O-antigen conversion in Pseudomonas aeruginosa PAO1 by bacteriophage D3.

The lysogenization of Pseudomonas aeruginosa PAO by phage D3 results in derivatives which are resistant to superinfection by phage D3c by virtue of the fact that homologous phage cannot adsorb to these cells. The serologically and morphologically unrelated phage E79 showed a markedly decreased adsorption rate to the lysogen PAO(D3). Since both of these phages are lipopolysaccharide specific, these results suggested lysogenic conversion of the phage receptor. The lipopolysaccharide was extracted from strain PAO by the hot phenol-water technique, but this procedure was ineffective with PAO(D3). We developed a technique involving cold trichloroacetic acid extraction, followed by ultracentrifugation, digestion of the high-speed pellet with proteinase K, and ultimate purification on CsCl step gradients. The lipopolysaccharide from the wild type had inactivating activity against D3 and E79, whereas that from PAO(D3) inactivated neither. Chromatographic analysis indicated that the convertant lipopolysaccharide was smooth, and quantitative chemical analyses of the two preparations showed no differences in the level of the major fatty acids, amino compounds, or neutral sugars. On the other hand, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the side chains had a decreased migration rate through the gel matrix. The application of 1H and 13C nuclear magnetic resonance spectroscopic analysis revealed that the PAO side chain is chemically identical to that of serotype O:2a,d, containing 2,3-(1-acetyl-2-methyl-2-imidazolino-5,4)-2,3-dideoxy-D-mannuronic acid, 2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, and 2-acetamido-2,6-dideoxy-D-galactose (D-fucosamine). The molecular basis of the conversion event was (i) the introduction of an acetyl group into position 4 of the fucosamine residue and a change in the bonding between trisaccharide repeating units from alpha 1 leads to 4 to beta 1 leads to 4.     

Biochemical characterization of WbpA, a UDP-N-acetyl-D-glucosamine 6-dehydrogenase involved in O-antigen biosynthesis in Pseudomonas aeruginosa PAO1

WbpA (PA3159) is an enzyme involved in the biosynthesis of unusual di-N-acetyl-d-mannosaminuronic acid-derived sugar nucleotides found in the O antigen of Pseudomonas aeruginosa PAO1 (serotype O5). The wbpA gene that encodes this enzyme was cloned into pET-28a, overexpressed as a histidine-tagged fusion protein, and purified by nickel chelation chromatography. Capillary electrophoresis was used to examine substrate conversion by WbpA, and the data revealed that WbpA is a UDP-N-acetyl-D-glucosamine 6-dehydrogenase (EC 1.1.1.136), which uses NAD(+) as a coenzyme. The enzyme reaction product was purified by HPLC and analyzed using NMR spectroscopy. Our results showed unequivocally that the product of the WbpA reaction is UDP-N-acetyl-d-glucosaminuronic acid. WbpA requires either NH(4)(+) or K(+) for activity and the accompanying anions exert secondary effects on activity consistent with their ranking in the Hofmeister series. Kinetic analysis showed positive cooperativity with respect to UDP-N-acetyl-d-glucosamine binding with a K(0.5) of 94 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.8. In addition, WbpA has a K(0.5) for NAD(+) of 220 microM, a k(cat) of 86 min(-1), and a Hill coefficient of 1.1. The oligomerization state of WbpA was analyzed by gel filtration, dynamic light scattering, and analytical ultracentrifugation, with all three techniques indicating that WbpA exists as a trimer in solution. However, tertiary structure predictions suggested a tetramer, which was supported by data from transmission electron microscopy. The electron micrograph of negatively stained WbpA samples revealed structures with 4-fold symmetry.     

Quorum sensing regulates denitrification in Pseudomonas aeruginosa PAO1

Anaerobic growth of Pseudomonas aeruginosa PAO1 was affected by quorum sensing. Deletion of genes that produce N-acyl-l-homoserine lactone signals resulted in an increase in denitrification activity, which was repressed by exogenous signal molecules. The effect of the las quorum-sensing system was dependent on the rhl quorum-sensing system in regulating denitrification.     

Studies on the Pseudomonas aeruginosa PAO1 bacteriophage receptors

Receptor for phage PIK specific for Pseudomonas aeruginosa strain PAO1 was studied. Phage PIK was strongly inactivated by lipopolysaccharide (LPS) in vitro, exhibiting a PhI50 of 4.8 micrograms/ml. Further it was noted that this inactivation by LPS was reduced to 50% by several mono- and disaccharides when tested in vitro. D-glucosamine, D-mannose and L-rhamnose were found to be most effective at the concentration of 0.045 M, 0.25 M and 0.35 M respectively. This suggests the possibility that phage PIK receptor in LPS contains D-mannose, L-rhamnose and D-glucosamine. Either one of the former two could be located at a terminal position alpha-linked to the adjacent residue or located internally in the polysaccharide chain linked through its C-4 position. A theoretical approach to the interpretation of phage cell interaction was also investigated.     

Basic characterization of a Pseudomonas aeruginosa PAO1 bacteriophage

A bacteriophage of Pseudomonas aeruginosa PAO1 was characterized. Bacteriophage PIK was found to adsorb on the cell wall of the host organism. Electron microscopy of the phage PIK revealed that it had a bipyramidal hexagonal prismatic head of 110 nm in diameter, a tail which was 158 nm long and a tail plate of 47 nm width. This paper describes its basic characters, and a quantitative study was made of its adsorption to exponential phase cells of two different strains of P. aeruginosa. PIK was found to contain double stranded DNA and it appears to be virulent towards its host, P. aeruginosa PAO1. It was classified into the group of phages possessing a contractile tail.     

Genome Diversity of Pseudomonas aeruginosa PAO1 Laboratory Strains

Pseudomonas aeruginosa PAO1 is the most commonly used strain for research on this ubiquitous and metabolically versatile opportunistic pathogen. Strain PAO1, a derivative of the original Australian PAO isolate, has been distributed worldwide to laboratories and strain collections. Over decades discordant phenotypes of PAO1 sublines have emerged. Taking the existing PAO1-UW genome sequence (named after the University of Washington, which led the sequencing project) as a blueprint, the genome sequences of reference strains MPAO1 and PAO1-DSM (stored at the German Collection for Microorganisms and Cell Cultures [DSMZ]) were resolved by physical mapping and deep short read sequencing-by-synthesis. MPAO1 has been the source of near-saturation libraries of transposon insertion mutants, and PAO1-DSM is identical in its SpeI-DpnI restriction map with the original isolate. The major genomic differences of MPAO1 and PAO1-DSM in comparison to PAO1-UW are the lack of a large inversion, a duplication of a mobile 12-kb prophage region carrying a distinct integrase and protein phosphatases or kinases, deletions of 3 to 1,006 bp in size, and at least 39 single-nucleotide substitutions, 17 of which affect protein sequences. The PAO1 sublines differed in their ability to cope with nutrient limitation and their virulence in an acute murine airway infection model. Subline PAO1-DSM outnumbered the two other sublines in late stationary growth phase. In conclusion, P. aeruginosa PAO1 shows an ongoing microevolution of genotype and phenotype that jeopardizes the reproducibility of research. High-throughput genome resequencing will resolve more cases and could become a proper quality control for strain collections.The metabolically versatile Pseudomonas aeruginosa is an opportunistic pathogen of plants, animals, and humans and is ubiquitously distributed in soil and aquatic habitats. The common reference strain is P. aeruginosa PAO1, a spontaneous chloramphenicol-resistant mutant of the original PAO strain (earlier called “P. aeruginosa strain 1”) that had been isolated in 1954 from a wound in Melbourne, Australia (9, 10). This PAO1 strain from Bruce Holloway''s laboratory has become the reference strain for Pseudomonas genetics and functional analyses of the physiology and metabolism of this gammaproteobacterium. A genetic map of its chromosome was generated by exploiting the mechanisms of gene exchange in bacteria, i.e., transduction and conjugation (11). With the advent of pulsed-field gel electrophoresis (PFGE), a physical map of the PAO1 genome was constructed (32) and later merged with the genetic map information (12). By 2000 the PAO1 strain had been completely sequenced (36). Thereafter, the genome annotation has been continually updated and the database content and functionality have been expanded to facilitate accelerated discovery of P. aeruginosa drug targets and vaccine candidates (38). Two near-saturation libraries of transposon insertion mutants have been constructed in P. aeruginosa PAO1 as a global resource for the scientific community (14, 22).Comparison of the genome sequence with the physical map revealed a large, 2.2-Mb inversion between the sequenced PAO1-UW strain (36) and the original PAO1 strain (9, 10), indicating that PAO1 sublines maintained worldwide in numerous laboratories and strain collections had diversified their genomic sequence. Mutational events were already reported in the 1970s (10), and more recently sequence variations of MexT, which regulates the MexEF-OprN multidrug efflux system, were described (18, 24). Furthermore, a PAO1 subline from a German strain collection (PAO1-D) and another, independent PAO1 subline from a Japanese strain collection (PAO1-J) that had been stored by research groups in Germany and Japan, respectively, were found to be quorum-sensing-negative mutants that carried point mutations in the regulatory gene lasR (6). In addition, spontaneous secretion-defective vfr mutants from a PAO1 population were observed after several cycles of static growth (2). Similarly, we noted a difference in virulence in a mouse infection model (see below) between the MPAO1 and PAO1-DSM sublines that had been utilized for the construction of the transposon library (14) and the physical map (32), respectively. PAO1-DSM was indistinguishable in its SpeI-DpnI-SwaI-PacI physical map from the PAO1 subline that had been stored in the Holloway laboratory (12). Hence, we decided to compare the genomic sequence of the initially sequenced PAO1 subline PAO1-UW (36) with that of MPAO1 and PAO1-DSM. Combined physical mapping and DNA sequencing-by-synthesis revealed numerous single-nucleotide polymorphisms (SNPs) and insertions-deletions (indels) in the chromosomes that were associated with differences in fitness, antimicrobial susceptibility, and virulence of the sublines.     

Characterization of nutrient-induced dispersion in Pseudomonas aeruginosa PAO1 biofilm

The processes associated with early events in biofilm formation have become a major research focus over the past several years. Events associated with dispersion of cells from late stage biofilms have, however, received little attention. We demonstrate here that dispersal of Pseudomonas aeruginosa PAO1 from biofilms is inducible by a sudden increase in carbon substrate availability. Most efficient at inducing dispersal were sudden increases in availability of succinate > glutamate > glucose that led to approximately 80% reductions in surface-associated biofilm biomass. Nutrient-induced biofilm dispersion was associated with increased expression of flagella (fliC) and correspondingly decreased expression of pilus (pilA) genes in dispersed cells. Changes in gene expression associated with dispersion of P. aeruginosa biofilms were studied by using DNA microarray technology. Results corroborated proteomic data that showed gene expression to be markedly different between biofilms and newly dispersed cells. Gene families that were upregulated in dispersed cells included those for flagellar and ribosomal proteins, kinases, and phage PF1. Within the biofilm, genes encoding a number of denitrification pathways and pilus biosynthesis were also upregulated. Interestingly, nutrient-induced dispersion was associated with an increase in the number of Ser/Thr-phosphorylated proteins within the newly dispersed cells, and inhibition of dephosphorylation reduced the extent of nutrient-induced dispersion. This study is the first to demonstrate that dispersal of P. aeruginosa from biofilms can be induced by the addition of simple carbon sources. This study is also the first to demonstrate that dispersal of P. aeruginosa correlates with a specific dispersal phenotype.     

Outer membrane protein shifts in biocide-resistant Pseudomonas aeruginosa PAO1

Benzisothiazolone (BIT), N-methylisothiazolone (MIT) and 5-chloro-N-methylisothiazolone (CMIT) are highly effective biocidal agents and are used as preservatives in a variety of cosmetic preparations. The isothiazolones have proven efficacy against many fungal and bacterial species including Pseudomonas aeruginosa. However, some species are beginning to exhibit resistance towards this group of compounds after extended exposure. This experiment induced resistance in cultures of Ps. aeruginosa exposed to incrementally increasing sub-minimum inhibitory concentrations (MICs) of the isothiazolones in their pure chemical forms. The induced resistance was observed as a gradual increase in MIC with each new passage. The MICs for all three test isothiazolones and a thiol-interactive control compound (thiomersal) increased by approximately twofold during the course of the experiment. The onset of resistance was also observed by reference to the altered presence of an outer membrane protein, designated the T-OMP, in SDS-PAGE preparations. T-OMP was observed to disappear from the biocide-exposed preparations and reappear when the resistance-induced cultures were passaged in the absence of biocide. This reappearance of T-OMP was not accompanied by a complete reversal of induced resistance, but by a small decrease in MIC. The induction of resistance towards one biocide resulted in the development of cross-resistance towards other members of the group and the control, thiomersal. It has been suggested that the disappearance of T-OMP from these preparations is associated with the onset of resistance to the isothiazolones in their Kathon form (CMIT and MIT).     

Sequence-Verified Two-Allele Transposon Mutant Library for Pseudomonas aeruginosa PAO1

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library.     

In vitro transcription analysis of rpoD in Pseudomonas aeruginosa PAO1

Thymidine kinase type II is an important part of the pyrimidine salvage pathway. The thymidine kinase gene from the thermophilic eubacterium Rhodothermus marinus was cloned, sequenced and overexpressed. The gene is 639 bp and encodes a protein of 213 amino acids with a calculated molecular mass of 23.6 kDa. It shows homology to other thymidine kinase proteins from eukaryotic and prokaryotic organisms. The recombinant protein is inhibited by dNTPs but not by dNDPs. It is a tetramer in its native state. Its optimum temperature of activity is 65 degrees C and it has a half life of 15 min at 90 degrees C. This is the first thymidine kinase to be described from a thermophilic bacterium.     

Characterization of the 2-ketogluconate utilization operon in Pseudomonas aeruginosa PAO1

The Pseudomonas aeruginosa protein PtxS negatively regulates its own synthesis by binding to the upstream region of its gene. We have recently identified a 14 bp palindromic sequence within the ptxS upstream region as the PtxS operator site (OP1). In this study, we searched the P. aeruginosa genomic sequence to determine whether this 14 bp sequence exists in other regions of the P. aeruginosa chromosome. Another PtxS operator site (OP2) was located 47 bp downstream of ptxS. DNA gel shift experiments confirmed that PtxS specifically binds to a 520 bp fragment that carries OP2. The DNA segment 3' of OP2 contains four open reading frames (ORF1-ORF4), which code for 29, 32, 48 and 35 kDa proteins respectively. The molecular weight of the products of ORFs 2 and 3 were confirmed by T7 expression experiments. Computer analyses suggest that ORF2 encodes an ATP-dependent kinase; ORF3, a transporter; and ORF4, a dehydrogenase. The predicted product of ORF1 showed no homology to previously identified proteins and contains all the conserved amino acids within the aldose 1-epimerase protein motif. Examination of the ptxs-ORF1 intergenic region (using promoter fusion experiments) showed that no potential promoter exists. An isogenic mutant defective in ORF1 was constructed in the P. aeruginosa strain PAO1. In contrast to its parent strain, the mutant failed to grow on a minimal medium in which 2-ketogluconate was the sole carbon source. Similarly, a previously constructed ptxS isogenic mutant of PAO1 did not grow in a minimal medium containing 2-ketogluconate as the sole carbon source. Furthermore, a plasmid carrying a fragment that contains ptxS and ORFs 1-4 complemented the defect of the previously described P. aeruginosa 2-ketogluconate-negative mutant. In the presence of 10 mM 2-ketogluconate, the in vitro binding of PtxS to a DNA fragment that carries either OP1 or OP2 was inhibited. These results suggest that: (i) ptxS together with the other four ORFs constitute the 2-ketogluconate utilization operon (kgu) in P. aeruginosa. Therefore, ORFs 1-4 were designated kguE, kguK, kguT and kguD respectively. (ii) PtxS regulates the expression of the kgu operon by binding to two operators (OP1 and OP2) within the operon; and (iii) 2-ketogluconate is the molecular inducer of the kgu operon or the molecular effector of PtxS.     

Pseudomonas aeruginosa PAO1 kills Caenorhabditis elegans by cyanide poisoning

In this report we describe experiments to investigate a simple virulence model in which Pseudomonas aeruginosa PAO1 rapidly paralyzes and kills the nematode Caenorhabditis elegans. Our results imply that hydrogen cyanide is the sole or primary toxic factor produced by P. aeruginosa that is responsible for killing of the nematode. Four lines of evidence support this conclusion. First, a transposon insertion mutation in a gene encoding a subunit of hydrogen cyanide synthase (hcnC) eliminated nematode killing. Second, the 17 avirulent mutants examined all exhibited reduced cyanide synthesis, and the residual production levels correlated with killing efficiency. Third, exposure to exogenous cyanide alone at levels comparable to the level produced by PAO1 killed nematodes with kinetics similar to those observed with bacteria. The killing was not enhanced if hcnC mutant bacteria were present during cyanide exposure. And fourth, a nematode mutant (egl-9) resistant to P. aeruginosa was also resistant to killing by exogenous cyanide in the absence of bacteria. A model for nematode killing based on inhibition of mitochondrial cytochrome oxidase is presented. The action of cyanide helps account for the unusually broad host range of virulence of P. aeruginosa and may contribute to the pathogenesis in opportunistic human infections due to the bacterium.     

Rhamnolipid surfactant production affects biofilm architecture in Pseudomonas aeruginosa PAO1

In response to certain environmental signals, bacteria will differentiate from an independent free-living mode of growth and take up an interdependent surface-attached existence. These surface-attached microbial communities are known as biofilms. In flowing systems where nutrients are available, biofilms can develop into elaborate three-dimensional structures. The development of biofilm architecture, particularly the spatial arrangement of colonies within the matrix and the open areas surrounding the colonies, is thought to be fundamental to the function of these complex communities. Here we report a new role for rhamnolipid surfactants produced by the opportunistic pathogen Pseudomonas aeruginosa in the maintenance of biofilm architecture. Biofilms produced by mutants deficient in rhamnolipid synthesis do not maintain the noncolonized channels surrounding macrocolonies. We provide evidence that surfactants may be able to maintain open channels by affecting cell-cell interactions and the attachment of bacterial cells to surfaces. The induced synthesis of rhamnolipids during the later stages of biofilm development (when cell density is high) implies an active mechanism whereby the bacteria exploit intercellular interaction and communication to actively maintain these channels. We propose that the maintenance of biofilm architecture represents a previously unrecognized step in the development of these microbial communities.     

A physical genome map of Pseudomonas aeruginosa PAO.

A complete macrorestriction map of the 5.9 Mb genome of Pseudomonas aeruginosa PAO (DSM 1707) was constructed by the combination of various one- and two-dimensional pulsed field gel electrophoresis techniques. A total of 51 restriction sites (36 SpeI sites, 15 DpnI sites) were placed on the physical map yielding an average resolution of 110 kb. Several genes encoding virulence factors and enzymes of metabolic pathways were located on the anonymous map by Southern hybridization. Distances between the gene loci were similar on the genetic and physical maps, suggesting an even distribution of genome mobility throughout the bacterial chromosome. The four rRNA operons were organized in pairs of inverted repeats. The two-dimensional macro-restriction techniques described herein are generally applicable for the genome mapping of any prokaryote and lower eukaryote which yields resolvable fragment patterns on two-dimensional pulsed field gels.     

Comparative analysis of two UDP-glucose dehydrogenases in Pseudomonas aeruginosa PAO1

UDP-glucose dehydrogenase (UGDH) catalyzes a two-step NAD(+)-dependent oxidation of UDP-glucose to produce UDP-glucuronic acid, which is a common substrate for the biosynthesis of exopolysaccharide. Searching the Pseudomonas aeruginosa PAO1 genome data base for a UGDH has helped identify two open reading frames, PA2022 and PA3559, which may encode a UGDH. To elucidate their enzymatic identity, the two genes were cloned and overexpressed in Escherichia coli, and the recombinant proteins were purified. Both the gene products are active as dimers and are capable of utilizing UDP-glucose as a substrate to generate UDP-glucuronic acid. The K(m) values of PA2022 and PA3559 for UDP-glucose are approximately 0.1 and 0.4 mM, whereas the K(m) values for NAD(+) are 0.5 and 2.0 mM, respectively. Compared with PA3559, PA2022 exhibits broader substrate specificity, utilizing TDP-glucose and UDP-N-acetylglucosamine with one-third the velocity of that with UDP-glucose. The PA2022 mutant and PA2022-PA3559 double mutant, but not the PA3559 mutant, are more susceptible to chloramphenicol, cefotaxime, and ampicillin. The PA3559 mutant, however, shows a reduced resistance to polymyxin B compared with wild type PAO1. Finally, real time PCR analysis indicates that PA3559 is expressed primarily in low concentrations of Mg(2+), which contrasts with the constitutive expression of PA2022. Although both the enzymes catalyze the same reaction, their enzymatic properties and gene expression profiles indicate that they play distinct physiological roles in P. aeruginosa, as reflected by different phenotypes displayed by the mutants.     

Genetics of O-antigen biosynthesis in Pseudomonas aeruginosa.

Pathogenic bacteria produce an elaborate assortment of extracellular and cell-associated bacterial products that enable colonization and establishment of infection within a host. Lipopolysaccharide (LPS) molecules are cell surface factors that are typically known for their protective role against serum-mediated lysis and their endotoxic properties. The most heterogeneous portion of LPS is the O antigen or O polysaccharide, and it is this region which confers serum resistance to the organism. Pseudomonas aeruginosa is capable of concomitantly synthesizing two types of LPS referred to as A band and B band. The A-band LPS contains a conserved O polysaccharide region composed of D-rhamnose (homopolymer), while the B-band O-antigen (heteropolymer) structure varies among the 20 O serotypes of P. aeruginosa. The genes coding for the enzymes that direct the synthesis of these two O antigens are organized into two separate clusters situated at different chromosomal locations. In this review, we summarize the organization of these two gene clusters to discuss how A-band and B-band O antigens are synthesized and assembled by dedicated enzymes. Examples of unique proteins required for both A-band and B-band O-antigen synthesis and for the synthesis of both LPS and alginate are discussed. The recent identification of additional genes within the P. aeruginosa genome that are homologous to those in the A-band and B-band gene clusters are intriguing since some are able to influence O-antigen synthesis. These studies demonstrate that P. aeruginosa represents a unique model system, allowing studies of heteropolymeric and homopolymeric O-antigen synthesis, as well as permitting an examination of the interrelationship of the synthesis of LPS molecules and other virulence determinants.     

Size of the Chromosome of Pseudomonas aeruginosa PAO

Electron microscope examination and velocity sedimentation analysis of the deoxyribonucleic acid released from Pseudomonas aeruginosa spheroplasts indicate that this organism carries the bulk of its genetic determinants in a single duplex deoxyribonucleic acid molecule having a molecular mass of 2.1 x 10(9) daltons.     

Regulation of proline catabolism in Pseudomonas aeruginosa PAO

Mutants of Pseudomonas aeruginosa deficient in the utilization of l-proline as the only carbon and nitrogen source have been found to be defective either in proline dehydrogenase activity or in both proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities of the bifunctional proline degradative enzyme. The latter type of mutants was unable to utilize l-ornithine, indicating that a single ¹-pyrroline-5-carboxylate dehydrogenase activity is involved in the degradation of ornithine and proline. Proline dehydrogenase and ¹-pyrroline-5-carboxylate dehydrogenase activities were strongly and coordinately induced by proline. It was excluded that ¹-pyrroline-5-carboxylate acted as an inducer of the bifunctional enzyme and it was shown that the low level induction observed during growth on ornithine was due to the intracellular formation of proline. The formation of the proline degradative enzyme was shown to be subject to catabolite repression by citrate and nitrogen control.Abbreviations EMS Ethylmethane sulfonate - NG N-methyl-N-nitro-N-nitrosoguanidine - P Minimal medium P - Pro-DH Proline dehydro-genase - P5C ¹-Pyrroline-5-carboxylate - P5C-DH ¹-Pyrroline-5-carboxylate dehydrogenase