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 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.     

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.     

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.     

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.     

The effect of nuclease on transformation efficiency in Serratia marcescens

No differences in the efficiency of transformation were observed from both plasmid and chromosomal DNA in Serratia marcescens 2170 and an extracellular nuclease defective isogenic strain. The efficiency of transformation was the same for Escherichia coli 5K and E. coli containing a recombinant plasmid conferring the ability to synthesize a S. marcescens nuclease. From these results we conclude that the extracellular nuclease of S. marcescens 2170 is not the main cause of the low efficiency of transformation observed in this bacterium.     

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.     

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.     

Molecular cloning and expression of an extracellular nuclease of Serratia marcescens in Escherichia coli

Abstract The gene encoding an extracellular nuclease of Serratia marcescens was cloned in Escherichia coli using the vector pBR322. Transformants were selected by their ability to grow in the presence of ampicillin, and nuclease-positive clones were detected on a commercially available DNase test agar. The production of a nuclease could be detected in recombinant strains and enzyme activity was found in culture supernatants of such strains. Deletion derivatives of the parental recombinant plasmid were constructed to define the region of DNA encoding the expression of the nuclease. The smallest DNA fragment found to produce the nuclease was determined to be 2.2 kb in length, although a somewhat smaller fragment appeared to be partially active.     

High-level secretion of Pseudomonas fluorescens type I secretion system-dependent lipase in Serratia marcescens

The type I secretion system-dependent lipase, TliA, of Pseudomonas fluorescens was successfully produced in quantity in Serratia marcescens by coexpressing its cognate ABC transporter, TliDEF. Compared with P. fluorescens and Escherichia coli, S. marcescens showed an outstanding capacity for the secretory production of TliA, which was done with the expression vectors available for use in E. coli, and no growth phase-dependency, which was unlike the typical feature of TOSS-mediated protein secretion. Among the S. marcescens tested, the highest amount of TliA (approximately 2600 units ml(-1)) was achieved by S. marcescens KCTC 2798 containing the expression plasmid pTliDEFA-223. Our results also suggest that strains of Serratia will provide a valuable opportunity for producing other extracellular TOSS-dependent proteins effectively as well as the TliDEF-dependent TliA in this study.     

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。     

GNA成熟蛋白基因亚克隆及其原核表达载体构建

雪花莲外源凝集素（GNA）对刺吸式昆虫和某些咀嚼式昆虫以及多种线虫均有毒性,从含GNA前体蛋白基因的质粒中亚克隆出GNA的成熟蛋白基因MGNA,将MGNA基因插入大肠杆菌表达载体pET22b的不同位点,再经测序验证,得到了三种不同表达形式的GNA原核表达载体;22bG1(分泌型融合GNA蛋白）,22bG2(包涵体型GNA蛋白）,22bG3(分泌型天然GNA蛋白）,这为进一步在大肠杆菌中表达GNA和将GNA制成生物农药奠定了基础。     

Cloning and expression in Escherichia coli of the Serratia marcescens metalloprotease gene: secretion of the protease from E. coli in the presence of the Erwinia chrysanthemi protease secretion functions.

The Serratia marcescens extracellular protease SM is secreted by a signal peptide-independent pathway. When the prtSM gene was cloned and expressed in Escherichia coli, the cells did not secrete protease SM. The lack of secretion could be very efficiently complemented by the Erwinia chrysanthemi protease B secretion apparatus constituted by the PrtD, PrtE, and PrtF proteins. As with protease B and alpha-hemolysin, the secretion signal was located within the last 80 amino acids of the protease. These results indicate that the mechanism of S. marcescens protease SM secretion is analogous to the mechanisms of protease B and hemolysin secretion.     

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.     

The three genes lipB, lipC, and lipD involved in the extracellular secretion of the Serratia marcescens lipase which lacks an N-terminal signal peptide.

The extracellular lipase of Serratia marcescens Sr41, lacking a typical N-terminal signal sequence, is secreted via a signal peptide-independent pathway. The 20-kb SacI DNA fragment which allowed the extracellular lipase secretion was cloned from S. marcescens by selection of a phenotype conferring the extracellular lipase activity on the Escherichia coli cells. The subcloned 6.5-kb EcoRV fragment was revealed to contain three open reading frames which are composed of 588, 443, and 437 amino acid residues constituting an operon (lipBCD). Comparisons of the deduced amino acid sequences of the lipB, lipC, and lipD genes with those of the Erwinia chrysanthemi prtDEC, prtEEC, and prtFEC genes encoding the secretion apparatus of the E. chrysanthemi protease showed 55, 46, and 42% identity, respectively. The products of the lipB and lipC genes were 54 and 45% identical to the S. marcescens hasD and hasE gene products, respectively, which were secretory components for the S. marcescens heme-binding protein and metalloprotease. In the E. coli DH5 cells, all three lipBCD genes were essential for the extracellular secretion of both S. marcescens lipase and metalloprotease proteins, both of which lack an N-terminal signal sequence and are secreted via a signal-independent pathway. Although the function of the lipD gene seemed to be analogous to those of the prtFEC and tolC genes encoding third secretory components of ABC transporters, the E. coli TolC protein, which was functional for the S. marcescens Has system, could not replace LipD in the LipB-LipC-LipD transporter reconstituted in E. coli. These results indicated that these three proteins are components of the device which allows extracellular secretion of the extracellular proteins of S. marcescens and that their style is similar to that of the PrtDEF(EC) system.     

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.     

Cloning of Escherichia coli and Pseudomonas aeruginosa phosphomannose isomerase genes and their expression in alginate-negative mutants of Pseudomonas aeruginosa.

The phosphomannose isomerase (pmi) gene of Escherichia coli was cloned on a broad-host-range cosmid vector and expressed in Pseudomonas aeruginosa at a low level. Plasmid pAD3, which harbors the E. coli pmi gene, contains a 6.2-kilobase-pair HindIII fragment derived from the chromosome of E. coli. Subcloning produced plasmids carrying the 1.5-kilobase-pair HindIII-HpaI subfragment of pAD3 that restored alginic acid production in a nonmucoid, alginate-negative mutant of P. aeruginosa. This fragment also complemented mannose-negative, phosphomannose isomerase-negative mutants of E. coli and showed no homology by DNA-DNA hybridization to P. aeruginosa chromosomal DNA. By using a BamHI constructed cosmid clone bank of the stable alginate producing strain 8830, we have been able to isolate a recombinant plasmid of P. aeruginosa origin that also restores alginate production in the alginate-negative mutant. This new recombinant plasmid, designated pAD4, contained a 9.9-kilobase-pair EcoRI-BamHI fragment with the ability to restore alginate synthesis in the alginate-negative P. aeruginosa. This fragment showed no homology to E. coli chromosomal DNA or to plasmid pAD3. Both mucoid and nonmucoid strains of P. aeruginosa had no detectable levels of phosphomannose isomerase activity as measured by mannose 6-phosphate-to-fructose 6-phosphate conversion. However, P. aeruginosa strains harboring the cloned pmi gene of E. coli contained measurable levels of phosphomannose isomerase activity as evidenced by examining the conversion of mannose 6-phosphate to fructose 6-phosphate.     

Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34.

A 238-kilobase-pair plasmid, pMOL30, confers resistance to cadmium, zinc, and cobalt salts in Alcaligenes eutrophus CH34. After Tn5 mutagenesis, restriction nuclease analysis, and Southern DNA-DNA hybridization, a 9.1-kilobase-pair EcoRI fragment was found to harbor all of these resistance properties and was cloned into the broad-host-range hybrid plasmid pRK290. When transferred to a plasmid-free derivative of CH34, the hybrid plasmid conferred the same degree of resistance as the parent plasmid pMOL30. In two other Alcaligenes strains, the hybrid plasmid was expressed, but to a lower degree than in CH34 derivatives.