Mapping of the multiple regulatory sites for putP and putA expression in the putC region of Escherichia coli

Summary The effects of regulatory proteins on the expression of putP and putA were studied using put-lacZ fusion genes. The expression of the putP-lacZ gene was activated by the glnG gene product and the catabolite gene activator protein (CAP). The putA gene product inhibited activation of putP-lacZ gene expression by CAP or the glnG gene product and its inhibition was greater in the absence of proline. The expression of the putA-lacZ gene was activated by CAP and repressed by the glnG gene product. The putA gene product acted as a repressor in the absence of proline, but not in its presence. Studies using put-lacZ fusion genes with upstream deletions showed that the region required for the activation of putP by CAP was within 234 bp upstream of the translational initiation site and that that for the activation of putP was within 107 bp upstream of the translational initiation site of the putA gene. This supported the suggested locations of CAP binding sites. The region required for induction of putP and putA expression by proline was located at the Hpal site 182 bp upstream of the translational starting site of putA, suggesting that a sequence of dyad symmetry located 1 bp to the left of the HpaI site is a candidate for the binding site of the putA gene product.Abbreviations AC L-azetidine-2-carboxylic acid - Ap ampicillin - CAP catabolite gene activator protein - NR_I nitrogen regulator I - RF DNA DNA replicative form - Str streptomycin - Tc tetracycline - TTC 2,3,5-triphenyl tetrazolium chloride - UV ultraviolet     

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12

Summary Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate(pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product).Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.Abbreviations pCMB p-chloromercuribenzoate - DM Davis and Mingioli - Ap ampicillin - NTG N-methyl-N-nitro-N-nitrosoguanidine - EMS ethylmethane sulfonate - Str streptomycin - Tet tetracycline - Ac l-azetidine-2-carboxylic acid - DHP 3, 4-dehydro-d,l-proline - MTT 3-(4,5-dimethyl-2)2,5-diphenyl tetrazolium bromide - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - Kan kanamycin - Spc spectinomycin     

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in Escherichia coli

The oxidation of l-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the ΔkatG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity.     

Molecular cloning of the SUF2 frameshift suppressor gene from Saccharomyces cerevisiae

A genetic approach to the molecular cloning of frameshift suppressor genes from yeast is described. These suppressors act by suppressing +1 G:C base-pair insertion mutations in glycine or proline codons. The cloning regimen involves an indirect screen for yeast transformants which harbor a functional suppressor gene inserted into the autonomously replicating “shuttle” vector YEp13, followed by transfer of the hybrid plasmid from yeast into Escherichia coli. Using this procedure a 10.7-kb DNA fragment carrying the SUF2 frameshift suppressor gene has been isolated. This suppressor acts specifically on +1 G:C insertions in proline codons. When inserted into an integrative vehicle and reintroduced into yeast by transformation, this fragment integrates by homologous recombination in the region of the SUF2 locus on chromosome III. A large proportion of the fragment overlaps with another cloned DNA segment which carries the closely linked CDC10 gene. The SUF2 fragment carries at least two tRNA genes. The SUF2 gene and one of the tRNA genes are located on a 0.85-kb restriction fragment within the 10.7-kb segment. A method is also described for the isolation of DNA fragments carrying alternative alleles of the SUF2 locus. Using this procedure, the wild-type suf2⁺ allele has been cloned.     

Purification and characterization of Put1p from Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the PUT1 and PUT2 genes are required for the conversion of proline to glutamate. The PUT1 gene encodes Put1p, a proline dehydrogenase (PRODH) enzyme localized in the mitochondrion. Put1p was expressed and purified from Escherichia coli and shown to have a UV-visible absorption spectrum that is typical of a bound flavin cofactor. A K_m value of 36 mM proline and a k_cat = 27 s⁻¹ were determined for Put1p using an artificial electron acceptor. Put1p also exhibited high activity using ubiquinone-1 (CoQ₁) as an electron acceptor with a k_cat = 9.6 s⁻¹ and a K_m of 33 μM for CoQ₁. In addition, knockout strains of the electron transfer flavoprotein (ETF) homolog in S. cerevisiae were able to grow on proline as the sole nitrogen source demonstrating that ETF is not required for proline utilization in yeast.     

Construction and Environmental Release of a Sinorhizobium meliloti Strain Genetically Modified To Be More Competitive for Alfalfa Nodulation

Highly efficient nitrogen-fixing strains selected in the laboratory often fail to increase legume production in agricultural soils containing indigenous rhizobial populations because they cannot compete against these populations for nodule formation. We have previously demonstrated, with a Sinorhizobium meliloti PutA⁻ mutant strain, that proline dehydrogenase activity is required for colonization and therefore for the nodulation efficiency and competitiveness of S. meliloti on alfalfa roots (J. I. Jiménez-Zurdo, P. van Dillewijn, M. J. Soto, M. R. de Felipe, J. Olivares, and N. Toro, Mol. Plant-Microbe Interact. 8:492–498, 1995). In this work, we investigated whether the putA gene could be used as a means of increasing the competitiveness of S. meliloti strains. We produced a construct in which a constitutive promoter was placed 190 nucleotides upstream from the start codon of the putA gene. This resulted in an increase in the basal expression of this gene, with this increase being even greater in the presence of the substrate proline. We found that the presence of multicopy plasmids containing this putA gene construct increased the competitiveness of S. meliloti in microcosm experiments in nonsterile soil planted with alfalfa plants subjected to drought stress only during the first month. We investigated whether this construct also increased the competitiveness of S. meliloti strains under agricultural conditions by using it as the inoculum in a contained field experiment at León, Spain. We found that the frequency of nodule occupancy was higher with inoculum containing the modified putA gene for samples that were analyzed after 34 days but not for samples that were analyzed later.     

A New Genetic Locus in Sinorhizobium meliloti Is Involved in Stachydrine Utilization

Stachydrine, a betaine released by germinating alfalfa seeds, functions as an inducer of nodulation genes, a catabolite, and an osmoprotectant in Sinorhizobium meliloti. Two stachydrine-inducible genes were found in S. meliloti 1021 by mutation with a Tn5-luxAB promoter probe. Both mutant strains (S10 and S11) formed effective alfalfa root nodules, but neither grew on stachydrine as the sole carbon and nitrogen source. When grown in the absence or presence of salt stress, S10 and S11 took up [¹⁴C]stachydrine as well as wild-type cells did, but neither used stachydrine effectively as an osmoprotectant. In the absence of salt stress, both S10 and S11 took up less [¹⁴C]proline than wild-type cells did. S10 and S11 appeared to colonize alfalfa roots normally in single-strain tests, but when mixed with the wild-type strain, their rhizosphere counts were reduced more than 50% (P ≤ 0.01) relative to the wild type. These results suggest that stachydrine catabolism contributes to root colonization. DNA sequence analysis identified the mutated locus in S11 as putA, and the luxAB fusion in that gene was induced by proline as well as stachydrine. DNA that restored the capacity of mutant S10 to catabolize stachydrine contained a new open reading frame, stcD. All data are consistent with the concept that stcD codes for an enzyme that produces proline by demethylation of N-methylproline, a degradation product of stachydrine.     

Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes

Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. K _m values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity. Received: 27 February 1997 / Accepted: 24 April 1997     

Dissecting the molecular mechanism of ion-solute cotransport: Substrate specificity mutations in theputP gene affect the kinetics of proline transport

Summary Rare mutations that alter the substrate specificity of proline permease cluster in discrete regions of theputP gene, suggesting that they may replace amino acids at the active site of the enzyme. IfputP substrate specificity mutations directly alter the active site of proline permease, the mutants should show specific defects in the kinetics of proline transport. In order to test this prediction, we examined the kinetics of threeputP substrate specificity mutants. One class of mutation increases theK _m over 120-fold but only decreases theV _max fourfold. SuchK _m mutants may be specifically defective in substrate recognition, thus identifying an amino acid critical for substrate binding. Another class of mutation decreases theV _max 80-fold without changing theK _m .V _max mutants appear to alter the rate of substrate translocation without affecting the substrate binding site. The last class of mutation alters both theK _m andV _max of proline transport. These results indicate that substrate specificity mutations alter amino acids critical for Na⁺/proline symport.     

Additional sex combs-like 1 belongs to the enhancer of trithorax and polycomb group and genetically interacts with Cbx2 in mice

The Additional sex combs (Asx) gene of Drosophila behaves genetically as an enhancer of trithorax and polycomb (ETP) in displaying bidirectional homeotic phenotypes, suggesting that is required for maintenance of both activation and silencing of Hox genes. There are three murine homologs of Asx called Additional sex combs-like1, 2, and 3. Asxl1 is required for normal adult hematopoiesis; however, its embryonic function is unknown. We used a targeted mouse mutant line Asxl1^tm1Bc to determine if Asxl1 is required to silence and activate Hox genes in mice during axial patterning. The mutant embryos exhibit simultaneous anterior and posterior transformations of the axial skeleton, consistent with a role for Asxl1 in activation and silencing of Hox genes. Transformations of the axial skeleton are enhanced in compound mutant embryos for the polycomb group gene M33/Cbx2. Hoxa4, Hoxa7, and Hoxc8 are derepressed in Asxl1^tm1Bc mutants in the antero-posterior axis, but Hoxc8 expression is reduced in the brain of mutants, consistent with Asxl1 being required both for activation and repression of Hox genes. We discuss the genetic and molecular definition of ETPs, and suggest that the function of Asxl1 depends on its cellular context.     

CcpA Regulates Arginine Biosynthesis in Staphylococcus aureus through Repression of Proline Catabolism

Staphylococcus aureus is a leading cause of community-associated and nosocomial infections. Imperative to the success of S. aureus is the ability to adapt and utilize nutrients that are readily available. Genomic sequencing suggests that S. aureus has the genes required for synthesis of all twenty amino acids. However, in vitro experimentation demonstrates that staphylococci have multiple amino acid auxotrophies, including arginine. Although S. aureus possesses the highly conserved anabolic pathway that synthesizes arginine via glutamate, we demonstrate here that inactivation of ccpA facilitates the synthesis of arginine via the urea cycle utilizing proline as a substrate. Mutations within putA, rocD, arcB1, argG and argH abolished the ability of S. aureus JE2 ccpA::tetL to grow in the absence of arginine, whereas an interruption in argJBCF, arcB2, or proC had no effect. Furthermore, nuclear magnetic resonance demonstrated that JE2 ccpA::ermB produced ¹³C₅ labeled arginine when grown with ¹³C₅ proline. Taken together, these data support the conclusion that S. aureus synthesizes arginine from proline during growth on secondary carbon sources. Furthermore, although highly conserved in all sequenced S. aureus genomes, the arginine anabolic pathway (ArgJBCDFGH) is not functional under in vitro growth conditions. Finally, a mutation in argH attenuated virulence in a mouse kidney abscess model in comparison to wild type JE2 demonstrating the importance of arginine biosynthesis in vivo via the urea cycle. However, mutations in argB, argF, and putA did not attenuate virulence suggesting both the glutamate and proline pathways are active and they, or their pathway intermediates, can complement each other in vivo.     

Isolation and expression analysis of proline metabolism-related genes in Chrysanthemum lavandulifolium

Proline plays a significant role in plant resistance to abiotic stresses, and its level is determined by a combination of synthesis, catabolism and transport. The primary proteins involved are Δ¹-pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (PDH) and proline transporter (ProT). To utilise proline metabolism to improve the stress resistance of Chrysanthemum × morifolium, we isolated two P5CS-homologous genes (ClP5CS1 and ClP5CS2), one PDH gene (ClPDH) and four ProT-homologous genes (ClProT1-4) (GenBANK accession numbers: KF743136–KF743142) from Chrysanthemum lavandulifolium, which is closely related to chrysanthemums and exhibits strong resistance to stresses. Expression analysis of these genes in different organs and under various stresses indicated that ClP5CSs showed substantial constitutive expression, while ClPDH was only strongly expressed in the capitulum and was inhibited under most stresses. The expression patterns of four ClProT genes presented characteristics of organ specificity and disparity under stresses. Above all, the expression of ClProT2 was restricted to above-ground organs, especially strong in the capitulum and could be obviously induced by various stress conditions. Promoters of ClPDH and ClProTs contained many cis-acting regulatory elements involved in stress responses and plant growth and development. High levels of free proline were found in flower buds, the capitulum under the non-stress condition and later periods of stress conditions except cold treatment. Interestingly, organ specificity and disparity also exist in the level of free proline under different stress conditions. Our study indicates that ClProTs play significant roles in proline accumulation and stress responses, and that ClProT2 could be used to genetically modify the stress resistance of chrysanthemums. In addition, proline metabolism might be closely related to plant flowering and floral development.     

The biosynthesis of proline in Escherichia coli phosphate-dependent glutamate γ-semialdehyde dehydrogenase (NADP), the second enzyme in the pathway

Extracts of Escherichia coli contain an enzyme which catalyzes the oxidation of glutamic γ-semialdehyde. This reaction is dependent upon the presence of inorganic phosphate, and utilizes NADP⁺ as a cofactor. The enzyme is subject to neither end-product inhibition nor repression by proline, and strains which lack the second enzyme in the pathway, lack this reaction.     

Intraspecific variation of the crotamine and crotasin genes in Crotalus durissus rattlesnakes

Crotamine is a small basic myotoxin peptide of Crotalus durissus venom, with β-defensin scafold and variable concentration in individual venoms. The crotamine gene was mapped to the end of chromosome 2 and the signal intensity differed significantly between the two homologues. In contrast to crotamine, the paralogous crotasin gene is scarcely expressed in the venom glands. In this study, we analyzed the crotamine concentrations in the venoms of a total of 23 rattlesnakes from diverse Brazilian localities by ELISA as well as the copy number of both crotamine and crotasin genes by real-time PCR. Crotamine was found to constitute 5–29% of venom proteins varying greatly among individual animals. The crotamine gene exists from 1 to 32 copies per haploid genome, whereas the crotasin gene is present from 1 to 7 copies. Furthermore, we observed that the crotamine concentration and crotamine gene copy number are positively correlated (r² = 0.68), implying the variation of crotamine in venom results from the variation of the gene copy number. Sequencing of 50 independent copies of crotamine and crotasin genes from four different rattlesnakes revealed the presence of six crotasin isoforms with a single amino acid difference from the original crotasin sequence, whereas only two additional crotamine isoforms were observed. Taken together, our results suggested that after duplication from a common ancestor gene, crotamine and crotasin may have diverged in such a way that the crotamine gene underwent repetitive duplication to increase its copy number, whereas the crotasin gene diversified its sequence.