Construction and genetic characterization of lambda specialized transducing particles carrying therglB gene ofEscherichia coli K-12

TherglB gene ofEscherichia coli codes for a restriction activity that cleaves the hydroxymethylated DNA of T2 and T4 phages. Earlier mapping data placed the gene at 98.39 min counterclockwise to thehsd operon. Genetic analysis of the in vivo gene fusions with fusion-transducing phages established the location of therglB gene next to thehsdS gene of thehsdRMS cluster. The methodology used in this study could be extended to similar in vivo physical mapping of closely linked genes.     

Short‐term high temperature treatment reduces viability and inhibits respiration and DNA repair enzymes in Araucaria angustifolia cells

We evaluated the effect of global warming on Araucaria angustifolia (Bert.) O. Kuntze, a critically endangered native tree of Southern Brazil, by studying the effects of short‐term high temperature treatment on cell viability, respiration and DNA repair of embryogenic cells. Compared with control cells grown at 25°C, cell viability was reduced by 40% after incubation at 30 and 37°C for 24 and 6 h, respectively, while 2 h at 40 and 42°C killed 95% of the cells. Cell respiration was unaffected at 30–37°C, but dramatically reduced after 2 h at 42°C. The in vitro activity of enzymes of the base excision repair (BER) pathway was determined. Apurinic/apyrimidine endonuclease, measured in extracts from cells incubated for 2 h at 42°C, was completely inactivated while lower temperatures had no effect. The activities of three enzymes of the mitochondrial BER pathway were measured after 30‐min preincubation of isolated mitochondria at 25–40°C and one of them, uracil glycosylase, was completely inhibited at 40°C. We conclude that cell viability, respiration and DNA repair have different temperature sensitivities between 25 and 37°C, and that they are all very sensitive to 40 or 42°C. Thus, A. angustifolia will likely be vulnerable to the short‐term high temperature events associated with global warming.     

Effects of temperature and novobiocin on the expression of calf prochymosin gene and on plasmid copy number in recombinantEscherichia coli

Escherichia coli strain HB101 harboring an expression plasmid bearing calf prochymosin gene under the control of thetac promoter was grown in the presence of IPTG with or without novobiocin at 28 and 40 °C, respectively. The differential rates of synthesis of prochymosin inclusion, and, for comparison, of β-lactamase and β-galactosidase, as well as plasmid copy number, were determined during the first hours of steady state growth. At 28 °C the induced expression of prochymosin gene was almost blocked. Addition of novobiocin did not alleviate this effect. In fact, it strengthtened it, and we conclude that both these additive inhibitory effects are a consequence of the decrease in negative superhelical tension of plasmid DNA to an insufficient level. At 40 °C the differential rate of prochymosin synthesis was markedly enhanced. Since the copy number of the expression plasmid increased approximately to the same extent, we conclude that an increase in gene dose is the cause. The stimulation of cloned heterologous gene expression at 40 °C and inhibition at 28 °C may be conveniently used in biotechnological-scale cultivations of some recombinant bacteria.     

Production of hepatitis B core antigen in a stirred tank bioreactor: The influence of temperature and agitation

The influence of temperature and agitation on the growth ofEscherichia coli expressing hepatitis B core antigen (HBcAg) in stirred tank bioreactor were investigated. The highest specific growth rate forE. coli (0.844 h⁻¹) was achieved at a temperature of 37°C and an agitation speed of 250 rpm. The activation energy for the growth of theE. coli strain W3110IQ in the stirred tank bioreactor was estimated to be 11 kcal/mol. The highest protein yield was achieved at a temperature of 44°C and an agitation speed of 250 rpm. The relative protein concentration at 44°C is 30 and 6% higher compared to that at 30 and 37°C, respectively.     

Amplification of synthesis and secretion of haemolysin using a run-away plasmid in Escherichia coli

Molin and co-workers have described the construction of a ‘run-away’ plasmid, pOU71 which could be useful for the amplification of cloned genes at high temperature when the plasmid replicates to high copy number.In this paper we describe the kinetics of synthesis of a plasmid-coded gene product, β-lactamase, concomitant with pOU71 amplification at 42°C. Maximum amplification was obtained by shifting a culture growing at 30–42°C for 60 min resulting in a 70- to 80-fold amplification for the β-lactamase gene product when the culture was returned to 30°C.The haemolytic determinant LE2001 from an Escherichia coli strain of human origin was cloned into plasmid pOU71 giving rise to plasmid pLG570. Using an identical amplification procedure a 20-fold amplification of the synthesis and secretion of haemolysin was achieved.     

Cloning of heterologous genes specifying detrimental proteins on pUC-derived plasmids inEscherichia coli

A system is described that enables the cloning of genes specifying detrimental proteins inEscherichia coli. The system is based on pUC plasmids and was developed for the expression of theBacillus subtilis csaA gene, which is lethal when expressed at high levels. Suppressor strains that tolerate the presence of plasmids for high-level expression ofcsaA were isolated, which contained small cryptic deletion variants of the parental plasmid in high copy numbers. The cryptic plasmids consisted mainly of the pUC replication functions and lacked thecsaA region and selectable markers. The co-resident, incompatible, cryptic plasmids enabled the maintenance of thecsaA plasmids by reducing their copy number 20-fold, which resulted in a concomitant 3- to 7-fold reduction in the expression of plasmid-encoded genes. Strains carrying these cryptic endogenous plasmids proved to be useful for the construction of pUC-based recombinant plasmids carrying other genes, such as theskc gene ofStreptococcus equisimilis, which cannot be cloned in high copy numbers inE. coli. Several strategies to reduce production levels of heterologous proteins specified by plasmids are compared.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^– strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.Deceased, December 15, 1995     

Enhanced thermotolerance for ethanol fermentation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain by overexpression of the gene coding for trehalose-6-phosphate synthase

The effect of overexpression of the trehalose-6-phosphate (T6P) synthase gene (TPS1) on ethanol fermentation of Saccharomyces cerevisiae has been studied at 30 and 38°C. The activity of T6P synthase and the accumulation of trehalose during ethanol fermentation were significantly improved by overexpression of TPS1, and especially at 38°C. Ethanol produced by transformants with and without TPS1 gene overexpression at 38°C was approx. 60 and 37 g/l, respectively. The fermentation efficiency of transformants with TPS1 gene overexpression at 38°C was similar to that at 30°C. The critical growth temperature was increased from 36 to 42°C by TPS1 gene overexpression. These results indicated that overexpression of the TPS1 gene had a beneficial effect on the fermentation capacity of the title yeast strain at high temperatures.     

Development of a serial bioreactor system for direct ethanol production from starch using<Emphasis Type="Italic">Aspergillus niger</Emphasis> and<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Aspergillus niger hyphae were found to grow with unliquefied potato starch under aerobic conditions, but did not grow under anaerobic conditions. The raw culture ofA. niger catalyzed saccharification of potato starch to glucose, producing approximately 12 g glucose/L/day/ The extracellular enzyme activity was decreased in proportion to incubation time, and approximately 64% of initial activity was maintained after 3 days. At 50°C,A. niger hyphae growth stopped, while the extracellular enzyme activity peaked. On the basis of theA. niger growth property and enzyme activity, we designed a serial bioreactor system composed of four different reactors. Fungal hyphae were cultivated in reactor I at 30°C, uniquefied starch was saccharified to glycose by a fungal hyphae culture in reactors II and III at 50°C, and glucose was fermented to ethanol bySaccharomyces cerevisiae in reactor IV. The total glucose produced by fungal hyphae in reactor I and saccharification in reactor II was about 42 g/L/day. Ethanol production in reactor IV was approximately 22 g/L/day, which corresponds to about 79% of the theoretical maximum produced from 55 g starch/L/day.     

MODULATION OF THE HEAT SHOCK UBIQUITIN POOL IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Immunoblotting experiments performed with an anti-ubiquitin antibody revealed that Skeletonema costatum (Grev.) Cleve cells contained free ubiquitin as well as ubiquitin conjugated to various endogenous proteins. A temperature shift from 18° to 30°C greatly increased the total amount of ubiquitin and particularly the ubiquitin fraction in high molecular mass conjugates. A solid-phase immunoassay indicated values of 0.031 ± 0.004 pmol·10^?6 cells for free ubiquitin and 0.046 ± 0.004 pmol·10^?6 cells for conjugated ubiquitin for cells grown at 18°C, and 0.056 ± 0.008pmol·10^?6cells and 0.21 ± 0.03 pmol·10^?6cells, respectively, after a temperature increase from 18° to 30°C. Cell-free extracts of S. costatum were equally able to form thiol ester linkages with ¹²⁵I-ubiquitin in an adenosine triphosphate–dependent manner at 18° C and at 30°C. Cell-free extracts were also able to conjugate ¹²⁵I-ubiquitin to endogenous proteins, but the ubiquitin conjugation rate at 30°C was lower than at 18°C. Incubation of S. costatum for 3 h at 30°C and then for 3 h at 18°C resulted in the formation of high amounts of ubiquitin conjugates, suggesting that partially inactive or denaturated proteins accumulate during heat stress. These denaturated proteins are then conjugated to ubiquitin very efficiently when the physiological temperature is restored. Thus, S. costatum cells contain ubiquitin and an active ubiquitin conjugation system responding to stress conditions (temperature stress). The intracellular concentration of ubiquitin conjugates is most likely limited by the availability of protein substrates to be conjugated rather than by ubiquitin-conjugating activity.     

Cloning of endoglucanase genes fromCellulomonas biazotea intoE. coli andS. cerevisiae using shuttle vector YEp24

We constructed aSmaI genomic library ofCellulomonas biazotea DNA inE. coli and in theS. cerevisiae shuttle vector, YEP 24. Three clone were identified that conferred the ability forE. coli orS. cerevisiae transformants to produce carboxymethylcellulase (CMCase). Cells transformed with these clones were compared with one another and with nontransformed cells for hyper-production of CMCase.In vivo andin vitro studies indicated that the CMCase genes were fully expressed and the enzyme activity was located extracellularly. The optimum pH and temperature for the CMCase thus cloned were pH 7 and 50°C, respectively, as was the case for the donor.     

Effects of incubation temperature and oxygen tension on nitrogenase activity of legume root nodules

Summary Acetylene reduction and H₂ evolution by legume root nodules from several plant species depended on incubation temperature; some nodules were active from 2 to 40°C. Acetylene reduction rates differed between plant species, with maximum activity at temperatures between 20 and 30°C forVicia faba, V. sativa, Trifolium pratense, T. subterraneum, Medicago truncatula and soybean, at 35°C forM. sativa and at 40°C for cowpea. OnlyM. sativa and cowpea reduced substantial amounts at 37.5°C. Temperatures from 2 to 10°C only slightly lessened activity ofT. subterraneum andV. sativa nodules. Nitrogenase functioned at temperatures which prevent establishment of other aspects of the symbiosis. The rate of acetylene reduction was constant for several hours at temperatures below 15°C, and activity continued for several days at 2°C for some species, but declined with time at warmer temperatures. Some nitrogenase was denatured at warmer temperatures, but the O₂ tension in the assay vial also affected activity. In closed assay vessels nodule respiration decreased the pO₂ and reduced nitrogenase activity. Activity was restored by adding O₂ or regassing assay vials with air or Ar/O₂. When the pO₂ was maintained, acetylene reduction and H₂ evolution by detached soybean nodules continued unchanged for 6 h.     

Suitability of some local agro-industrial wastes as carrier materials for cyanobacterial inoculant

Survival and nitrogenase efficiency ofNostoc commune andN. austinii were evaluated monthly in four carrier materials (sugarcane bagasse, wheat straw, wheat bran and peat) at 10, 30 and 40 °C. Survival, as well as nitrogenase activity, of both species was much better in peat, followed by wheat bran, sugarcane bagasse than in wheat straw at 10 and 30 °C up to three months, the activity ofN. commune being better thanN. austinii. None of the materials tested was found to be superior to peat as carrier ofNostoc species but the results indicated that wheat bran and sugarcane bagasse can be used as inoculant carriers with relative success. Storage of inoculants in these carriers is feasible at 30 °C up to three months.     

Overproduction of the hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype

The geneshsdM andhsdS for M.EcoKI modification methyltrasferase and the complete set ofhsdR,hsdM andhsdS genes coding for R.EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation inhsdS gene, were cloned in pBR322 plasmid and introduced intoE. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. ThehsdS _ts-1 mutation, mapped previously in the distal variable region of thehsdS gene with C^{1 245}-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42°C. In clones transformed with the wholehsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutanthsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of thehsdS _ts-1 mutation on restriction and modification.     

Expression of a DNA methylation (dam) gene inEscherichia coli K-12

Plasmid pMQ3, carrying thedam gene ofEscherichia coli on a 6.1 Kb fragment, shows a tenfold increase in relative DNA adenine methylase activity, while plasmid pdam118, with a 1.14 Kbdam insert, shows only a twofold increase, although both plasmids were derived from plasmid pLC13–42. Since a copy number effect did not seem to be the cause of this difference, we have subcloned pMQ3 in order to determine whether the additional chromosomal DNA present in this plasmid is responsible for the enhancement of methylase activity. We show that the 346 base pairs upstream ofdam contain sequences necessary for expression. DNA sequence analysis has revealed that in pdam118 only the 118 bases 5-prime to thedam gene are present in other constructs and that the additional upstream material is pBR322 DNA. This shows that pdam118 carries a DNA duplication.     

Mortality fecundity and longevity of parasitoids of the spotted tentiform leafminer,Phyllonorycter blancardella [Lep.: Gracillariidae] at constant temperatures in the laboratory

The results of laboratory tests showed that mortality of adult eulophids, primarily,Sympiesis sericeicornis (Nees),S. marylandensis Girault andPnigalio flavipes (Ashmead), was significantly (P<0.05) lower than that of adultPholetesor ornigis (Weed) when exposed to temperatures between 20° and 36°C for 48 h. However, adultP. ornigis lived longer than those of the eulophids at 15°C, but were shorter liver at 33°C. The fecundity ofP. ornigis was little affected at temperatures of 15°, 20°, 24° and 33°C. Exposure of adultP. ornigis to 30°C for 16 h resulted in reduced longevity of both sexes but did not affect fecundity or the proportion of females ovipositing. Mortality of pupae of the eulophids was significantly lower than that of pupae ofP. ornigis at temperatures of 20°, 30° and 33°C. The sex ratio of surviving adults was not affected by temperature.      

Genetic location ofrra mutation and its role in alleviation of rgl restriction

Restriction of glucosyl-free HMC-DNA mediated by RglB is alleviated inrecBC sbcA strains ofEscherichia coli K12. Mutation in the unlinkedrra gene reverses thisrecBC sbcA-mediated alleviation. The map position ofrra is 90.16 min on the standard map, and therra ⁺ gene product counteracts Rgl restriction. The activation of therra gene is controlled by thesbcA gene, and this regulation does not seem to require the involvement of other gene functions.