Nature of Escherichia coli cell lysis by culture supernatants of Bacillus species.

Escherichia coli cells were found to be sensitive to lysis by the supernatants of a variety of protease-positive Bacillus species when treated at 45 degrees C but not when treated at 37 degrees C. Different E. coli strains manifested different lysis sensitivities when treated at 45 degrees C. When the lysis rates of E. coli cells at various stages of growth were investigated, post-exponential-phase cells were shown to be most sensitive to lysis. The lysis rate was inversely related to cell viability, and susceptibility appeared to be at least partly due to lysis of dead E. coli cells. A close relation was observed between levels of lysis activity and proteolytic activity. A Bacillus subtilis mutant lacking alkaline and neutral protease activity failed to lyse E. coli cells. It was concluded that Bacillus proteases played a major role in the observed E. coli lysis.     

Isolation and characterization of autolysis-defective mutants of Escherichia coli that are resistant to the lytic activity of seminalplasmin.

Two temperature-sensitive autolysis-defective mutants of Escherichia coli were isolated and shown to be resistant to lysis induced by seminalplasmin, an antimicrobial protein from bovine seminal plasma, as well as to lysis induced by ampicillin, D-cycloserine and nocardicin, at 37 or 42 degrees C but not at 30 degrees C. The mutants were, however, sensitive to inhibition of RNA synthesis by seminalplasmin even at the nonpermissive temperature. Temperature-resistant revertants of the mutants were sensitive to lysis induced by the various antibiotics at 37 or 42 degrees C. The mutations in both strains were mapped at 58 min on the E. coli linkage map. The lysis resistance of the mutants was phenotypically suppressed by the addition of NaCl. Partial suppression of the lysis-resistant phenotype was also observed in a relA genetic background.     

Effect of temperature on RNA synthesis in Escherichia coli CRT 266 (dna Bts) following infection with bacteriophage T3]

Infection of the temperature-sensitive E. coli CRT 266 (dnaBts) with T3-phages at the temperature of 30 degrees C and 35 degrees C, respectively, induced T3-specific RNA synthesis with a maximum rate at 7 min (30 degrees C) and 4.5 min (35 degrees C) after infection. At temperatures above 40 degrees C no T3-induced RNA synthesis could be observed. Infection of E. coli CR 34--45 (dnaB+) with T3 phages at 30 degrees C, 35 degrees C and at temperatures above 40 degrees C, however, produced T3-specific RNA synthesis. The maximum of T3-induced RNA synthesis could be observed between 7 min and 3 min depending on the temperature during infection. The inability to form T3-specific RNA after infection of E. coli CRT 266 at nonpermissive temperatures may be a cause for the absence of the formation of T3 phages and lysis of the host cells.     

Lysis of Escherichia coli by Ethylenediaminetetraacetate and Phospholipases as Measured by β-Galactosidase Activity

A permeaseless mutant of Escherichia coli, which produces beta-galactosidase constitutively, was treated briefly with ethylenediaminetetraacetate and then with the phospholipases of Bacillus cereus. Cell lysis occurred, as indicated by an increase in beta-galactosidase activity and a decrease in absorbancy of the cell suspension. The susceptibility of the cells to attack by ethylenediaminetetraacetate and the phospholipases was markedly affected by the age of the cells when harvested. The results suggest that permeability changes may be associated with the activity of a phospholipase that specifically degrades phosphatidyl ethanolamine. A sonic-treatment method for determining the total beta-galactosidase content of E. coli cells, which is independent of their age when harvested, is described.     

Lysis of Escherichia coli by the bacteriophage phi X174 E protein: inhibition of lysis by heat shock proteins.

Lysis of Escherichia coli by the cloned E protein of bacteriophage phi X174 was more rapid than expected when bacteria were shifted from 30 to 42 degrees C at the time of E induction. Since such treatment also induces the heat shock response, we investigated the effect of heat shock proteins on lysis. An rpoH mutant was more sensitive to lysis by E, but a secondary suppressor mutation restored lysis resistance to parental levels, which suggests that the sigma 32 subunit itself did not directly increase lysis resistance. At 30 degrees C, mutants in five heat shock genes (dnaK, dnaJ, groEL, groES, and grpE) were more sensitive to lysis than were their wild-type parents. The magnitude of lysis sensitivity varied with mutation and strain background, with dnaK, dnaJ, and groES mutants consistently exhibiting the greatest sensitivities. Extended protection against lysis occurred when overproduction of heat shock proteins was induced artificially in cells that contained a plasmid with the rpoH+ gene under control of the tac promoter. This protective effect was completely abolished by mutations in dnaK, dnaJ, or groES but not by grpE or groEL mutations. Altered membrane behavior probably explains the contradiction whereby an actual temperature shift sensitized cells to lysis, but production of heat shock proteins exhibited protective effects. The results demonstrate that E-induced lysis can be divided into two distinct operations which may now be studied separately. They also emphasize a role for heat shock proteins under non-heat-shock conditions and suggest cautious interpretation of lysis phenomena in systems where E protein production is under control of a temperature-sensitive repressor.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor.

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

Effects of temperature on Escherichia coli overproducing beta-lactamase or human epidermal growth factor

The effects of temperature on strains of Escherichia coli which overproduce and excrete either beta-lactamase or human epidermal growth factor were investigated. E. coli RB791 cells containing plasmid pKN which has the tac promoter upstream of the gene for beta-lactamase were grown and induced with isopropyl-beta-D-thiogalactopyranoside in batch culture at 37, 30, 25, and 20 degrees C. The lower temperature greatly reduced the formation of periplasmic beta-lactamase inclusion bodies, increased significantly the total amount of beta-lactamase activity, and increased the purity of extracellular beta-lactamase from approximately 45 to 90%. Chemostat operation at 37 and 30 degrees C was difficult due to poor cell reproduction and beta-lactamase production. However, at 20 degrees C, continuous production and excretion of beta-lactamase were obtained for greater than 450 h (29 generations). When the same strain carried plasmid pCU encoding human epidermal growth factor, significant cell lysis was observed after induction at 31 and 37 degrees C, whereas little cell lysis was observed at 21 and 25 degrees C. Both total soluble and total human epidermal growth factor increased with decreasing temperature. These results indicate that some of the problems of instability of strains producing high levels of plasmid-encoded proteins can be mitigated by growth at lower temperatures. Further, lower temperatures can increase for at least some secreted proteins both total plasmid-encoded protein formed and the fraction that is soluble.     

A set of UV-inducible autolytic vectors for high throughput screening

A high throughput screening scheme is often a prerequisite for directed evolution of enzymes or metagenomic analysis of DNA samples. For assaying intracellular enzymes of interest (e.g. when Escherichia coli is used), it requires cell lysis in many cases, chemical or enzymatic, which can be tedious and cost-consuming. In this study, a set of UV-inducible autolytic vectors was constructed to offer a simpler means of cell lysis that is free of additional liquid handling. The SRRz lysis gene cassette from bacteriophage Lambda was cloned downstream of a UV-inducible promoter, the recA promoter or the umuDC promoter, and further inserted into the backbone of pUC18, and transformed into E. coli BL21 cells. The SRRz expression and cell lysis was induced by UV irradiation. For both the recA and umuDC promoters, at 30 degrees C the lysis efficiency was found to be consistent and above 60% as measured using beta-galactosidase as the reporter. However, at 37 degrees C the lysis profiles were found to be erratic. UV lysis in 96-well plates also produced consistent lysis results that were comparable to those obtained by lysozyme treatment, demonstrating the utility of these autolytic vectors in high throughput screening. This set of artificial SRRz autolysis units should be transferable to other vectors. Surprisingly, it was found that the E. coli BL21(DE3) was also partially disrupted under UV irradiation, with a lysis efficiency of 44.5% at 30 degrees C, and 22.5% at 37 degrees C.     

Display of bacterial lipase on the Escherichia coli cell surface by using FadL as an anchoring motif and use of the enzyme in enantioselective biocatalysis

We have developed a novel cell surface display system by employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. strain TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. The localization of the truncated FadL-lipase fusion protein on the cell surface was confirmed by confocal microscopy and Western blot analysis. Lipase activity was mainly detected with whole cells, but not with the culture supernatant, suggesting that cell lysis was not a problem. The activity of cell surface-displayed lipase was examined at different temperatures and pHs and was found to be the highest at 50 degrees C and pH 9 to 10. Cell surface-displayed lipase was quite stable, even at 60 and 70 degrees C, and retained over 90% of the full activity after incubation at 50 degrees C for a week. As a potential application, cell surface-displayed lipase was used as a whole-cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic acid could be produced with the enantiomeric excess of 99% and the enantiomeric ratio of 292, which are remarkably higher than values obtained with crude lipase or cross-linked lipase crystal. These results suggest that FadL may be a useful anchoring motif for displaying enzymes on the cell surface of E. coli for whole-cell biocatalysis.     

Ampicillin induced septum formation in Bacillus cereus

Cells of Bacillus cereus grown in the presence of subinhibitory concentrations of ampicillin at either 30 degrees or 45 degrees C exhibited an increase in the numbers of centres of septum formation per unit cell length. Under identical conditions of cultivation, cells of Escherichia coli grew as aseptate filaments. In general, untreated B. cereus cells grown at 45 degrees C were longer than those grown at 30 degrees C. The strain of E. coli used was unaffected in terms of filamentation by elevated growth temperature. Results are discussed in terms of the presence and availability of penicillin binding proteins and autolysins involved in cell growth, division and separation.     

Requirement for a functional host cell autolytic enzyme system for lysis of Escherichia coli by bacteriophage phi X174

Escherichia coli VC30 is a temperature-sensitive mutant which is defective in autolysis. Strain VC30 lyses at 30 degrees C when treated with beta-lactam antibiotics or D-cycloserine or when deprived of diaminiopimelic acid. The same treatments inhibit growth of the mutant at 42 degrees C but do not cause lysis. Strain VC30 was used here to investigate the mechanism of host cell lysis induced by bacteriophage phi X 174. Strain VC30 was transformed with plasmid pUH12, which carries the cloned lysis gene (gene E) of phage phi X174 under the control of the lac operator-promoter, and with plasmid pMC7, which encodes the lac repressor to keep the E gene silent. Infection of strain VC30(pUH12)(pMC7) with phage phi X174 culminated in lysis at 30 degrees C. At 42 degrees C, intracellular phage development was normal, but lysis did not occur unless a temperature downshift to 30 degrees C was imposed. Similarly, induction of the cloned phi X174 gene E with isopropyl-beta-D-thiogalactoside resulted in lysis at 30 degrees C but not at 42 degrees C. Temperature downshift of the induced culture to 30 degrees C resulted in lysis even in the presence of chloramphenicol. These results indicate that host cell lysis by phage phi X174 is dependent on a functional cellular autolytic enzyme system.     

Deletion of a cytotoxic, N-terminal putative signal peptide results in a significant increase in production yields in Escherichia coli and improved specific activity of Cel12A from Rhodothermus marinus

The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65 degrees C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately three-fold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90 degrees C and unfolding temperatures up to 103 degrees C.     

Escherichia coli Mutants Tolerant to Beta-Lactam Antibiotics

Two types of Escherichia coli mutants tolerant to beta-lactam antibiotics were isolated. One is E. coli chi2452, which showed a tolerant response against beta-lactam antibiotics when grown at 42 degrees C, and the others are the mutants C-80 and C-254, selected from mutagenized E. coli chi1776 by cycles of exposure to ampicillin, cephaloridine, and starvation of the nutritionally required diaminopimelic acid. Beta-lactam antibiotics caused rapid loss of viability and lysis in cultures of chi1776 or in chi2452 grown at 32 degrees C. In contrast, the same antibiotics caused only a reversible inhibition of growth in mutants C-80 and C-254 or in cultures of chi2452 grown at 42 degrees C. Beta-lactam antibiotics that show high affinity for penicillin-binding proteins 2 or 3 (mecillinam and cephalexin, respectively) induced similar morphological effects (ovoid cell formation and filament formation) in both parent and mutant strains. In contrast, beta-lactam antibiotics which have a high affinity for penicillin-binding protein 1 (e.g., cephaloridine or cefoxitin), which cause rapid lysis in the parental strains, caused cell elongation in the tolerant bacteria. In contrast to the parental cells, autolytic cell wall degradation was not triggered by beta-lactam treatment of chi2452 cells grown at 42 degrees C or in mutants C-80 and C-254. The total autolytic activity of mutants C-80 and C-254 was less than 30% that of the parent strain. However, virtually identical autolytic activities were found in cells of chi2452 grown either at 42 or 32 degrees C. Possible mechanisms for the penicillin tolerance of E. coli are considered on the basis of these findings.     

Temperature-sensitive beta-lactam-tolerant mutants of Escherichia coli

Seven temperature-sensitive penicillin-tolerant mutants of Escherichia coli strain LD5 (thi lysA dapD) were isolated and characterized. Treatment with beta-lactams caused lysis of the mutants at 30 degrees C. Although growth of the mutants at 42 degrees C was inhibited by beta-lactams, no lysis occurred. The mutants were also slightly tolerant to D-cycloserine at 42 degrees C but lysed readily when deprived of diaminopimelate or when treated with moenomycin. The minimum inhibitory concentrations of various antibiotics were the same for the mutants and their parent. The mutations conferring penicillin tolerance were phenotypically suppressed in the presence of a variety of compounds which may act as chaotropic or antichaotropic agents. No defects in penicillin-binding proteins and peptidoglycan hydrolases were detected. Temperature-resistant revertants of the mutants were no longer tolerant to penicillin-induced autolysis at 42 degrees C. The mutations in five isolates were localized to the 56 to 61 min region of the E. coli linkage map and to the 44 to 51 min region in the case of two other isolates.     

A continuous thermal lysis procedure for the large-scale preparation of plasmid DNA

There is an increasing interest and need for the development of scaleable process for the preparation of plasmid DNA for vaccines and gene therapy. In this report, we describe a streamline modified process of plasmid extraction based on boiling lysis in order to simplify the operation and process large volumes of Escherichia coli cultures. The bacteria, harvested using a hollow fiber cartridge after fermentation, were treated with lysozyme at 37 degrees C prior to passing through a heat-exchanger coil. Subsequently, the supernatant was separated from lysed bacteria using a 65 microm nylon filter. The employment of a peristaltic pump and two heating coils at constant temperature without the use of centrifugation enabled the process protocol to be constant and controllable. A relatively low lysis temperature of approximately 70-80 degrees C and a buffer modified for the high-density cultures were also optimized for the process. Prior to thermal lysis, a pre-treatment step with the lysozyme for 20 min at 37 degrees C was one of the crucial steps contributing to the high plasmid quantity and quality from batch to batch. After harvesting 17 L of E. coli cultures (OD600 = 50), the plasmid can be extracted within 45 min with this streamline protocol. The plasmid yields are approximately 100mg/L culture, which makes it attractive and promising for the large-scale preparation of plasmid.     

Heat-inducible autolytic vector for high-throughput screening

In directed evolution, a high-throughput screening system is often a prerequisite for sampling the enzyme variants. When the target enzyme is expressed intracellularly, for example when Escherichia coli is used as the host, chemical or enzymatic disruption of cell membrane is often required in many cases, which can be tedious, time-consuming, and costly. In this study, a set of heat-inducible autolytic vectors were constructed to solve this problem, in which the SRRz lysis gene cassette from bacteriophage lambda was placed downstream of heat-inducible promoters, lambda cI857/pR promoter and its mutant, c1857/pR(M). The artificial autolytic units were inserted into the backbone of pUC18 (away from the multiple cloning sites). For the wild promoter; cI857/pR, the SRRz lysis cassette was expressed by temperature up-shift from 28 degrees to 38 degrees C, and the lysis efficiency of transformed bacterial cells was found to be consistent and could reach 96.3% as measured by the reporter beta3-galactosidase assay. In order to obtain a higher cell growth rate, the mutant promoter cI857/pR(M) was utilized to allow bacteria growth at 35 degrees C and lysis at 42 degrees C. However; this heat-inducible system showed significant inconsistency in terms of lysis efficiency. Bacillus subtilis 168 lipase A gene was further inserted into the multiple cloning sites of the autolytic vector containing cI857/pR, and 93.7% of the expressed lipase activity was found in the culture medium upon heat induction, demonstrating the utility of the vector for expression and rapid extracellular assay of heterologous enzymes.     

Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A

AIMS: The aim of this research was to investigate the production of bacteriocins by Bacillus spp. isolated from native soils of south of Brazil. METHODS AND RESULTS: A bacteriocin produced by the bacterium Bacillus cereus 8 A was identified. The antimicrobial activity was produced starting at the exponential growth phase, although maximum activity was at stationary growth phase. A crude bacteriocin obtained from culture supernatant fluid was inhibitory to a broad range of indicator strains, including Listeria monocytogenes, Clostridium perfringens, and several species of Bacillus. Clinically relevant bacteria such as Streptococcus bovis and Micrococcus luteus were also inhibited. Bacteriocin was stable at 80 degrees C, but the activity was lost when the temperature reached 87 degrees C. It was resistant to the proteolytic action of trypsin and papain, but sensitive to proteinase K and pronase E.Bacteriocin activity was observed in the pH range of 6.0-9.0. CONCLUSIONS: A bacteriocin produced by Bacillus cereus 8 A was characterized, presenting a broad spectrum of activity and potential for use as biopreservative in food. SIGNIFICANCE AND IMPACT OF STUDY: The identification of a bacteriocin with large activity spectrum, including pathogens and spoilage microorganisms, addresses an important aspect of food safety.     

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine

A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.     

Production and release of polyphosphate by a genetically engineered strain of Escherichia coli.

A recombinant strain of Escherichia coli MV1184, which contains plasmid-borne genes encoding the phosphate-specific transport (Pst) system and polyphosphate (polyP) kinase, accumulated high levels of Pi and released polyP into the medium. PolyP could be separated from the culture supernatant by DEAE-Toyopearl 650M chromatography and identified by high-resolution 31P nuclear magnetic resonance spectroscopy. Once E. coli recombinants accumulated high levels of polyP, they released polyP concomitantly with Pi uptake. PolyP release did not accompany the decrease in the cell density, indicating that it is not simply a result of cell lysis. PolyP release ceased when Pi became depleted in the medium and resumed upon addition of Pi to the medium. When Pi uptake was inhibited by 0.1 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP), no polyP release was observed. Furthermore, neither Pi uptake nor polyP release occurred when cells were incubated at 4 degrees C. These findings suggest that the occurrence of polyP release is a possible mechanism that limits a further increase in the cellular polyP concentration in E. coli recombinants. High-resolution 31P nuclear magnetic resonance spectroscopy also detected a surface pool of polyP in intact cells of the E. coli recombinant. The polyP resonance increased when cells were treated with EDTA and broadened upon the addition of a shift reagent, praseodymium. Although the mechanism of surface polyP accumulation is unclear, surface polyP seems to serve as the source for polyP release.     

Temperature-sensitive autolysis-defective mutants of Escherichia coli.

Two independently isolated temperature-sensitive autolysis-defective mutants of Escherichia coli LD5 (thi lysA dapD) were characterized. The mutants were isolated by screening the survivors of a three-step enrichment process involving sequential treatments with bactericidal concentrations of D-cycloserine, benzyl-penicillin, and D-cycloserine at 42 degrees C. Cultures of the mutants underwent autolysis during beta-lactam treatment, D-cycloserine treatment, or diaminopimelic acid deprivation at 30 degrees C. The same treatments at 42 degrees C inhibited growth but did not induce lysis of the mutants. The minimum inhibitory concentrations of selected beta-lactam antibiotics and D-cycloserine were identical for the parent and mutant strains at both 30 and 42 degrees C. Both mutants failed to form colonies at 42 degrees C, and both gave rise to spontaneous temperature-resistant revertants. The revertants exhibited the normal lytic response when treated with D-cycloserine and beta-lactams or when deprived of diaminopimelic acid at 42 degrees C. The basis for the autolysis-defective phenotype of these mutants could not be determined. However, a nonspecific in vitro assay for peptidoglycan hydrolase activity in cell-free extracts indicated that both mutants were deficient in a peptidoglycan hydrolase. Both mutations were localized to the 56- to 61-min region of the E. coli chromosome by F' complementation.