Prototrophic Strains Research Articles

Diversity of biofilms produced by quorum-sensing-deficient clinical isolates of Pseudomonas aeruginosa

The quorum-sensing (QS) systems control several virulence attributes of Pseudomonas aeruginosa. Five QS-deficient P. aeruginosa clinical isolates (CI) that were obtained from wound (CI-1), tracheal (CI-2, CI-3, CI-4) and urinary tract (CI-5) infections had previously been characterized. In this study, a flow-through continuous-culture system was utilized to examine in detail the biofilms formed by these isolates in comparison with the P. aeruginosa prototrophic strain PAO1. Analysis of the biofilms by confocal laser scanning microscopy and COMSTAT image analysis at 1 and 7 days post-inoculation showed that the isolates produced diverse biofilms. In comparison with PAO1, the CI produced biofilms that scarcely or partially covered the surface at day 1, although CI-1 produced larger microcolonies. At day 7, CI-2 and CI-4 produced mature biofilms denser than that produced by PAO1, while the biofilm formed by CI-1 changed very little from day 1. CI-1 was defective in both swarming and twitching motilities, and immunoblotting analysis confirmed that it produced a reduced level of PilA protein. The twitching-motility defect of CI-1 was not complemented by a plasmid carrying intact pilA. In the 48 h colony biofilm assay, the CI varied in susceptibility to imipenem, gentamicin and piperacillin/tazobactam. These results suggest that: (1) the isolates produced biofilms with different structures and densities from that of PAO1; (2) biofilm formation by the isolates was not influenced by either the isolation site or the QS deficiencies of the isolates; (3) the behaviour of CI-1 in the different biofilm systems may be due to its lack of swarming motility and type IV pilus-related twitching motility.

Isolation of recombinant strains with enhanced pectinase production by protoplast fusion between Penicillium expansum and Penicillium griseoroseum

Protoplast fusion between complementary auxotrophic and morphological mutant strains of Penicillium griseoroseum and P. expansum was induced by polyethylene glycol and calcium ions (Ca2+). Fusant strains were obtained in minimal medium and a prototrophic strain, possibly diploid, was chosen for haplodization with the fungicide benomyl. Different recombinant strains were isolated and characterized for occurrence of auxotrophic mutations and pectinolytic enzyme production. The fusant prototrophic did not present higher pectinase production than the parental strains, but among 29 recombinants analyzed, four presented enhanced enzyme activities. The recombinant RGE27, which possesses the same auxotrophic and morphologic mutations as the P. griseoroseum parental strain, presented a considerable increase in polygalacturonase (3-fold) and pectin lyase production (1.2-fold).

Comparative genomics of the lactic acid bacteria

Lactic acid-producing bacteria are associated with various plant and animal niches and play a key role in the production of fermented foods and beverages. We report nine genome sequences representing the phylogenetic and functional diversity of these bacteria. The small genomes of lactic acid bacteria encode a broad repertoire of transporters for efficient carbon and nitrogen acquisition from the nutritionally rich environments they inhabit and reflect a limited range of biosynthetic capabilities that indicate both prototrophic and auxotrophic strains. Phylogenetic analyses, comparison of gene content across the group, and reconstruction of ancestral gene sets indicate a combination of extensive gene loss and key gene acquisitions via horizontal gene transfer during the coevolution of lactic acid bacteria with their habitats.

Biotin Sensing in Saccharomyces cerevisiae Is Mediated by a Conserved DNA Element and Requires the Activity of Biotin-Protein Ligase

Biotin is a water-soluble vitamin that functions as a prosthetic group in carboxylation reactions. In addition to its role as a cofactor, biotin has multiple roles in gene regulation. We analyzed biotin effects on gene expression in the yeast Saccharomyces cerevisiae and demonstrated by microarray, Northern, and Western analyses that all yeast genes encoding proteins involved in biotin metabolism are up-regulated following biotin depletion. Many of these genes contain a palindromic promoter element that is necessary and sufficient for mediating the biotin response and functions as an upstream-activating sequence. Mutants lacking the plasma membrane biotin transporter Vht1p display constitutively high expression levels of biotin-responsive genes. However, they react normally to biotin precursors that do not require Vht1p for uptake. The biotin-like effect of precursors with regard to gene expression requires their intracellular conversion to biotin. This demonstrates that Vht1p does not act as a sensor for biotin and that intracellular biotin is crucial for gene expression. Mutants with defects in biotin-protein ligase, similar to vht1delta mutants, also display aberrantly high expression of biotin-responsive genes. Like vht1delta cells, they have reduced levels of protein biotinylation, but unlike vht1delta mutants, they possess normal levels of free intracellular biotin. This indicates that free intracellular biotin is irrelevant for gene regulation and identifies biotin-protein ligase as an important element of the biotin-sensing pathway in yeast.

5-fluoro-orotic acid induces chromosome alterations in genetically manipulated strains of Candida albicans

We previously reported the occurrence of chromosome alterations in a Candida albicans prototrophic strain 3153A treated with 5-fluoro-orotic acid (5-FOA). In this study we investigated the mutagenic properties of 5-FOA with two derivatives of C. albicans strain CAF4-2 (ura3/ura3), each containing an ectopic copy of URA3 gene (ura3/ ura3 URA3) on a different chromosome. As expected, after the ura3/ura3 URA3 constructs were applied to 5-FOA containing solid medium, the “pop-outs” that lost URA3 appeared. However most of the “pop-outs” acquired various chromosome alterations. Thus constructs exposed to 5-FOA should be examined for chromosome alterations or the use of 5-FOA should be avoided.

5-fluoro-orotic acid induces chromosome alterations in genetically manipulated strains of Candida albicans

We previously reported the occurrence of chromosome alterations in a Candida albicans prototrophic strain 3153A treated with 5-fluoro-orotic acid (5-FOA). In this study we investigated the mutagenic properties of 5-FOA with two derivatives of C. albicans strain CAF4-2 (ura3/ura3), each containing an ectopic copy of URA3 gene (ura3/ ura3 URA3) on a different chromosome. As expected, after the ura3/ura3 URA3 constructs were applied to 5-FOA containing solid medium, the "pop-outs" that lost URA3 appeared. However most of the "pop-outs" acquired various chromosome alterations. Thus constructs exposed to 5-FOA should be examined for chromosome alterations or the use of 5-FOA should be avoided.

Sequential gene deletions in Hypocrea jecorina using a single blaster cassette

In Hypocrea jecorina (anamorph: Trichoderma reesei) multiple gene deletions are limited by the number of readily available selection markers. We have therefore constructed a blaster cassette which enables successive gene knock-outs in H. jecorina. This 3.5 kb pyr4 blaster cassette contains the H. jecorina pyr4 marker gene encoding orotidine-5'-monophosphate (OMP) decarboxylase flanked by two direct repeats of the Streptoalloteichus hindustanus bleomycin gene (Sh ble), which facilitate the excision of the blaster cassette by homologous recombination after each round of deletion. Functionality of this pyr4 blaster cassette was demonstrated by deletion of the glk1 encoding glucokinase and hxk1 encoding hexokinase. 1.4-1.8 kb of the non-coding flanking regions of both target genes were cloned into the respective blaster cassettes and transformation of a pyr4 negative H. jecorina strain with the two cassettes resulted in 10-13% of the transformants in the deletion of one of the two kinase genes. For excision of the pyr4 blaster cassettes, Deltaglk1 strains were selected for growth in the presence of 5-fluoroorotic acid. Recombination between the two Sh ble elements resulted in uridine auxotrophic strains which retained their respective glucokinase negative phenotype. Subsequent transformation of one of these auxotrophic Deltaglk1 strains with the hexokinase blaster cassette resulted in pyr4 prototrophic strains deleted in both glk1 and hxk1. Deltaglk1 strains showed reduced growth on d-glucose and d-fructose whereas Deltahxkl strains showed reduced compact growth on d-glucose but were unable to grow on d-fructose as carbon source. The double Deltaglk1Deltahxk1 deletion strain was completely unable to grow on either d-glucose or d-fructose.

Structural Basis for Autoinhibition and Mutational Activation of Eukaryotic Initiation Factor 2α Protein Kinase GCN2*[boxs

The GCN2 protein kinase coordinates protein synthesis with levels of amino acid stores by phosphorylating eukaryotic translation initiation factor 2. The autoinhibited form of GCN2 is activated in cells starved of amino acids by binding of uncharged tRNA to a histidyl-tRNA synthetase-like domain. Replacement of Arg-794 with Gly in the PK domain (R794G) activates GCN2 independently of tRNA binding. Crystal structures of the GCN2 protein kinase domain have been determined for wild-type and R794G mutant forms in the apo state and bound to ATP/AMPPNP. These structures reveal that GCN2 autoinhibition results from stabilization of a closed conformation that restricts ATP binding. The R794G mutant shows increased flexibility in the hinge region connecting the N- and C-lobes, resulting from loss of multiple interactions involving Arg794. This conformational change is associated with intradomain movement that enhances ATP binding and hydrolysis. We propose that intramolecular interactions following tRNA binding remodel the hinge region in a manner similar to the mechanism of enzyme activation elicited by the R794G mutation.

Identification of residues in the WD-40 repeat motif of the F-box protein Met30p required for interaction with its substrate Met4p

The SCF family of ubiquitin-ligases consists of a common core machinery, namelySkp1p, Cdc53p, Hrt1p, and a variable component, the F-box protein that is responsible for substrate recognition. The F-box motif, which consists of approximately 40 amino acids, connects the F-box protein to the core ubiquitin-ligase machinery. Distinct SCF complexes, defined by distinct F-box proteins, target different substrate proteins for proteasome-dependent degradation. As part of the SCF(Met30p) complex, the F-box protein Met30p selects the substrate Met4p, a transcriptional activator for MET biosynthetic genes that mediate sulfur uptake and biosynthesis of sulfur containing compounds. When cells are grown in the absence of methionine, Met4p evades degradation by the SCF(Met30p) complex and activates the MET biosynthetic pathway. However, overproduction of Met30p represses MET gene expression and induces methionine auxotrophy in an otherwise methionine prototrophic strain. Here we demonstrate that overproduction of the C-terminal portion of Met30p, which is composed almost entirely of seven WD-40 repeat motifs, is necessary and sufficient to induce methionine auxotrophy and complement the temperature sensitive (ts) met30-6 mutation. Furthermore, we show that this region of Met30p is important for binding Met4p and that mutations that disrupt this interaction prevent both the induction of methionine auxotrophy and complementation of the met30-6 mutation. These assays have been exploited to identify residues that are important for the interaction of Met30p with its substrate. Since the C-terminal domain of Met30p lacks the F-box and cannot support the ubiquitination of Met4p, our results indicate that the recruitment of Met4p to the SCF(Met30p) complex itself results in inactivation of Met4p, independently of its ubiquitination.

5-Fluoro-orotic acid induces chromosome alterations inCandida albicans

Treatment of a prototrophic laboratory strain of Candida albicans with 5-fluoro-orotic acid (5-FOA) produced two major types of mutants with chromosomal alterations, 5-FOA-resistant (FoaR) and those remaining sensitive (FoaS). Both major types remained Ura+. FoaR mutants, produced after a long exposure, contained either a duplication of chromosome 4b or an inner enlargement of chromosome 5b. The average mutant frequency was approximately 1.0 x 10(-5). The reverse mutation of FoaR to FoaS also caused the loss of either the extra chromosome 4b or the enlarged chromosome 5b, revealing a causal relationship between the resistance and the specific chromosome constitution. The cells remained sensitive after a relatively short 24 h exposure to 5-FOA medium, but the treatment induced non-specific changes in lengths of various chromosomes. Furthermore, FoaR type mutants acquired a notable chromosomal and phenotypic instability. Our results indicate the necessity of electrokaryotyping of strains that have been exposed to 5-FOA, especially with studies of gene function and with DNA microarray assays.

A laboratory yeast strain suitable for spirit production

Yeast strains of the species Saccharomyces cerevisiae currently in use for the production of consumable alcohols such as beer, wine and spirits are genetically largely undefined. This prevents the use of standard genetic manipulations, such as crossings and tetrad analysis, for strain improvement. Furthermore, it complicates the application of the majority of modern methods developed in yeast molecular biology. Here we used two haploid laboratory strains with suitable auxotrophic markers for the construction of a genetically well defined, prototrophic diploid production strain. This strain was tested for its fermentative and sensory performances in comparison to commercially available yeasts. Three different fruit mashes (cherries, plums and pears) were fermented in a 90 kg scale. These were then subjected to distillation and used for the production of spirits with a final ethanol content of 40% (v/v). Fermentation parameters assayed included growth, sugar utilization, ethanol production and generation of volatile compounds, higher alcohols and glycerol. The spirits were also tested for their sensory performances and the data obtained statistically consolidated. Our results clearly demonstrate that this laboratory strain does not display any disadvantage compared with commercial yeasts in spirit production for any of the parameters tested, yet it offers the potential to apply both classical breeding and modern molecular genetic techniques for adjusting yeast physiology to special production schemes.

Visualization of protoplast fusion and quantitation of recombination in fused protoplasts of auxotrophic strains of Escherichia coli

Protoplast fusion has been used to combine genes from different organisms to create strains with desired properties. A recently developed variant on this approach, genome shuffling, involves generation of a genetically heterogeneous population of a single organism, followed by recursive protoplast fusion to allow recombination of mutations within the fused protoplasts. These are powerful techniques for engineering of microbial strains for desirable industrial properties. However, there is a prevailing opinion that it will be difficult to use these methods for engineering of Gram-negative bacteria because the outer membrane makes protoplast fusion more difficult. Here we describe the successful use of protoplast fusion in Escherichia coli. Using two auxotrophic strains of E. coli, we obtained prototrophic strains by recombination in fused protoplasts at frequencies of 0.05–0.7% based on the number of protoplasts subjected to fusion. This frequency is three–four orders of magnitude better than those previously reported for recombination in fused protoplasts of Gram-negative bacteria such as E. coli and Providencia alcalifaciens.

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.

Yeast strains designed for screening of reversal agents and genetic suppressors of multidrug resistance

Multidrug resistance in yeast results from over-expression of drug efflux transporter genes due to gain-of-function mutations in transcription factors. To suppress multidrug resistance at the level of gene expression, we have developed a yeast-based screening system for the detection of compounds down-regulating the major multidrug ABC transporter Pdr5p expressed under the control of Pdr3p transcription factor. Here, we report the construction and properties of the improved set of yeast strains designed along with such screening also for a global analysis of genetic suppressors of multidrug resistance. The basic components of this system, the P GAL1-PDR3 and P PDR5-pma1(D378N) fusion genes, were individually or simultaneously integrated into corresponding chromosomes of a hypersensitive S. cerevisiae strain deleted in the PDR1 and PDR3 genes. This resulted in increased mitotic stability of a set of new test strains compared with the original prototrophic strain ZK11-1 developed previously. In addition, some of the strains designed are auxotrophic for leucine, uracil and histidine allowing them to be used in genetic screens for positive selection of multicopy or loss-of-function genetic suppressors of multidrug resistance.

Flux to acetate and lactate excretions in industrial fermentations: physiological and biochemical implications.

The efficiency of carbon conversion to biomass and desirable end products in industrial fermentations is diminished by the diversion of carbon to acetate and lactate excretions. In this study, the use of prototrophic and mutant strains of Escherichia coli, as well as enzyme active site directed inhibitors, revealed that flux to acetate excretion is physiologically advantageous to the organism as it facilitates a faster growth rate (mu) and permits growth to high cell densities. Moreover, the abolition of flux to acetate excretion was balanced by the excretion of lactate as well as 2-oxoglutarate, isocitrate and citrate, suggesting a 'bottle-neck' effect at the level of 2-oxoglutarate in the Krebs cycle. It is proposed that the acetate excreting enzymes, phosphotransacetylase and acetate kinase, constitute an anaplerotic loop or by-pass, the primary function of which is to replenish the Krebs cycle with reduced CoA, thus relieving the bottle-neck effect at the level of 2-oxoglutarate dehydrogenase. Furthermore, flux to lactate excretion plays a central role in regenerating proton gradient and maintaining the redox balance within the cell. The long-held view that flux to acetate and lactate excretions is merely a function of an 'over-flow' in central metabolism should, therefore, be re-evaluated.

Saccharomyces cerevisiae multidrug transporter Qdr2p (Yil121wp): localization and function as a quinidine resistance determinant.

This work reports the functional analysis of Saccharomyces cerevisiae open reading frame YIL121w, encoding a member of a family of drug:H(+) antiporters with 12 predicted membrane-spanning segments (DHA12 family). Like its close homologue Qdr1p, Yil121wp was localized at the plasma membrane, and its increased expression also led to increased tolerance to the antiarrhythmia and antimalarial drug quinidine. The quinidine resistance phenotype was confirmed for different yeast strains and growth media, including a prototrophic strain, and YIL121w was named the QDR2 gene. Both QDR1 and QDR2 were also implicated in yeast resistance to the herbicide barban (4-chloro-2-butynyl [3-chlorophenyl] carbamate), and the genes are functionally interchangeable with respect to both resistance phenotypes. The average intracellular pH of a yeast population challenged with quinidine added to the acidic growth medium was significantly below the intracellular pH of the unstressed population, suggesting plasma membrane permeabilization by quinidine with consequent increase of the H(+) influx rate. For the same extracellular quinidine concentration, internal acidification was more intense for the Deltaqdr2 deletant compared with the parental strain. Although QDR2 transcription was not enhanced in response to quinidine, the results confirmed that Qdr2p is involved in the active export of quinidine out of the cell, thus conferring resistance to the drug.

Characterization of the Exoglucanase-Encoding Gene PaEXG2 and Study of Its Role in the Biocontrol Activity of Pichia anomala Strain K

ABSTRACT The PaEXG2 gene, encoding an exo-beta-1,3-glucanase, was isolated from the biocontrol agent Pichia anomala strain K. PaEXG2 has the capacity for coding an acidic protein of 427 amino acids with a predicted molecular weight of 45.7 kDa, a calculated pI of 4.7, and one potential N-glycosylation site. PaEXG2 was disrupted by the insertion of the URA3 marker gene, encoding orotidine monophosphate decarboxylase in strain KU1, a uracil auxotroph derived from strain K. Strain KU1 showed inferior biocontrol activity and colonization of wounds on apples, compared to the prototrophic strain. Antagonism and colonization were recovered after the restoration of prototrophy by transformation with the URA3 gene. Integrative transformation was shown to be mostly ectopic in strain K descendants (only 4% of integration by homologous recombination). PaEXG2 disruption abolished all detectable extracellular exo-beta-1,3-glucanase activity in vitro and in situ but did not affect biocontrol of Botrytis cinerea on wounded apples.

Ubiquitin Pathway Proteins Influence the Mechanism of Action of the Novel Immunosuppressive Drug FTY720 in Saccharomyces cerevisiae

FTY720 is an immunosuppressive drug in clinical development for transplant graft protection in humans. This agent is of particular interest because, unlike currently available regimes, it acts to sequester lymphocytes without causing cytotoxicity or blocking differentiation and growth potential. In an effort to elucidate the mechanism of action of FTY720, and identify its downstream effectors, we have screened genomic libraries and spontaneous mutants of the model system Saccharomyces cerevisiae for resistance to FTY720. We identified several proteins and pathways as being involved in the mechanism of action of FTY720. We show specifically that the two amino acid transporters TAT1 and TAT2, the two ubiquitin proteases UBP5 and UBP11, and the heat shock protein CAJ1 confer growth resistance to FTY720 when overexpressed. Another amino acid transporter, GNP1, and the ubiquitin structural gene UBI4 as well as the ubiquitin ligase RSP5, and its binding protein BUL1 confer growth resistance in a mutated form. Supporting the importance of amino acid transport in the growth resistance phenotype of S. cerevisiae to the immunosuppressive agent FTY720, a prototrophic strain was more resistant to FTY720 than the isogenic auxotroph. To further explore these results, the effects on amino acid uptake and protein degradation were measured in the presence of FTY720. Due to the high conservation of these proteins and pathways between yeast and humans, these results may provide valuable insights into the mechanism of action of FTY720 in lymphocyte sequestration in humans.

Ca2+ -dependence and inhibition of transformation by trifluoperazine and chlorpromazine in Thermoactinomyces vulgaris.

Ca(2+) enhanced the transformation frequency of Thermoactinomyces vulgaris (stock no. 1278) of an auxotrophic strain by the chromosomal DNA isolated from a prototrophic strain (stock no. 1227). The number of transformants showed a marked increase with increasing concentration of CaCl(2) upto 0.05 mM; and above this concentration, the transformation frequency decreased significantly. Antipsychotic drugs that are potent calmodulin inhibitors, like trifluoperazine and chlorpromazine, when applied in the concentration range of 0.01-0.04 mM along with optimal CaCl(2) concentration to the cultures of the recipient cells, resulted in a significant inhibition in the frequency of Ca(2+)-stimulated transformation. The results of present investigation suggest the involvement of a Ca(2+)-dependent protein activator in the development of Ca(2+)-mediated competence, which could have played an important role in the enhancement of genetic transformation in this aerobic spore forming thermophilic actinomycete.

Expression of selenomethionyl proteins in a prototrophic strain of Escherichia coli

Expression of selenomethionyl proteins in a prototrophic strain of Escherichia coli