Construction of an Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp.

A plasmid transformation system for Rhodococcus sp. strain H13-A was developed by using an Escherichia coli-Rhodococcus shuttle plasmid constructed in this study. Rhodococcus sp. strain H13-A contains three cryptic indigenous plasmids, designated pMVS100, pMVS200, and pMVS300, of 75, 19.5, and 13.4 kilobases (kb), respectively. A 3.8-kb restriction fragment of pMVS300 was cloned into pIJ30, a 6.3-kb pBR322 derivative, containing the E. coli origin of replication (ori) and ampicillin resistance determinant (bla), as well as a Streptomyces gene for thiostrepton resistance, tsr. The resulting 10.1-kb recombinant plasmid, designated pMVS301, was isolated from E. coli DH1(pMVS301) and transformed into Rhodococcus sp. strain AS-50, a derivative of strain H13-A, by polyethylene glycol-assisted transformation of Rhodococcus protoplasts and selection for thiostrepton-resistant transformants. Thiostrepton-resistant transformants were also ampicillin resistant and were shown to contain pMVS301, which was subsequently isolated and transformed back into E. coli. The cloned 3.8-kb fragment of Rhodococcus DNA in pMVS301 contains a Rhodococcus origin of replication, since the hybrid plasmid was capable of replication in both genera. The plasmid was identical in E. coli and Rhodococcus transformants as determined by restriction analysis and was maintained as a stable, independent replicon in both organisms. Optimization of the transformation procedure resulted in transformation frequencies in the range of 10(5) transformants per micrograms of pMVS301 DNA in Rhodococcus sp. strain H13-A and derivative strains. The plasmid host range extends to strains of Rhodococcus erythropolis, R. globulerus, and R. equi, whereas stable transformants were not obtained with R. rhodochrous or with several coryneform bacteria tested as recipients. A restriction map demonstrated 14 unique restriction sites in pMVS301, some of which are potentially useful for molecular cloning in Rhodococcus spp. and other actinomycetes. This is the first report of plasmid transformation and of heterologous gene expression in a Rhodococcus sp.     

Genetic transformation of the ruminal bacteria Butyrivibrio fibrisolvens and Streptococcus bovis by electroporation

Electroporation methods for introduction of plasmid DNA into the ruminal bacteria Butyrivibrio fibrisolvens and Streptococcus bovis were developed. Electroporation of the strictly anaerobic B. fibrisolvens was carried out in an anaerobic glovebox with a buffer of 10% (v/v) glycerol and 1 mM MgCl₂ in distilled water. Streptococcus bovis electroporation could be carried out aerobically with a buffer of 10% (v/v) glycerol in distilled water. The Escherichia coli/Bacillus subtilis shuttle vector pBS42 could be transformed into B. fibrisolvens strain H17c, selecting for chloramphenicol resistance. The Streptococcus sanguis/E. coli shuttle vector pVA838 could replicate and express erythromycin resistance in Strep. bovis. Both vectors were stable in each organism in the absence of antibiotic selection. While the efficiency was low (<10²/μg DNA), the results demonstrate a means to introduce cloned genes into these organisms.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

Electrotransformation of meat lactobacilli. Effect of several parameters on their efficiency of transformation

Intact cells of several lactobacilli isolated from Spanish dry fermented sausages ( Lactobacillus curvatus, Lact. sake, Lact. plantarum and Lact. bavaricus ) were transformed by electroporation. With pNZ12 as a vector, transformation efficiencies of 2.4 times 10⁵, 3.8 times 10³ and 8.8 times 10² transformants μg^-1 DNA were observed for Lact. curvatus CTC435, Lact. sake CTC335 and Lact. bavaricus CTC232, respectively.
Effects of variation in experimental parameters on transformation efficiency were evaluated. Strains, vectors and buffers were the determinant parameters. The growth phase of the culture, cell concentration, voltage, use of cell wall weakening agents and the purity of the vector influenced the transformation efficiency in most strains.     

Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1.

The degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine (2-chloro-4-ethyl-amino-6-isopropylamino-1,3,5-triazine) is associated with an indigenous plasmid in Rhodococcus sp. strain TE1. Plasmid DNA libraries of Rhodococcus sp. strain TE1 were constructed in a Rhodococcus-Escherichia coli shuttle vector, pBS305, and transferred into Rhodococcus sp. strain TE3, a derivative of Rhodococcus sp. strain TE1 lacking herbicide degradation activity, to select transformants capable of growing on EPTC as the sole source of carbon (EPTC+). Analysis of plasmids from the EPTC+ transformants indicated that the eptA gene, which codes for the enzyme required for EPTC degradation, residues on a 6.2-kb KpnI fragment. The cloned fragment also harbored the gene required for atrazine N dealkylation (atrA). The plasmid carrying the cloned fragment could be electroporated into a number of other Rhodococcus strains in which both eptA and atrA were fully expressed. No expression of the cloned genes was evident in E. coli strains. Subcloning of the 6.2-kb fragment to distinguish between EPTC- and atrazine-degrading genes was not successful.     

Cloning and expression of the s-triazine hydrolase gene (trzA) from Rhodococcus corallinus and development of Rhodococcus recombinant strains capable of dealkylating and dechlorinating the herbicide atrazine.

We used degenerate oligodeoxyribonucleotides derived from the N-terminal sequence of the s-triazine hydrolase from Rhodococcus corallinus NRRL B-15444R in an amplification reaction to isolate a DNA segment containing a 57-bp fragment from the trzA gene. By using the nucleotide sequence of this fragment, a nondegenerate oligodeoxyribonucleotide was synthesized and used to screen a genomic library of R. corallinus DNA for fragments containing trzA. A 5.3-kb PstI fragment containing trzA was cloned, and the nucleotide sequence of a 2,450-bp region containing trzA was determined. No trzA expression was detected in Escherichia coli or several other gram-negative bacteria. The trzA gene was subcloned into a Rhodococcus-E. coli shuttle vector, pBS305, and transformed into several Rhodococcus strains. Expression of trzA was demonstrated in all Rhodococcus transformants. Rhodococcus sp. strain TE1, which possesses the catabolic gene (atrA) for the N-dealkylation of the herbicides atrazine and simazine, was able to dechlorinate the dealkylated metabolites of atrazine and simazine when carrying the trzA gene on a plasmid. A plasmid carrying both atrA and trzA was constructed and transformed into three atrA- and trzA-deficient Rhodococcus strains. Both genes were expressed in the transformants. The s-triazine hydrolase activity of the recombinant strains carrying the trzA plasmid were compared with that of the R. corallinus strain from which it was derived.     

Construction and characterization of Bordetella pertussis RecA− mutants

Abstract Antibiotic drug-resistance cassettes (DRCs) were used to insertionally inactivate the wild-type Bordetella pertussis recA gene cloned into a suicide vector. The mutant allele was mobilized by conjugal gene transfer from Escherichia coli strain SM10 into different genetic backgrounds of B. pertussis . Southern hybridization studies of one of these mutants showed that it contained a DRC integrated within a recA gene situated within a Cla I genomic DNA fragment. Selected mutants were assayed to quantify recombinational and DNA repair deficiencies. These mutants were shown to be highly sensitive to both chemically and physically induced DNA damage. Gene transfer studies of another RecA⁻ mutant also indicated that it was defective in intergenic recombination. No difference in hemolytic activity or production of capsule was detected between the RecA⁻ mutants and their corresponding wild-type strains. The results of this investigation corroborate previous studies with the cloned B. pertussis recA gene, and demonstrate that the expression of the B. pertussis recA gene in the original host promotes both DNA repair and recombination.     

Ds excision from extrachromosomal geminivirus vector DNA is coupled to vector DNA replication in maize

Analysis of transposition products generated after Activator (Ac) excision from the P locus in maize suggest that Ac excises either during or after replication of the P locus. The frequency of excision of the non-autonomous Ac derivative, Dissociation ( Ds ), from extrachromosomal replicating and nonreplicating vector DNAs in transfected black mexican sweet maize protoplasts was compared to assess directly a role of extrachromosomal vector DNA replication in Ds excision. Replicating (rep⁺) and non-replicating (rep⁻) vector DNAs comprised a Ds element that harbored a geminivirus, wheat dwarf virus (WDV), origin of replication and WDV genes required for viral DNA replication (rep⁺) or mutant, inactive derivatives of these genes (rep⁻). Excision of Ds was detected only in those cell nuclei co-transfected with the replicating Ds -vector DNA and a transposase expression vector. Quantitative reconstruction experiments showed that Ds excised at least 3 × 10⁵-fold more frequently from replicating vector DNA as compared with nonreplicating vector DNA. Therefore, these results provide direct evidence for a coupling of Ds excision from extrachromosomal vector DNA to vector DNA replication in maize.     

A new pathway for bacterial catabolism of 3-hydroxybenzoic acid

Abstract This paper is the first to describe the transformation of 3-hydroxybenzoate (3-HBA) by Pseudomonas putida BS893 by a new pathway via 2,3-dihydroxybenzoate (2,3-DBA) and catechol. We have compared the intermediates and appropriate enzyme activities in P. putida BS893 (pBS241) and in a cured derivative BS662 (Bph⁻) thereof, for the ascertainment of plasmid or chromosomal genetic control over 3-HBA-catabolism. The results presented show that catabolism of 3-HBA in P. putida BS893 (pBS241) is controlled by chromosomal genes.     

Physiology of biosurfactant synthesis by Rhodococcus species H13-A

The physiology of biosurfactant synthesis by a soil isolate, identified as a Rhodococcus species, is described. The biosurfactant is a surface-active glycolipid produced during the stationary growth phase of Rhodococcus species H13-A on n-alkanes and fatty alcohols in response to limiting ammonium ion concentrations. Hexadecane-grown cells produced increasing amounts of extracellular glycolipid when the carbon to nitrogen ratio (C/N) was increased from 1.7 to 3.4. The increase in extracellular glycolipid in hexadecane-grown cells correlated with a decrease in the interfacial tension of the spent growth medium to values less than 5?mN/m. Significant levels of extracellular glycolipid were not detected in the spent growth medium of cells grown on triglycerides, fatty acids, ethanol, organic acids, or carbohydrates. Rhodococcus species H13-A contains the three indigenous plasmids pMVS100, pMVS200, and pMVS300, with neither pMVS200 nor pMVS300 being involved in glycolipid synthesis or hexadecane dissimilation. The role of pMVS100 remains undetermined. Key words: biosurfactants, glycolipids, trehalose lipids, Rhodococcus.     

Site-directed mutagenesis of the aspartokinase gene lysC and its characterization in Brevibacterium flavum

Using overlap extension polymerase chain reaction (PCR), five transformants of Escherichia coli containing site-directed mutagenized lys Cβ gene were generated and analysed. Exchange of C to A and C to T at nucleotide 1118 of the mutated lys Cβ gene causes a substitution of serine³⁰¹ in the wild-type enzyme for tyrosine³⁰¹ and phenylalanine³⁰¹ in the mutant enzymes, respectively. Enzyme assays showed that Brevibacterium flavum cells harbouring pSUMN18 with mutated lys Cβ genes exhibited 16–20 fold lower specific activities of aspartokinase as compared to that of host containing wild-type lys C gene. The mutation introduced into lys Cβ of B. flavum CCRC18271 resulted in partial feedback-resistant aspartokinase activity.     

Transformation of group A streptococci by electroporation

Abstract The introduction, via electroporation, of free plasmid DNA into three strains of Streptococcus pyogenes is described. The method is very simple and rapid and efficiencies vary from 1 × 10³ to 4 × 10⁴ per μg of DNA. The method was also used to introduce an integrative plasmid and transformants were obtained, albeit at a somewhat lower frequency (2 × 10²). Some of the plasmids used in this study are derivatives of the Lactococcus lactis subsp. cremoris Wg2 plasmid pWV01. These broad host range vectors replicate in Gram-positives as well as Gram-negatives (viz. Escherichia coli ). Here we show that they also replicate in S. pyogenes and S. sanguis .     

Electrotransformation of the stable L-form of Proteus mirabilis

Abstract To improve the transformability of stable protoplast type L-forms of Proteus mirabilis for recombinant plasmid DNA, conditions for efficient electrotransformation were explored. Exposing cells from the exponential phase of growth at a density of 6−8 × 10⁹/ml in electrotransformation buffer having a conductivity of 1.4 mS/cm to a field strength of 6.5 kV/cm for a mean pulse duration time of 1.2 ms reproducibly yielded transformation efficiencies in the order of 5 × 10⁴ transformants per μg of DNA. Compared to the polyethylene glycol method for transformation, electrotransformation appeared to be the method of choice for introduction of plasmid DNA into L-form cells.     

Screening and selection of surfactant-producing bacteria from waste lubricating oil

Forty-four strains with the ability to grow on waste lubricating oil as sole carbon source were isolated from soil-contaminated samples and identified. Of these, four Rhodococcus and one Bacillus strain were selected for their capacity to lower the surface tension of culture medium from 55 mN m^-1 to less than or equal to 40 mN m^-1 and the interfacial tension between the culture medium and kerosene from 21 mN m^-1 to less than 5 mN m^-1, respectively. The surface-active compounds, identified by means of TLC and specific reagents, from cultures of Rhodococcus strains and Bacillus were glycolipids and lipopeptide, respectively. The percentage loss of waste lubricating oil after 120 h of incubation ranged from 7.87% to 45.8% depending on the strain selected compared with uninoculated control. Decreases in paraffinic compounds and accumulation of new products, mainly with amide groups, were observed by direct and differential infrared spectrometry when selected strains grew on waste lubricating oil.     

Zinc ions promote prestalk-to-stalk and prespore-to-stalk conversions in Dictyostelium discoideum

Abstract To clarify the mechanism of stalk cell differentiation in Dictyostelium discoideum (strain NC4), we have examined the effects of Zn²⁺ on in vitro cell differentiation of prestalk and prespore cells isolated from normally formed slugs. Prestalk cells did not differentiate into stalk cells under submerged conditions, but in the presence of the stalk-inducing factor-1 (DIF-1) at 100 nM or Zn²⁺ at 5 mM, a small number of the cells (< 15%) differentiated into stalk cells. Interestingly, Zn²⁺ in combination with DIF-1 induced the prestalk-to-stalk conversion at high efficiencies (approx. 60%). Furthermore, isolated prespore cells were also converted to stalk cells at high efficiencies (approx. 50%) in the presence of both DIF-1 and Zn²⁺, while the conversion poorly occurred in the absence of Zn²⁺. These results indicate that Zn²⁺ may mimic some cellular interaction(s) which are required for stalk cell formation in this strain.     

Molecular cloning and expression in Saccharomyces cerevisiae and Neurospora crassa of the invertase gene from Neurospora crassa

A plasmid (named pCN2) carrying a 7.6 kb BamHI DNA insert was isolated from a Neurospora crassa genomic library raised in the yeast vector YRp7. Saccharomyces cerevisiae suc ⁰ and N. crassa inv strains transformed with p NC2 were able to grow on sucrose-based media and expressed invertase activity. Saccharomyces cerevisiae suc ⁰ ( p NC2) expressed a product which immunoreacted with antibody raised against purified invertase from wild type N. crassa , although S. cerevisiae suc ⁺ did not. The cloned DNA hybridized with a 7.6 kb DNA fragment from BamHI -restricted wild type N. crassa DNA. Plasmid pNC2 transformed N. crassa Inv^- to Inv⁺ by integration either near to the endogenous inv locus (40% events) or at other genomic sites (60% events). It appears therefore that the cloned DNA piece encodes the N. crassa invertase enzyme. A 3.8 kb XhoI DNA fragment, derived from pNC2, inserted in YRp7, in both orientation, was able to express invertase activity in yeast, suggesting that it contains an intact invertase gene which is not expressed from a vector promoter.     

Electrotransfection of Propionibacterium freudenreichii TL 110

The first highly efficient protocol is described for the electrotransfection of Propionibacterium freudenreichii with DNA phage. The transfection efficiency is 7 times 10⁵ transfectants per μg of DNA under optimal conditions. Optimized parameters included the field strength (12.5 kV, 200 Ohms, 25 μF), phage DNA concentration (1 μg ml^-1) and cell density (1.5 times 10¹⁰ cells ml^-1). Growth in the presence of glycine and harvesting of cells during the early exponential growth phase increased the transfection efficiency. This electrotransfection protocol is of importance for the genetic improvement of dairy propionibacteria.     

Cloning and manipulation of the Corynebacterium pseudotuberculosis recA gene for live vaccine vector development

Abstract Corynebacterium pseudotuberculosis is an intracellular bacterial pathogen causing a chronic abscessing disease in sheep and goats called caseous lymphadenitis. We are developing this bacterial species as a live vector system to deliver vaccine antigens to the animal immune system. Foreign genes expressed in bacterial hosts can be unstable so we undertook to delete the C. pseudotuberculosis chromosomal recA gene to determine whether a recA ⁻ background would reduce the frequency of recombination in cloned DNA. Homologous DNA recombination within an isogenic recA ⁻ C. pseudotuberculosis was 10–12-fold lower than that in the recA ⁺ parental strain. Importantly, the recA mutation had no detectable affect upon the virulence of C. pseudotuberculosis in a mouse model. Taken together these results suggest that a recA ⁻ background may be useful in the further development of C. pseudotuberculosis as a vaccine vector.     

Conjugal transfer at natural population densities in a microcosm simulating an estuarine environment

Abstract Estuarine microcosms were used to follow conjugal transfer of a broad host range IncP1 plasmid from a Pseudomonas putida donor to indigenous bacteria. Donor cells were added at a concentration similar to the natural abundance of bacteria in the water column (10⁶ cells ml⁻¹). Transfer was not detected in any of the test microcosms (calculated limit of detection of 10⁻⁷ and 10⁻⁴ transconjugants donor⁻¹ in water column and sediment, respectively), with the exception of transfer to an isogenic recipient (added at 10⁵ cells ml⁻¹) in sediments of controls that had been inoculated with both donors and recipients. The same plasmid was transferred with high efficiencies (10⁻¹ to 10⁻³) to a variety of recipients in filter and broth matings. These results suggest that if conjugal gene transfer occurred, it was at efficiencies that were not detectable in estuarine microcosms simulating natural population densities.     

Meeting US biofuel goals with less land: the potential of Miscanthus

Biofuels from crops are emerging as a Jekyll & Hyde – promoted by some as a means to offset fossil fuel emissions, denigrated by others as lacking sustainability and taking land from food crops. It is frequently asserted that plants convert only 0.1% of solar energy into biomass, therefore requiring unacceptable amounts of land for production of fuel feedstocks. The C₄ perennial grass Miscanthus × giganteus has proved a promising biomass crop in Europe, while switchgrass ( Panicum virgatum ) has been tested at several locations in N. America. Here, replicated side-by-side trials of these two crops were established for the first time along a latitudinal gradient in Illinois. Over 3 years of trials, Miscanthus × giganteus achieved average annual conversion efficiencies into harvestable biomass of 1.0% (30 t ha⁻¹) and a maximum of 2.0% (61 t ha⁻¹), with minimal agricultural inputs. The regionally adapted switchgrass variety Cave-in-Rock achieved somewhat lower yields, averaging 10 t ha⁻¹. Given that there has been little attempt to improve the agronomy and genetics of these grasses compared with the major grain crops, these efficiencies are the minimum of what may be achieved. At this 1.0% efficiency, 12 million hectares, or 9.3% of current US cropland, would be sufficient to provide 133 × 10⁹ L of ethanol, enough to offset one-fifth of the current US gasoline use. In contrast, maize grain from the same area of land would only provide 49 × 10⁹ L, while requiring much higher nitrogen and fossil energy inputs in its cultivation.