Two-level factorial screening for influence of temperature, pH, and aeration on production of Serratia marcescens nuclease

Two high-nuclease-yielding mutants of Serratia marcescens, derived by chemical mutagenesis (W280, W355), and two strains with the pBR322 plasmid 403-SD2, carrying a nuclease gene and a chloramphenicol resistance gene [Escherichia coli CSH50(403-SD2) and S. marcescens CH30(403-SD2)] were investigated for nuclease production in a factorial shake flask experiment, with temperature (30 and 37 degrees C), pH (with or without CaCO3 tablets), and aeration (with or without baffles) as variable conditions. Yields varied 10-fold depending on the conditions investigated.     

Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.     

Fermentation studies of the secretion of Serratia marcescens nuclease by Escherichia coli.

The secretion of a Serratia marcescens nuclease was followed by fermentation with Escherichia coli. A plasmid, p403-SD2, carrying a 1.3-kilobase-pair insert with a 0.4-kilobase-pair region upstream of the nuclease gene caused a growth-phase-regulated expression of nuclease in E. coli in the same way as that seen in S. marcescens. Deletion of the regulatory gene generating plasmid p403-Rsa1 resulted in a constitutive expression of the nuclease. Anaerobiosis stimulated the expression from p403-SD2 in stationary growth phase by a factor of 10 compared with expression stimulated by cultivation in aerobic conditions; no such effect was found for plasmid p403-Rsa1. Different nutritional factors caused the expression level and the amount of extracellular nuclease to vary more when nuclease was expressed from plasmid p403-SD2 than when it was expressed from plasmid p403-Rsa1. A correlation between the regulatory gene and the extracellular secretion of nuclease is proposed.     

Two-Level factorial screening of new plasmid/strain combinations for prodution of recombinant-DNA products

This article treats the basic problem of selection of experimental conditions for microbiological experiments for evaluation of newly isolated bacterial strains, mutants, or plasmid/strain combinations. For this purpose shake flask experiments in a 2(10-4)confounded factorial design at resolution IV with four blocks of 16 flasks were used. The design was used for testing of two new strain/plasmid combinations (E. coli MT 102/403-SD2 and W 3110/403-SD2) i.e., both strains with the same plasmid 403-SD2. Both strains were integrated in the design, so both strains were tested with nine factors (temperature, aeration, glucose, initial pH, pH regulation, reduced aeration, chloramphenicol, acetate, and glycerol). With both strains the interaction between initial pH and reduced aeration had a significant influence on the yield of the recombinant-DNA product nuclease. There was more than a factor of 10 between lowest and highest yield of product. In this interactive system the strains reacted differently. MT 102/403-SD2 had highest yields at high initial pH (8.4) and no reduction in aeration, whereas W 3110/403-SD2 had highest yields of nuclease at low initial pH (7.4) and reduced aeration (rubber stopper inserted after cultivation for 12 h). These data (and previous work) clearly demonstrate that it is impossible to suggest a simple set of experimental conditions for testing of new plasmid/strain combinations. It is clear that the exclusive application of a standardized growth technique e.g., LB-medium at 37 degrees C at an unspecified and uncontrolled aeration level, may lead to wrong conclusions on properties and potentials of now plasmid/strain combinations and may lead to rejection of useful strains or plasmids.     

灵杆菌非特异性核酸酶的原核表达、纯化及活性分析

以灵杆菌基因组DNA为模板,PCR扩增非特异性核酸酶 (Non-specific nuclease,NU) 基因,并克隆到pMAL-c4X载体上构建重组表达载体pMAL-c4X-NU。经测序及 BLASTN发现其与灵杆菌Serratia marcescens核酸酶基因的同源性为97％。将构建的表达载体pMAL-c4X-NU转入大肠杆菌BL21,经IPTG诱导实现了胞内表达78 kDa的麦芽糖结合蛋白-NU融合蛋白 (Maltose-binding protein-NU,MBP-NU),其最佳诱导表达条件为37 ℃,0.75 mmol/L IPTG诱导1.5 h。用Amylose resin纯化得到了目的蛋白。活性检测表明MBP-NU具有同时降解DNA和RNA的活性,在37 ℃、pH 8.0时活性最高,比活力为1.11×106 U/mg,目标蛋白的纯化效率可达10.875 mg/L。纯化的目标蛋白中无蛋白酶活性存在。0.5 mmol/L乙二胺四乙酸 (Ethylene diamine tetraacetic acid,EDTA)、1 mmol/L苯甲基磺酰氟 (Phenylmethanesulfonyl fluoride,PMSF) 以及150 mmol/L KCl对MBP-NU的活性几乎无影响,因此MBP-NU可作为蛋白质纯化过程中核酸的高效降解酶。     

Differential secretion of isoforms of Serratia marcescens extracellular nuclease.

Extracellular secretion of the Serratia marcescens nuclease occurs in a two-step process: (i) rapidly to the periplasm via a signal sequence-dependent pathway and then (ii) slowly to the extracellular growth medium without cell lysis. There are two major isoforms of the nuclease in the culture supernatant of S. marcescens. We have isolated, purified, and determined the sequences of both isoforms. The first isoform, the mature nuclease (Sm2), is the result of signal sequence processing. The second isoform (Sm1) has three additional amino acids missing from the N terminus of the mature nuclease. Sm1 starts to appear extracellularly only during prolonged growth of a culture (16 to 48 h), probably because of cell lysis. However, pulse-chase experiments show that it is made early with Sm2 but is not secreted efficiently.     

Nuclease biosynthesis and growth of Serratia marcescens in the presence of 2-(p-aminobenzenesulfonamide)-thiazole

The biosynthesis of nuclease in Serratia marcescens has been studied under the conditions of purine synthesis inhibition with 2-(p-aminobenzenesulfonamide)-thiazole. The addition of this sulfonamide to S. marcescens at different growth stages is found to inhibit both culture growth and nuclease synthesis.     

Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis.

By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).     

The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli

We are studying exoproteins of the enteric bacterium Serratia marcescens as a model system for the release of extracellular proteins from the cell. In this work we report the cloning of the gene for a secreted nuclease from S. marcescens and its complete nucleotide sequence. Following expression of the nuclease gene in both S. marcescens and Escherichia coli we were able to demonstrate the presence of the nuclease extracellularly in both organisms. Cell lysis did not occur and there was no concurrent release of cytoplasmic or periplasmic proteins. No accessory genes appeared to be required for extracellular secretion of the nuclease from E. coli. We can conclude that E. coli is capable of secreting certain proteins extracellularly, and may be a suitable host organism for the genetic analysis of extracellular protein secretion when provided with a suitable protein to export.     

A conditional suicide system in Escherichia coli based on the intracellular degradation of DNA.

The potential risks associated with the intentional or unintentional release of genetically engineered microorganisms led to the construction of biological containment systems by which bacteria are killed in a controlled suicide process. In previously published suicide systems, cell killing was caused by proteins destroying the cell membrane or cell wall. Here a conditional cell killing system based on the intracellular degradation of cellular DNA is presented. The nuclease gene used was that of the extracellular nuclease of Serratia marcescens. The nuclease gene was deleted for the leader-coding sequence, and the truncated gene was put under the control of the lambda pL promoter. Following thermoinduction of the nuclease gene cassette in Escherichia coli, cell survival dropped to 2 x 10(-5), and more than 80% of the radioactively labeled DNA was converted to acid-soluble material within 2.5 h in the absence of cell lysis. The majority (84%) of clones which survived thermoinduced killing turned out to be as sensitive to a second thermoinduction as the original strain. The other clones showed somewhat slower killing kinetics or slightly higher final levels of survivors. The suicide system described combines the regulated killing of cells with the destruction of intracellular DNA otherwise potentially available for horizontal gene transfer processes.     

Identification, genetic analysis and characterization of a sugar-non-specific nuclease from the cyanobacterium Anabaena sp. PCC 7120

A nuclease that could be recovered from the supernatant of cultures, as well as from cell-free extracts, of the cyanobacterium Anabaena sp. PCC 7120 was identified as a 29 kDa polypeptide by its ability to degrade DNA after electrophoresis in DNA-containing SDS-polyacrylamide gels. Some clones of a gene library of strain PCC 7120 established in Escherichia coli were found to produce the 29 kDa nuclease. The nucA gene encoding this nuclease was subcloned and sequenced. The deduced polypeptide, NucA, had a molecular weight of 29,650, presented a presumptive signal peptide in its N-terminal region and showed homology to the products of the nuc gene from Serratia marcescens and the NUC1 gene from Saccharomyces cerevisiae. The NucA protein from Anabaena itself, or from the cloned nucA gene expressed in E. coli, catalysed the degradation of both RNA and DNA, had the potential to act as an endonuclease, and functioned best in the presence of Mn2+ or Mg2+. An Anabaena nucA insertional mutant was generated which failed to produce the 29 kDa nuclease.     

Expression of Serratia marcescens extracellular proteins requires recA.

A previously described regulatory mutation which abolishes expression of the extracellular nuclease of Serratia marcescens is shown to be a mutation of the Serratia recA gene. The defect in nuclease expression could be restored by introducing a plasmid carrying the recA gene of Escherichia coli. The DNA sequence of the Serratia gene is very similar to that of the E. coli gene. The putative LexA-binding site of the Serratia recA gene is almost identical to that of E. coli, along with the promoter. A similar LexA-binding site can also be found upstream of the nuclease gene. As expected from this finding, we show that nuclease expression can be induced by SOS-inducing agents such as mitomycin C. Although inducible in S. marcescens, the nuclease was expressed only at the uninduced levels in E. coli and could not be induced by mitomycin C. The extracellular chitinase and lipase were similarly affected by the mutations altering nuclease expression and were also induced by mitomycin C.     

Genetic analysis of extracellular proteins of Serratia marcescens.

Serratia marcescens, a gram-negative enteric bacterium, is capable of secreting a number of proteins extracellularly. The types of activity found in the growth media include proteases, chitinases, a nuclease, and a lipase. Genetic studies have been undertaken to investigate the mechanisms used for the extracellular secretion of these exoproteins by S. marcescens. Many independent mutations affecting the extracellular enzymes were isolated after chemical and transposon mutagenesis. Using indicator media, we have identified loci involved in the production or excretion of extracellular protease, nuclease, or chitinase by S. marcescens. None of the mutations represented general extracellular-excretion mutants; in no case was the production or excretion of multiple exoproteins affected. A variety of loci were identified, including regulatory mutations affecting nuclease and chitinase expression. A number of phenotypically different protease mutants arose. Some of them may represent different gene products required for the production and excretion of the major metalloprotease, a process more complex than that for the other S. marcescens exoproteins characterized to date.     

Isolation of isoforms and crystallization of endonucleases of Serratia marcescens

Two isoforms of an extracellular endonuclease, nuclease Sm1 and nuclease Sm2, were isolated from the culture filtrate of Serratia marcescens strain B10 M1 by the ligand-exchange chromatography on iminodiacetate-agarose in Cu2(+)-form, and chromatography on phosphocellulose and DEAE-Toyopearl 650S. The pI for nucleases Sm1 and Sm2 were found to be 7.1 and 6.7, respectively. The amino acid analysis and N-terminal amino acid sequencing of the proteins showed a significant degree of homology between the enzymes. The secondary structure of nuclease Sm2 was calculated. Crystals of nuclease Sm2 were obtained with the space group P2(1)2(1)2(1), a 69.0; b 106.7; c 74.8 A.     

Evaluation of the Serratia Marcescens Nuclease (NucA) as a Transgenic Cell Ablation System in Porcine

The efficiency of the Serratia marcescens nuclease encoded by the NucA gene, with or without a nuclear localization signal (NLS), and the commonly used diphtheria toxin A (DTA) were compared for their ability to ablate cells in culture. Constructs containing the test genes driven by the β-actin promoter coupled with enhancer elements from the cytomegalovirus promoter and rabbit β-globin gene (pCAG) and the blasticidin resistance gene driven by the phosphoglycerate kinase (PGK) promoter were generated and electroporated into porcine fetal fibroblasts. Three independent replicates were completed. Following blasticidin selection, the number of surviving colonies was counted to assess the efficiency of the toxic gene. Both NucA and DTA proved to be effective in killing porcine fibroblasts compared to controls. However, the efficiency of cell ablation was significantly higher with DTA than with NucA or NucANLS (p < 0.05). Gene expression analysis of surviving colonies indicated that survival is related to low or absent expression of the toxic genes. These results indicate that the NucA gene, while capable of mammalian cell ablation, is less efficient than DTA.     

Secretion of nuclease across the outer membrane of Serratia marcescens and its energy requirements.

Extracellular secretion of Serratia marcescens nuclease occurs as a two-step process via a periplasmic intermediate. Unlike other extracellular proteins secreted by gram-negative bacteria by the general secretory pathway, nuclease accumulates in the periplasm in its active form for an unusually long time before its export into the growth medium. The energy requirements for extracellular secretion of nuclease from the periplasm were investigated. Our results suggest that the second step of secretion across the outer membrane is dependent upon the external pH; acidic pH effectively but reversibly blocks extracellular secretion. However, electrochemical proton gradient, and possibly ATP hydrolysis, are not required for this step. We suggest that nuclease uses a novel mechanism for the second step of secretion in S. marcescens.     

Crystallization and preliminary crystallographic analysis of a novel nuclease from Serratia marcescens.

Crystals have been obtained of the extracellular endonuclease from the bacterial pathogen Serratia marcescens. This magnesium-dependent enzyme is equally active against single and double-stranded DNA, as well as RNA, without any apparent base preference. The Serratia nuclease is not homologous with staphylococcal nuclease, the only other broad specificity endonuclease for which a structure exists, nor is it homologous with other nucleases that have been solved by X-ray diffraction. The structure of this enzyme should, therefore, provide new information about this class of enzyme. At present we have succeeded in obtaining large, high quality crystals using ammonium sulfate. They crystallize in the orthorhombic space group P2(1)2(1)2(1), with cell dimensions a = 106.7 A, b = 74.5 A, c = 68.9 A, and diffract to beyond 2 A. Low-resolution native data sets have been recorded and a search is under way for heavy-atom derivatives.