Multiple Genes for the Last Step of Proline Biosynthesis in Bacillus subtilis

Abstract

The complete Bacillus subtilis genome contains four genes (proG, proH, proI, and comER) with the potential to encode ⌬ 1 -pyrroline-5-carboxylate reductase, a proline biosynthetic enzyme. Simultaneous defects in three of these genes (proG, proH, and proI) were required to confer proline auxotrophy, indicating that the products of these genes are mostly interchangeable with respect to the last step in proline biosynthesis.The pathway of proline synthesis from glutamate, the most common mechanism of proline biosynthesis, comprises three enzymatic steps (Fig. 1). The corresponding genes of Escherichia coli, proB, proA, and proC, encode ␥-glutamyl kinase, ␥-glutamyl phosphate reductase, and ⌬ 1 -pyrroline-5-carboxylate (P5C) reductase, respectively (21). The proBA-dependent pathway of proline synthesis was shown to function also in Bacillus subtilis; mutations within the proBA locus cause auxotrophy for proline (8,29). While B. subtilis has a single proAlike gene, a second proB-like gene, proJ of the proHJ locus (B. R. Belitsky and A. L. Sonenshein, GenBank accession number AF006720) has been found. In a manner unique to this bacterium, either ProB-like enzyme can provide enough ␥-glutamyl kinase activity to support growth in the absence of exogenous proline (unpublished results). Apparently, previously described mutations to auxotrophy in the proBA locus either affect proA or are proB alleles that are polar on proA expression. No proC mutant of B. subtilis has been described, and four genes have the potential to encode ProC-like proteins with P5C reductase activity: proH (also called orf257 and proC), comER (also called comED), proI (also called yqjO), and ykeA (here renamed proG) (1,14,20). The four genes are located at 172.3°, 225.5°, 211.2°, and 116.1°on the chromosomal map (http://genolist.pasteur.fr/SubtiList [26]) and code for proteins of 271, 273, 278, and 272 amino acids, respectively (the originally reported coding region of proH [1,20] was extended by resequencing the proH 3Ј end [GenBank accession number AF006720]). ProH and ProI are 42% identical to each other and up to 35% identical to many other P5C reductases from bacteria, archaea, and eukaryotes. ProG and ComER have more limited similarity to other P5C reductases and to each other. The functions of the four B. subtilis genes are not known. In this work we sought to identify the gene(s) responsible for the last step of proline biosynthesis.Construction and properties of a proG (ykeA) null mutant. To create pBB1081, the 1.56-kb PvuII-EcoRI fragment from pCM103 (23) containing most of the proG gene and the 5Ј end of the dppA gene was cloned between the PstI (blunt-ended) and EcoRI sites of pJPM1, a derivative of pBS (Stratagene) containing a chloramphenicol resistance marker (27). Methods for plasmid isolation, agarose gel electrophoresis, use of restriction and DNA modification enzymes, DNA ligation, PCR, Southern hybridization with digoxigenin-labeled DNA probes, and electroporation of E. coli JM107 or DH5␣ cells were as described by Sambrook et al....

T-box-mediated control of the anabolic proline biosynthetic genes of Bacillus subtilis

“…For the construction of a strain defective in all three genes encoding the enzyme for the last step in proline biosynthesis ( Fig. 1), chromosomal DNA of the B. subtilis strain BB1951 (Belitsky et al, 2001) carrying the D(proG : : ble) allele was transformed into strain JSB13, and zeocin-resistant transformants were selected; this yielded strain JSB41 [D(proI : :…”

“…Proline also serves as an important osmostress protectant (Bremer, 2002). For this task, proline can either be taken up by B. subtilis from the environment via the osmotically inducible OpuE transporter (Spiegelhalter & Bremer, 1998;von Blohn et al, 1997) or be produced in very large quantities from the precursor glutamate (Belitsky et al, 2001;Whatmore et al, 1990). Proline belongs to a selected group of organic osmolytes, the compatible solutes, which are amassed by many micro-organisms to adapt to high-osmolarity growth conditions (Kempf & Bremer, 1998).…”

“…1) (Belitsky et al, 2001). The activity of the ProB enzyme from B. subtilis is subjected to feedback control by proline (Chen et al, 2006), thereby tying proline production to proline consumption by the protein biosynthetic activities of the cell (Csonka & Leisinger, 2007).…”

“…The activity of the ProB enzyme from B. subtilis is subjected to feedback control by proline (Chen et al, 2006), thereby tying proline production to proline consumption by the protein biosynthetic activities of the cell (Csonka & Leisinger, 2007). Genetic studies have shown that in addition to the anabolic proline biosynthetic pathway formed by the ProB-ProA-ProI enzymes, a second route for proline biosynthesis is present in B. subtilis (Belitsky et al, 2001) that functions for the supply of the very large amounts of proline required for its use as an osmostress protectant (Bremer, 2002). This osmoadaptive proline biosynthetic route is formed by the ProJProA-ProH enzymes, and relies on isoenzymes for the first and last steps of the anabolic proline biosynthetic route (Fig.…”

“…In addition to the ProI and ProH enzymes, the ProG protein also possesses D 1 -pyrroline-5-carboxylase reductase activity ( Fig. 1), although the precise physiological function of the ProG protein is unresolved (Belitsky et al, 2001).…”