This arrangement could increase the transfer frequency by raising the probability of contact between the relaxase and the membrane-embedded, coupling protein for type IV secretion.

Fig. 1.

Two sets of plasmids (A and B) containing a cloned copy of mobB. The parental vector is indicated in each case. In set A, expression of mobB is from a T5 promoter under lac control. MobB and derivatives all contain a terminal 6×his tag. In set B, the gene for tetracycline resistance (tet) has been inactivated by mobB, and transcription is from the tet promoter. Below the general plasmid maps are the different MobB proteins encoded by each set. Changes in the protein (see Fig. 2 for details) are indicated in parentheses: deletions are designated according to the missing amino acids, restriction enzymes refer to sites for these enzymes introduced into the coding sequence, and MD-MobX refers to the putative membrane domain of MobX.

Fig. 2.

Primary amino acid sequence of MobB protein, showing the changes in this sequence caused by the different mutations, with a dash indicating a missing amino acid. Changes due to new restriction sites are labeled by the restriction enzyme in parentheses. The membrane domain of MobB is indicated by the rectangle above the sequence. Fragments of MobX and FrdD cloned to form a hybrid protein with MobB are underlined. The membrane domains are also indicated by the rectangles. The domain of MobX is putative and based on amino acid sequence (25); the domain of FrdD is from Westenberg et al. (35).

Fig. 3.

Effect of different MobB proteins, expressed in trans, on the transfer frequency of R1162ΔmobB and R1162mobBΔ(Q48-L101). Frequencies are expressed as transconjugants per donor cell. The results are the averages of three to six independent determinations. The vertical bars indicate the standard deviations.

Fig. 4.

Western gel analysis of His-tagged MobB proteins. In addition to the plasmid encoding MobB-his, a second plasmid encoding FrdD-his was also present. FrdD is the membrane anchor subunit of fumarate reductase and is a well-characterized integral membrane protein (35). Membrane (M) and cytoplasmic (C) fractions are shown.

Fig. 5.

Increase in cell density after induced overexpression of mobB or a mutated allele, cloned into pQE60 (Fig. 1). The increase in OD was measured at 2 h postinduction. The results are the averages of three to five independent determinations. The vertical bars indicate the standard deviations.

Fig. 6.

Transfer frequencies of pUT2070 (R1162ΔmobB) from donor strains also containing cloned mobB. The differences in the complementing MobB protein are shown on the horizontal axis. The results are the averages of three independent trials with the standard deviation indicated by the vertical bars.

Fig. 7.

Transformation of MV10(pUT2070) with pACYC184 or with a derivative containing a cloned mobB. Transformed cultures were diluted 1:100 (no MobB, no mutation, or K8A K9A) or left undiluted, and 50 μl was spotted onto a plate containing chloramphenicol and streptomycin (25 μg of each).