Disease relevance of trfA

• In a previous study of mutations in trfA, the gene encoding the replication initiation protein of the broad host-range plasmid RK2, a carboxyl-terminal deletion of 3 amino acids of the TrfA protein was found to be completely nonfunctional for RK2 replication in Escherichia coli and other Gram-negative bacteria [1].
• The RK2 delta trfA mutant was found to be conjugation proficient, but unable to replicate in the RK2 hosts Acinetobacter calcoaceticus, Caulobacter crescentus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Pseudomonas putida, Rhizobium meliloti, or Rhodobacter sphaeroides [2].
• Here we describe the isolation of four trfA mutants temperature sensitive for replication in Pseudomonas aeruginosa [3].
• Utilizing deletion and site-specific mutagenesis to alter the trfA operon, we compared the replication of an RK2-origin plasmid in several distantly related gram-negative bacteria when supported by both TrfA-44 and TrfA-33, TrfA-33 alone, or TrfA-44/98L (a mutant form of the TrfA-44 protein) alone [4].
• Further characterization of the point and deletion trfA mutants in vivo revealed that a subset was capable of supporting RK2 replication in other gram-negative bacteria, including Pseudomonas putida, Agrobacterium tumefaciens, and Azotobacter vinelandii [5].

High impact information on trfA

• For replication, plasmid RK2 encodes a vegetative replication origin, oriV RK2, and a gene, trfA, whose polypeptide product(s) is essential for oriV RK2 activity [6].
• The trfA gene is transcribed as part of a polycistronic operon which also includes kilD [6].
• The trfAp-1 mutation reduces RNA polymerase binding and open complex formation at trfAp but does not stimulate melting at trbAp in vitro [7].
• korA function of promiscuous plasmid RK2: an autorepressor that inhibits expression of host-lethal gene kilA and replication gene trfA [8].
• Two TrfA proteins, 33 and 44 kDa in molecular mass (designated TrfA-33 and TrfA-44, respectively), are expressed from the trfA gene of RK2 through the use of two alternative in-frame start codons within the same open reading frame [9].

Biological context of trfA

• Interestingly, in contrast to all other IncP-1beta plasmids analysed so far, the oriV region between trfA and klcA is not interrupted by accessory genes, and there is no indication that previously inserted accessory genes have subsequently been deleted [10].
• The nucleotide sequence of this region shows the same two operator-like palindromes present in the previously sequenced promoters of trfA and korA [8].
• The positions of the trfA and trfB promoters of broad host range IncP plasmid RK2 (identical to RP1, RP4, R68 and R18 ) were identified by RNA polymerase protection studies, and the nucleotide sequences of the promoter regions determined [11].
• In addition, we describe the isolation and characterization of a mutant trfA promoter which has become insensitive to repression by trfB as a result of a point mutation within the inverted repeat sequence previously predicted to be the trfB protein binding site [12].
• These results show that trfA is essential for replication in these hosts and indicate that the broad host-range of RK2 does not involve multiple replicons [2].

Associations of trfA with chemical compounds

• The glycine 85 codon in trfA mRNA is GGU, and a change of this to GGC did not significantly affect expression [3].

Other interactions of trfA

• The kilB locus (which is unclonable in the absence of korB) of broad-host-range plasmid RK2 (60 kb) lies between the trfA operon (co-ordinates 16.4 to 18.2 kb), which encodes a protein essential for vegetative replication, and the Tra2 block of conjugative transfer genes (co-ordinates 20.0 to 27.0 kb) [13].
• KorA is known to repress transcription from seven promoters on the plasmid genome but at the trfA promoter, for the vegetative replication genes, it also causes derepression of trbAp, the first promoter for the operon whose genes are required primarily for mating pair formation prior to conjugative transfer [14].

Analytical, diagnostic and therapeutic context of trfA

• In this work site-directed mutagenesis of the trfA gene was carried out to construct TrfA proteins altered in the three carboxyl-terminal positions [1].
• Dissection of the switch between genes for replication and transfer of promiscuous plasmid RK2: basis of the dominance of trfAp over trbAp and specificity for KorA in controlling the switch [15].

References