The Rhodobacter sphaeroides 2.4.1 rho gene: expression and genetic analysis of structure and function

Abstract

The gene which encodes transcription termination factor Rho from Rhodobacter sphaeroides 2.4.1, the gram-negative facultative photosynthetic bacterium, has been cloned and sequenced. The deduced protein shows a high level of sequence similarity to other bacterial Rho factors, especially those from proteobacteria. However, several amino acid substitutions in the conserved ATP-binding site have been identified. When expressed in Escherichia coli, the R. sphaeroides rho gene relieves Rho-dependent polarity of the trp operon, indicating interference with the transcription termination machinery of E. coli. A truncated version of R. sphaeroides Rho (Rho') is toxic to a bacterium related to R. sphaeroides, Paracoccus denitrificans, and is lethal to R. sphaeroides. We suggest that toxicity is due to the ability of Rho' to form inactive heteromers with the chromosomally encoded intact Rho. We localized a minimal amino acid sequence within Rho which appears to be critical for its toxic effect and which we believe may be involved in protein-protein interactions. This region was previously reported to be highly conserved and unique among various Rho proteins. The lethality of rho' in R. sphaeroides together with our inability to obtain a null mutation in rho suggests that Rho-dependent transcription termination is essential in R. sphaeroides. This is analogous to what is observed for gram-negative E. coli and contrasts with what is observed for gram-positive Bacillus subtilis. The genetic region surrounding the R. sphaeroides rho gene has been determined and found to be different compared with those of other bacterial species. rho is preceded by orf1, which encodes a putative integral membrane protein possibly involved in cytochrome formation or functioning. The gene downstream of rho is homologous to thdF, whose product is involved in thiophene and furan oxidation.