Cloning, genetic characterization, and nucleotide sequence of the hemA-prfA operon of Salmonella typhimurium

Abstract

The first step in heme biosynthesis is the formation of 5-aminolevulinic acid (ALA). Mutations in two genes, hemA and hemL, result in auxotrophy for ALA in Salmonella typhimurium, but the roles played by these genes and the mechanism of ALA synthesis are not understood. I have cloned and sequenced the S. typhimurium hemA gene. The predicted polypeptide sequence for the HemA protein shows no similarity to known ALA synthases, and no ALA synthase activity was detected in extracts prepared from strains carrying the cloned hemA gene. Genetic analysis, DNA sequencing of amber mutations, and maxicell studies proved that the open reading frame identified in the DNA sequence encodes HemA. Another surprising finding of this study is that hemA lies directly upstream of prfA, which encodes peptide chain release factor 1 (RF-1). A hemA::Kan insertion mutation, constructed in vitro, was transferred to the chromosome and used to show that these two genes form an operon. The hemA gene ends with an amber codon, recognized by RF-1. I suggest a model for autogenous control of prfA expression by translation reinitiation.

Purification and partial characterisation of barley glutamyl‐tRNAGlu reductase, the enzyme that directs glutamate to chlorophyll biosynthesis

“…The cloned DNA fragment was later sequenced, and shown to have an open reading frame which at the amino acid level showed 28.4% identity with the E. coli hemA gene product (Majumdar et al, 1991). Complementation cloning has been used to isolate the hemA gene from Salmonella typhimurium (Elliott, 1989), Bacillus subtilis (Petricek et al, 1990), Synechocystis (Grimm, 1992;Verkamp et al, 1992), Arabidopsis thaliana (Ilag et al, 1994), Coxiella burnetii (EMBL X78969), Clostridium josui (EMBL D28503) and barley (Grimm, B. and Bougri, O., unpublished results). Fig.…”

“…(Ilaget al, 1994); synts, Synechocystis (Grimm, 1992); ecoli, E. coli (Verkamp and Chelm, 1989); salty, S. Qphimuriurn (Elliott, 1989); coxbr, Coxiellu burnetii (Willems et al, 1994, EMBL database); chvib, C. vibriofonne (Majumdar et al, 1991); cljoj, C. josui (Fujino et al, 1994, EMBL database); and conse, consensus sequence. Amino acids are included in the consensus sequence, if they occur in six or more of the aligned sequences, or if the changes are considered conservative.…”

HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination

“…However, no direct evidence for such activity has been reported to date. In addition, whereas the tripartite operon structure is conserved only among some members of the ␥ subclass of proteobacteria (2,3), the link between the last two genes, prfA and hemK, is more widely conserved in eubacteria (4,5), implying a possible relationship of HemK to translation.…”

Light regulation of chlorophyll biosynthesis at the level of 5-aminolevulinate formation in Arabidopsis.

“…In an attempt to delineate the mechanism of action, genes encoding GluTR have been isolated by complementation of the E. coli hemA mutation from E. coli (Drolet et al, 1989;Li et al, 1989;Verkamp and Chelm, 1989), Sa/mone//a fyphimurium (Elliott, 1989), Bad / u s subfilis (Petricek et al, 1990), Chlorobium vibrioforme (Majumdar et al, 1991), and Synechocystis sp strain PCC 6803 (Grimm, 1992;Verkamp et al, 1992). However, corresponding genes from higher plants have not been reported.…”