Effects of Mutations in the Pseudomonas putida miaA Gene: Regulation of the trpE and trpGDC Operons in P. putida by Attenuation

Abstract

Tn5 insertion mutants defective in regulation of the Pseudomonas putida trpE and trpGDC operons by tryptophan were found to contain insertions in the P. putida miaA gene, whose product (in Escherichia coli) modifies tRNA Trp and is required for attenuation. Nucleotide sequences upstream of trpE and trpG encode putative leader peptides similar in sequence to leader peptides found in other bacterial species, and the phenotypes of the mutants strongly suggest that transcription of these operons is regulated solely by attenuation.The arrangements of trp genes and their patterns of regulation differ widely among prokaryotes (8). The Escherichia coli trp operon, which consists of five contiguous genes-trpE, trp(G) D, trpC(F), trpB, and trpA-encoding seven enzymatic activities, is negatively regulated by TrpR in the presence of tryptophan and by the trp attenuator in the presence of acylated tRNA Trp (28). However, in Pseudomonas putida, Pseudomonas aeruginosa, and Pseudomonas syringae, the trpB and trpA genes, which encode the subunits of tryptophan synthase, comprise a separate operon that is positively regulated. In the presence of indoleglycerol phosphate (InGP), a substrate for tryptophan synthase, the trpBA operon is activated by the product of a gene, trpI, that is unique to these three species (1,6,19). Furthermore, trpE and trpGDC are transcribed independently (13,14).Isolation of unlinked mutations causing constitutive expression of P. putida and P. aeruginosa trpE, trpG, trpD, and trpC suggested that these genes, like their E. coli counterparts, were regulated by a TrpR-like repressor (5,20). However, we have isolated phenotypically similar mutants induced by Tn5 insertion; the mutations all disrupt the P. putida miaA homolog, whose product (in E. coli) modifies tRNA Trp and is required for attenuation (12,18,30). Assays of trp enzyme synthesis in the mutant strains strongly suggest that transcription of the trpE and trpGDC operons is regulated by attenuation and not by repression.Selection of P. putida 5-MT r mutants. P. putida strain M (22) was mutated by transposition of a mini-Tn5 lacZ1 transposon from the suicide plasmid pUT/mini-Tn5lacZ1, which encodes kanamycin resistance (11). Mutants were selected at 30°C on M9 minimal medium containing 200 g of 5-methyl-DL-tryptophan (5-MT)/ml, 20 g of kanamycin/ml, and 100 g of rifampin (to which P. putida, but not E. coli, is resistant)/ml. Five Kanr 5-MT r mutants (MTR1 through MTR5), chosen randomly from among 19 mutants that remained after a preliminary screening of more than 100 original isolates from several experiments, were assayed for expression of anthranilate synthase II (AS II) (trpG gene product); in the presence of exogenous tryptophan, all of the mutants were derepressed (data not shown). Strains MTR1 through MTR5 were also defective in utilization of phenylalanine (0.1%) as the sole carbon and energy source. Thus, the insertions appeared to cause defects in one or more general regulatory functions, rather than in a single function specific for trp ...

Ancient Origin of the Tryptophan Operon and the Dynamics of Evolutionary Change

“…Thus, in Rhizobium meliloti, the trpAa•trpAb operon is regulated by transcription attenuation but not the trpBD operon or the trpCEbEa operon (7). However, such a generalization may not be justified in consideration of P. aeruginosa and its close relatives P. putida, P. fluorescens, P. syringae, and Azotobacter vinelandii, in which transcription of the trpEbEa operon is activated by trpI (6,14) and the free-standing trpAa and the trpAbBD operon are regulated by attenuation (67).…”

“…67,2003 TRYPTOPHAN OPERON IN PROKARYOTES 305 have extended this nomenclature to the Trp pathway (89,92) (see Fig. 1 and Table 1).…”

“…The trpAa and trpAbBD operons are regulated by attenuation mechanisms employing leader peptides (67), whereas the trpEbtrpEa operon is controlled by an indoleglycerol phosphate-activated regulatory protein encoded by trpI (6). trpC is not known to be regulated in any way.…”

“…Therefore, if Helicobacter had VOL. 67,2003 TRYPTOPHAN OPERON IN PROKARYOTES 323 been the trp operon donor, one would expect Campylobacter to also have the fusion-containing trp operon. As pointed out before, the modern Helicobacter operon lacks repression control by trpR, presumably because trpR of the alien enteric lineage donor was unlinked to the transferred operon.…”

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84

“…One hypothesis for the impaired growth is that disruption of MiaA altered the biosynthesis and utilization of primary metabolites such as amino acids. For example, the absence of a functional MiaA in P. putida resulted in 30-fold higher expression of the tryptophan biosynthetic genes trpE and trpGCD regardless of the presence or absence of exogenous tryptophan [55]. In P. chlororaphis 30-84, the transcripts of genes involved in the biosynthesis and metabolism of anthranilate and tryptophan (aroF, antA, trpE, trpGD and iaaM) were significantly more abundant (two to eightfold) in 30-84MiaA than in the wild-type (Table 4).…”