Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.     

枯草芽孢杆菌耐盐突变株proA基因克隆及proBA基因渗透压调节功能研究

利用TaKaRaLAPCRTM试剂盒扩增枯草芽孢杆菌 931 5 1耐盐突变株proA基因的未知下游序列。根据测序结果 ,设计引物 ,克隆出发菌株和突变株全长proBA基因。将出发菌株和突变株的proBA基因分别转化大肠杆菌JM83(proBA- ) ,均能够与其功能互补。SDS PAGE分析其表达产物 ,有两条分子量分别约为 4 0kD和 4 5kD的新蛋白带出现。测定 4种转化子 (分别含有出发菌株和突变株proB基因的大肠杆菌 1 1 2 5 2转化子及proBA基因的大肠杆菌JM83转化子 )的耐盐能力。发现含有突变株proB或proBA基因转化子的耐盐能力 ,均比相应的含有出发菌株proB或proBA基因的转化子高。另外含有出发菌株和突变株的proBA基因转化子的耐盐能力 ,也均比相应的仅含proB基因的转化子高 ,表明枯草芽孢杆菌的ProA比大肠杆菌的ProA更为有效。测定所有JM83转化子胞内自由脯氨酸 ,发现其含量随盐浓度的上升而提高 ,其中含突变菌株proBA基因的转化子提高更为显著     

Analysis of the Escherichia coli proBA locus by DNA and protein sequencing

A 2.9 kb DNA fragment carrying the Escherichia coli proBA region, which encodes the first two enzymes of the proline biosynthetic pathway, was subcloned onto an expression plasmid carrying both the bacteriophage lambda PL promoter (lambda PL) and the lambda gene encoding a thermolabile cI repressor protein (cI857). Derepression of the lambda PL promoter by thermal inactivation of the cI857 repressor protein resulted in the simultaneous overproduction of the proB (gamma-glutamyl kinase) and proA (gamma-glutamyl phosphate reductase) gene products. Nucleotide sequence analysis of the proBA locus allowed gene assignments consistent with the NH2 and COOH-terminal analyses and amino acid compositions of homogeneous preparations of the proB and proA proteins. The contiguous nature of the proB and proA genes suggests that the two genes constitute an operon in which proB precedes proA.     

Identification and characterization of the proBA operon of Streptococcus bovis.

A genomic DNA library of the rumen bacterium Streptococcus bovis was constructed in Escherichia coli, and recombinant plasmids able to complement proA and proB mutations of the host were found. Southern hybridization and restriction analysis showed that a 3.5-kb fragment of S. bovis DNA contained two genes, organized in an operon and coding for enzymes functionally similar to the glutamyl phosphate reductase-glutamyl kinase enzyme complex that in E. coli catalyzes the first step of proline biosynthesis. Complementation of the E. coli mutations was observed with the fragment inserted in both orientations, which suggested that the S. bovis proBA operon was transcribed from its own promoter. Genetic and biochemical data suggested that the proline biosynthetic pathway of S. bovis is similar to the one previously characterized for E. coli.     

Expression of Bacillus subtilis proBA genes and reduction of feedback inhibition of proline synthesis increases proline production and confers osmotolerance in transgenic Arabidopsis

Proline accumulation has been shown to correlate with tolerance to drought and salt stresses in plants. We attempt to introduce the wild-type, mutant, and fusion proBA genes derived from Bacillus subtilis into Arabidopsis thaliana under the control of a strong promoter cauliflower mosaic virus 35S (CaMV35S). The transgenic plants produced higher level of free proline than control and the overproduction of proline resulted in the increased tolerance to osmotic stress in transgenic plants. Besides, the mutation in proBA genes, which were proved to lead gamma-glutamyl kinase (gamma-GK) reduces sensitivity to the end-product inhibition and the fusion of proB and proA also result in increasing proline production and confer osmotolerance in transgenic lines.     

Mutations in the Corynebacterium glutamicum proline biosynthetic pathway: a natural bypass of th proA step.

Two chromosomal loci containing the Corynebacterium glutamicum ATCC 17965 proB and proC genes were isolated by complementation of Escherichia coli proB and proC auxotrophic mutants. Together with a proA gene described earlier, these new genes describe the major C. glutamicum proline biosynthetic pathway. The proB and proA genes, closely linked in most bacteria, are in C. glutamicum separated by a 304-amino-acid open reading frame (unk) whose predicted sequence resembles that of the 2-hydroxy acid dehydrogenases. C. glutamicum mutants that carry null alleles of proB, proA, and proC were constructed or isolated from mutagenized cultures. Single proC mutants are auxotrophic for proline and secrete delta1-pyrroline-5-carboxylate, which are the expected phenotypes of bacterial proC mutants. However, the phenotypes or proB and proA mutants are unexpected. A proB mutant has a pleiotropic phenotype, being both proline auxotrophic and affected in cell morphology. Null proA alleles still grow slowly under proline starvation, which suggests that a proA-independent bypass of this metabolic step exists in C. glutamicum. Since proA mutants are complemented by a plasmid that contains the wild-type asd gene of C. glutamicum, the asd gene may play a role in this bypass.     

枯草芽孢杆菌抗脯氨酸结构类似物突变株的筛选及突变株proBA基因的克隆和序列分析

利用亚硝基胍对枯草芽孢杆菌93151进行诱变处理,获得了耐NaCl浓度达14％的突变株,同时发现该突变株也是一个抗脯氨酸反馈抑制突变菌株,其胞内自由脯氨酸的含量随着盐浓度的提高显著增加,说明其对渗透压的耐受能力与胞内自由脯氨酸的含量紧密相关。利用PCR方法克隆突变株的proBA基因,得到一个约2.3kb的DNA片段,序列分析表明该片段含有一完整的proB基因和部分proA基因,与野生菌株的proB基因相比,突变株proB基因中有3个碱基发生了改变,其中一个碱基的变化（从起始密码子开始第781位由T→A）导致了一个氨基酸发生改变（Ser→Thr),另外两个碱基变化为沉默位点突变。将该proB基因转入大肠杆菌脯氨酸营养缺陷型菌株,能够与其功能互补。同时对部分proA基因序列分析发现,其与proB基因头尾重叠。在proA基因起始密码子上游第14个碱基处有一个类似于SD的序列,其所编码的氨基酸序列与枯草芽孢杆菌168的同源性为77％。     

Identification and disruption of the proBA locus in Listeria monocytogenes: role of proline biosynthesis in salt tolerance and murine infection

Intracellular accumulation of the amino acid proline has previously been linked to the salt tolerance and virulence potential of a number of bacteria. Taking advantage of the proBA mutant Escherichia coli CSH26, we identified a listerial proBA operon coding for enzymes functionally similar to the glutamyl kinase (GK) and glutamylphosphate reductase (GPR) enzyme complex which catalyzes the first and second steps of proline biosynthesis in E. coli. The first gene of the operon, proB, is predicted to encode GK, a 276-residue protein with a calculated molecular mass of 30.03 kDa and pl of 5.2. Distal to the promoter and overlapping the 3' end of proB by 17 bp is proA, which encodes GPR, a 415-residue protein with a calculated molecular mass of 45.50 kDa (pl 5.3). Using this information, we created a chromosomal deletion mutant by allelic exchange which is auxotrophic for proline. This mutant was used to assess the contribution of proline anabolism to osmotolerance and virulence. While inactivation of proBA had no significant effect on virulence in mouse assays (either perorally or intraperitoneally), growth at low (2 to 4% NaCl) and high (>6% NaCl) salt concentrations in complex media was significantly reduced in the absence of efficient proline synthesis. We conclude that while proline biosynthesis plays little, if any, role in the intracellular life cycle and infectious nature of Listeria monocytogenes, it can play an important role in survival in osmolyte-depleted environments of elevated osmolarity.     

Sub-cloning of the wild-type proAB region of the Escherichia coli genome

The genes proA and proB encoding the first two enzymes of the proline biosynthetic sequence in Escherichia coli were subcloned from a ColE1 hybrid plasmid containing 23.3 kilobases of genomic DNA. proA and proB are contiguous and constitute a single operon transcribed in the direction proB-proA. The pro operon is contiguous with the gene phoE. Hybridization experiments showed no homology between proAB of E. coli and the other regions of the E. coli genome or with the DNA of several other bacterial species.     

Cloning of genes required for amino acid biosynthesis from Leptospira interrogans serovar icterohaemorrhagiae

Leptospira interrogans belongs to a large family of important pathogens, which is part of the order Spirochaetales, a distinct group of eubacteria. In order to obtain a better understanding of the genetic organization of this species, we have constructed a DNA library of the serovar icterohaemorrhagiae, using the Escherichia coli vector pUC13. We have isolated Leptospira DNA fragments containing the genetic information required to complement strains of E. coli with defects in proline and leucine biosynthesis. While a 3.9 kb fragment which complemented proA also complemented proB, a 15 kb fragment complementing leuB could not complement other leu mutations. The L. interrogans origin of the cloned DNA fragments was confirmed by DNA-DNA hybridization. The hydridization was specific to the pathogenic species and was not seen with the saprophytic species L. biflexa.     

Cloning of genes for proline biosynthesis in Escherichia coli

Restriction map of Escherichia coli chromosome fragment (7.4 MD) carrying proAB genes was constructed. Localization of proA and proB genes on the cloned chromosome fragment was determined by complementation test and the measuring of glutamylkinase activity (proB gene product). ProA and proB genes were cloned separately on multicopy plasmids of alternative orientation and their expression being, probably, under the control of their own regulatory regions, studied.     

Characterization of a gamma-glutamyl kinase from Escherichia coli that confers proline overproduction and osmotic tolerance.

Mutation(s) in the proBA operon of Escherichia coli confers proline overproduction and enhanced osmotic tolerance in enteric bacteria (L. N. Csonka, Mol. Gen. Genet. 182:82-86, 1981; M. J. Mahan and L. N. Csonka, J. Bacteriol. 156:1249-1262, 1983). A glutamate-dependent ATPase assay was developed and used to determine proB-encoded gamma-glutamyl kinase activity in the absence of glutamate-gamma-semialdehyde dehydrogenase. This assay indicated that the feedback insensitivity of mutant gamma-glutamyl kinase was independent of glutamate-gamma-semialdehyde dehydrogenase. However, the capacity of glutamate-gamma-semialdehyde dehydrogenase from the osmotolerant mutant to interact with the kinase was altered in thermal stability, suggesting that mutations in both proB and proA may be required for osmotolerance.     

Mutations in the listerial proB gene leading to proline overproduction: effects on salt tolerance and murine infection

The observed sensitivity of Listeria monocytogenes to the toxic proline analogue L-azetidine-2-carboxylic acid (AZ) suggested that proline synthesis in Listeria may be regulated by feedback inhibition of gamma-glutamyl kinase (GK), the first enzyme of the proline biosynthesis pathway, encoded by the proB gene. Taking advantage of the Epicurian coli mutator strain XL1-Red, we performed random mutagenesis of the recently described proBA operon and generated three independent mutations in the listerial proB homologue, leading to proline overproduction and salt tolerance when expressed in an E. coli (DeltaproBA) background. While each of the mutations (located within a conserved 26-amino-acid region of GK) was shown to confer AZ resistance (AZ(r)) on an L. monocytogenes proBA mutant, listerial transformants failed to exhibit the salt-tolerant phenotype observed in E. coli. Since proline accumulation has previously been linked to the virulence potential of a number of pathogenic bacteria, we analyzed the effect of proline overproduction on Listeria pathogenesis. However, our results suggest that as previously described for proline auxotrophy, proline hyperproduction has no apparent impact on the virulence potential of Listeria.     

Expression of Campylobacter genes for proline biosynthesis in Escherichia coli

Cloned DNA from Campylobacter jejuni was found to complement auxotrophic defects in proline metabolism in several strains of Escherichia coli. A 4.4-kilobase fragment of Campylobacter DNA encodes the genes analogous to the proA and B genes of E. coli and Salmonella typhimurium.     

渗透压调节基因proBA的融合表达对大肠杆菌耐高渗胁迫能力的影响

枯草芽孢杆菌 9315 1的渗透压调节基因proB和proA以重叠基因的方式组织 ,但是表达两个单独的蛋白质ProB和ProA。通过引物设计 ,在抗脯氨酸反馈抑制耐盐突变菌株 9315 1 14的proBA基因重叠区引入一个限制性酶切位点 ,分别扩增出proB和proA基因 ,并构建融合的proBA基因。SDS PAGE分析显示有一条新的分子质量约为85kD的蛋白带出现。相对于表达未融合的proB和proA ,proBA融合基因的表达明显提高了宿主菌大肠杆菌JM83胞内游离脯氨酸含量和其耐高渗胁迫能力     

Proline excretion and indirect suppression in Escherichia coli and Salmonella typhimurium

The last step in proline biosynthesis in Escherichia coli K-12, Salmonella typhimurium LT7, and a number of other enterobacterial isolates is regulated so that no proline is excreted, even if excess Delta(1)-pyrroline-5-carboxylate, the immediate precursor of proline, is added to a culture. In proline auxotrophs blocked at an early step in proline biosynthesis (proA or proB), reversion to prototrophy is often due to a mutation in the arginine pathway which diverts N-acetyl glutamate gamma-semialdehyde to proline synthesis, thus bypassing the proA or proB block. In such double mutants (proAB, argD), the last step in proline synthesis appears to be unregulated, since proline is excreted. Feedback inhibition and repression of the arginine pathway overcomes indirect suppression (restoring the Pro(-) phenotype), but proline regulation is not restored; double mutants still excrete proline when fed Delta(1)-pyrroline-5-carboxylate exogeneously. A new class of proline analogue-resistant mutant, due to mutation at argD, is also described.     

Proline biosynthesis by cell-free extracts of Escherichia coli and potential errors arising from the use of a bioradiological assay procedure.

1. The growth of Escherichia coli proline auxotrophs on medium containing L-proline (50 microgram/ml) induces catabolic enzymes. A bioradiological assay system for proline, using proB cells of E. coli, might give erroneous results owing to proline catabolism by the proline auxotrophs on which the assay depends. 2. Differential utilization of proline and 1-pyrroline-5-carboxylate by the proB cells for the synthesis of protein, and failure of the method to distinguish between these two possible products of the proline-biosynthetic enzymes, might also give rise to error. 3. The proline-dependent incorporation of [14C]phenylalanine into the protein of proline-starved proB auxotrophs was to some degree directly influenced by the presence of crude cell extract from E. coli, even though this was not supplied with substrate and cofactors, and could thus not itself synthesize proline. 4. The kinetics of proline biosynthesis by cell-free extracts were linear and biphasic, only the last phase being affected by the concentrations of substrate and extract. This phenomenon is not understood. 5. Proline biosynthesis is inhibited, not only by high concentrations of ATP, but also by aspartate, glycine, alanine and serine, aspartate having the greatest effect. 6. Attempts at complementation in vitro between extracts of proline auxotrophic mutants were not successful, suggesting the possibility that strain X680 (proA) and/or X278 (proB) may be a double mutant. 7. The enzymes of proline biosyntehsis are co-eluted from a column of Bio-Gel A1.5M in a position corresponding to a mol.wt. of 350000. 8. Comparisons between rates of proline biosynthesis in vivo and in vitro were made.     

Isolation of a spontaneous mutant of Escherichia coli superproducing proline and resistant to elevated concentrations of NaCl

We obtained a spontaneous mutant of Escherichia coli that was characterized by both proline superproduction and the resistance to osmotic stress. The selection of mutants was carried out among 2.5.10(5) clones survived upon plating strain SU1604 containing the sex factor F104, with a chromosome fragment carrying genes proB and proA, on solid modium with the proline analogue L-azetidine-2-carboxylate (AzT). The obtained mutant AztR clones were used as donors in replica crossing with a pro- recipient, followed by subsequent selection of AztR Pro+ exconjugants. Analysis of growth of 456 exconjugants in liquid minimal medium with NaCl at a concentration of 0.6 M helped to identify 9 mutants with increased salt tolerance, as compared with control. From these, we selected one mutant (denoted as SU1604/F'104S) which demonstrated the highest salt tolerance correlating with higher production of proline. Analysis of the mutant's properties suggests that it belongs to the group of Osm mutants.