Analysis of the Serratia marcescens proBA operon and feedback control of proline biosynthesis

Abstract

The proBA operon, coding for y-glutamyl b a s e (GK) and y-glutamyl phosphate reductase (GPR), was cloned from the chromosome of the wild-type strain of Serratia marcescens Sr41. From our sequence data the proB (1101 bp) and proA (1472 bp) genes were shown to code for two proteins of M, 39169 and 44640, respectively. Analysis of expression of the ZacZstructural gene fused with theproBA promoter showed that theproBA operon is not subject to proline-mediated feedback repression. Amplification of the proBA operon enabled us to determine GK activity, which was inhibited in the presence of a low concentration of L-proline. Comparison of the amino acid sequences of the S. marcescens GK and GPR proteins with those of the GI( and GPR proteins from E. coZi revealed extensive similarities.

Isolation, characterization, and chromosomal location of a gene encoding the Δ1‐pyrroline‐5‐carboxylate synthetase in Arabidopsis thaliana

“…In eubacteria, ~'-glutamyl kinase and T-glutamyl phosphate reductase which are encoded by the proBA operon form a multimolecular complex allowing a direct and protected transfer of the very labile ~,-glutamyl phosphate intermediate [13,14,30]. Interestingly in yeast, ?…”

“…To identify A. thaliana P5C synthetase cDNA(s), degenerate oligonucleotide primers in conserved sequences between P5CS cDNA from V aconitifolia [8], proB and proA sequences from E. eoli [13] and Serratia marcescens [14] (primer 1 = 5'-ATWGCYGATGCCCTKGAAGC-AAA-3'; primer 2 --5'-TCWSWGAATCTKGTGCTKGCRTT-3') were used in polymerase chain reactions (PCR) to amplify a DNA tYagment from genomic DNA ofA. thaliana ecotype Columbia.…”

“…Sequence comparison with the databases were performed using BLAST through the NCBI E-mail server [18]. Protein secondary structure analysis was determined by using the PredictProtein program (EMBL Heidelberg, Germany) [19,20] with multiple sequence alignments of the seven proteins showing highest similarity, P5CS from V. aconitifolia [8], y-glutamyl kinase and y-glutamyl phosphate reductase from E. coli [13], S. marcescens [14] and Bacillus subtilis [21]. Inclusion of protein homologs increases the prediction accuracy of the secondary structure to 70% [19].…”

Organization of threonine biosynthesis genes from the obligate methylotroph Methylobacillus flagellatus The GenBank accession numbers for the aspC-hom-thrC-thyA gene cluster and thrB gene sequences from M. flagellatus reported in this paper are L78665 and L78666, respectively.

“…Some or all genes for threonine biosynthesis have been cloned from several Gram-positive bacteria, several species of Corynebacterium (Mateos et al, 1987a, b ;Kalinowski et al, 1991 ;Follettie et al, 1993 ;Malumbres et al, 1994 ;), several species of Mycobacterium (Cirillo et al, 1994), Lactobacillus lactis (Le et al, 1996), Bacillus spp. (Bondaryk & 1985 ; Parsot & Cohen, 1988 ;Malumbres et al, 1995), Pseudomonas aeruginosa (Clepet et al, 1992), Thermus flavus (Nishiyama et al, 1995) and enterobacteria (Cohen & Saint-Girons, 1987 ;Omori et al, 1993). Organization of threonine biosynthesis genes in Haemophilus influenzae became known upon completion of its genome sequencing (Fleischmann et al, 1995).…”

Complete sequence and organization of the Serratia marcescens biotin operon

“…For monitoring the expression of biuA and bioB, ZacZ transcriptional/ translational fusions were constructed. Since amplification of the regulatory site of the genes in a high-copy-number plasmid might cause abnormal expression because of the absence of its repressor, we used pLGlacZ339, a low-copy-number promoter analysis vector, consisting of the pSClOl replicon and the promoterless facZ structural gene (Omori et al, 1991).…”