Double Auxotrophic Mutants Research Articles

Intravenous Immunization with Triple Auxotrophs of Mycobacterium tuberculosis : A novel vaccine strategy against tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis ( Mtb ), remains a leading infectious cause of mortality worldwide despite widespread use of the BCG vaccine and the availability of sterilizing pharmacopoeia. Recent research indicates that the intravenous administration of BCG confers sterilizing immunity against Mtb pulmonary challenge in non-human primates. However, while BCG is relatively safe, complications such as disseminated BCGosis have been observed in immunocompromised individuals. Double auxotrophic mutants of Mtb lacking the ability to synthesize leucine and pantothenate are safe and sterilized in immunocompromised mice and SIV-infected Rhesus macaques. We examined how immunization with a Mtb triple auxotrophic strain, mc 2 7902, which cannot synthesize leucine, pantothenate, and arginine, protects immunocompetent mice from a virulent Mtb infection. The route of immunization was a crucial factor for protection with mc 2 7902 with intravenous immunization being 100 times more effective in protecting immunocompetent mice from Mtb challenge when compared to conventional subcutaneous vaccination with BCG. To further increase the safety of the attenuated auxotroph for vaccine purposes, the type VII secretion system Esx1 responsible for BCG attenuation was deleted in mc 2 7902. When tested by prime-boost immunization of immunocompetent mice followed by aerosol challenge with virulent Mtb , mc 2 7902 Δ esx1 provided similar protection to mc 2 7902. This robust protection against Mtb infection conferred by mc 2 7902 and mc 2 7902 Δ esx1 in a mouse model paves the way for new TB vaccine development using highly attenuated, auxotrophic Mtb strains.

A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella

ABSTRACT Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialized animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.

SteadyCom: Predicting microbial abundances while ensuring community stability.

Genome-scale metabolic modeling has become widespread for analyzing microbial metabolism. Extending this established paradigm to more complex microbial communities is emerging as a promising way to unravel the interactions and biochemical repertoire of these omnipresent systems. While several modeling techniques have been developed for microbial communities, little emphasis has been placed on the need to impose a time-averaged constant growth rate across all members for a community to ensure co-existence and stability. In the absence of this constraint, the faster growing organism will ultimately displace all other microbes in the community. This is particularly important for predicting steady-state microbiota composition as it imposes significant restrictions on the allowable community membership, composition and phenotypes. In this study, we introduce the SteadyCom optimization framework for predicting metabolic flux distributions consistent with the steady-state requirement. SteadyCom can be rapidly converged by iteratively solving linear programming (LP) problem and the number of iterations is independent of the number of organisms. A significant advantage of SteadyCom is compatibility with flux variability analysis. SteadyCom is first demonstrated for a community of four E. coli double auxotrophic mutants and is then applied to a gut microbiota model consisting of nine species, with representatives from the phyla Bacteroidetes, Firmicutes, Actinobacteria and Proteobacteria. In contrast to the direct use of FBA, SteadyCom is able to predict the change in species abundance in response to changes in diets with minimal additional imposed constraints on the model. By randomizing the uptake rates of microbes, an abundance profile with a good agreement to experimental gut microbiota is inferred. SteadyCom provides an important step towards the cross-cutting task of predicting the composition of a microbial community in a given environment.

L-Tryptophan Production by Auxotrophic and Analogue Resistant Mutants of Aureobacterium flavescens

A number of tyrosine plus phenylalanine double auxotrophic mutants were isolated by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) treatment of a locally isolated strain of Aureobacterium flavescens of which 11A39 and 11A17 were selected on the basis of their tryptophan production in a mineral salt medium over other isolated mutant strains. The mutational block in the aromatic amino acid biosynthetic pathway of the selected double auxotrophs were determined. By controlling pH of the production medium to near neutrality, the active growth period could be extended up to 72 h and more tryptophan was accumulated compared to pH unregulated culture where the active growth ceased after 48 h. Further improvement of the tryptophan production has been achieved by stepwise isolation of a mutant strain resistant to the tryptophan analogues p-fluorotryptophan (FT) and 5-methyl tryptophan (MT) from the 11A39. Demand for L-tryptophan as food additive and therapeutic agent is increasing day by day throughout the World, particularly in the underdeveloped and developing countries like India. Still to date India depends on other countries for L-tryptophan. The aim of this work is to develop a potent high yielding, feed back insensitive mutant strain and optimization of its medium pH for maximum production of tryptophan.

Abstract 580: Bone marrow cells trafficking after Salmonella typhimurium A1-R tumor targeting

Abstract Our laboratory has developed the tumor-targeting S. typhimurium double auxotrophic mutant termed A1-R. The A1-R mutant requires leucine and arginine, which disable it from growing in tumor tissue but the bacteria maintain high tumor virulence. In the present study, we used fluorescent imaging to observe bone marrow cells (BMC) trafficking in Lewis lung cancer-bearing nude mice after A1-R treatment. BMC were obtained from transgenic nude mice ubiquitously expressing green fluorescent protein (GFP). The GFP bone marrow cells were injected into the tail vein of red fluorescent protein (RFP)-expressing Lewis lung carcinoma-bearing nude mice. GFP-BMC trafficking to the tumor, lung, liver, and spleen were imaged at various time points after treatment with S. typhimurium A1-R. Untreated tumor-bearing mice injected with GFP-BMC were imaged as controls. Fluorescence imaging showed that there were more GFP-BMCs trafficking to tumor, lung, liver, and spleen in A1-R treated mice compared to untreated mice at 5 hours after A1-R infection. At day-2 after infection, there were significantly more GFP-BMCs in the tumor, and at day 4 after A1-R infection, BMCs appeared to die within the A1-R-treated tumors. GFP dendritic cells were observed in the liver of A1-R infected mouse at day 2 and day 4 after A1-R infection. These results suggest that immune cells in nude mice may be involved in the bacteria-induced antitumor response. The immune cells may also play a role in clearing the bacteria from normal organs in the host. In future experiments, we will identify the types of immune cells which play various roles in A1-R tumor targeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 580.

Abstract 597: Bacteria targeting and tumor vascular destruction by S. typhimurium A1-R depend on the vascularity of the tumor site

Abstract Our laboratory has previously developed a tumor-targeting double-auxotrophic mutant of S. typhimurium termed A1-1 (Cancer Research 66, 7647-7652, 2006; Proc. Natl. Acad. Sci. USA 104, 10170-10174, 2007). The A1-R mutant has high tumor virulence but requires leucine and argentine which disables the bacteria from growing in normal tissue. Our previous studies suggested that the rich vascularity of tumors may contribute to bacterial tumor targeting by A1-R. The goal of the present study was to determine the effect of A1-R on the tumors growing at different sites with different vascularity. In this study, we administered A1-R to Lewis lung carcinoma-bearing mice with tumor growing at three different sites. We observed that tumor location influences the efficacy of targeting of A1-R to the Lewis lung tumor. RFP-expressing Lewis lung cancer cells (1×107 cells/ml) were transplanted subcutaneously in the ear (50 µl), back skin (100 µl), and foot pad (25 µl) of nestin-GFP transgenic nude mice, which selectively express GFP in nascent blood vessels. The tumor-bearing mice were then treated with A1-R bacteria (5×107) via tail-vein injection. Bacteria tumor targeting and vascular damage were evaluated by fluorescence imaging with the Olympus IV100 and OV100 small-animal imaging systems designed for fluorescence imaging. Tumors in the ear had the fastest growth rate and were the most sensitive to bacteria targeting and hemorrhaged severely the day after A1-R injection. Tumors growing in the back were the second most sensitive to bacteria targeting and appeared necrotic 3 days after A1-R injection. Tumors growing in the food pad were the least sensitive to A1-R. Tumor vascularity correlated positively with A1-R targeting with the ear being the most vascular site and the foot pad the least vascular site. The present study suggests that bacteria tumor targeting and hemorrhage of the tumor depend on the vascularity of the tumor site. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 597.

Transformation of the white-rot basidiomycete Coriolus hirsutus using the ornithine carbamoyltransferase gene.

An efficient transformation system for the basidiomycete Coriolus hirsutus was developed. A double-auxotrophic mutant of C. hirsutus, deficient both in ornithine carbamoyltransferase (OCTase) and 3-isopropylmalate dehydrogenase (3-IPM dehydrogenase), was transformed to Arg+ with each allelic type of the C. hirsutus genomic OCTase gene (arg1) newly cloned. The transformation frequency of 10(3)-10(4) transformants per mug DNA per 10(6)-10(7) oidial protoplasts was reached. Southern blots showed that the transforming DNA was integrated into chromosomal DNA with multi-copies. The Arg+ phenotype of the transformants was stably inherited through mitosis.

Breeding of high-pyruvate-producing Torulopsis glabrata and amino acid auxotrophic mutants

An efficient method for the isolation of pyruvate-producing mutants of Torulopsis glabrata IFO 0005 was established. On mutagenesis of the parent strain, mutants requiring polypeptone for complete growth were isolated. Seven mutants among one hundred auxotrophs produced higher concentrations of pyruvate than the parent strain. Among them, arginine ( l-Arg) auxotrophic mutants and an isoleucine ( l-Ile) and valine ( l-Val) double auxotrophic mutant exhibited higher fermentative production of pyruvate from glucose than the parent strain. T. glabrata X-15 and X-17 required l-Arg for complete growth. T. glabrata X-68 absolutely required l-Ile and l-Val for complete growth. These three strains (X-15, X-17 and X-68) have more than 10% higher yields of pyruvate than the parent strain. Among them, the best strain regarding pyruvate productivity, T. glabrata X-15, accumulated 59.5 g/ l free pyruvic acid (yield, 60.1%; conversion to pyruvic acid of added glucose in 43 h) on a 3- l jar-fermentor scale. This yield with strain X-15 represented a 12% increase compared to that obtained with the parent strain.

Cellulolytic activity of wild type and mutantTrichoderma pseudokoningii

Strains of Trichoderma pseudokoningii are promising objects for genetic studies and cellulase production. Auxotrophic mutants with deficiencies in the biosynthesis of aminoacids, nucleotides and vitamins (up to five markers) in addition to morphological aspects like conidial colour were obtained from two strains of double auxotrophic mutants using UV radiation. In order to compare the cellulolytic capabilities of the T. pseudokoningii (wild type strain), some of its mutants and T. reesei QM9414 we performed semiquantitative cellulase assays and quantitative determination of the enzymes exoglucanase and endoglucanase. The semiquantitative test showed that the strains with minimal mycelial growth rate were better producers. Both tests revealed that two of the studied mutants, TG3 and TG4 presented a yield higher than the wild type, reaching 30% more exoglucanase and 70% more endoglucanase. These results indicate that the wild type was improved for cellulase production. Highly significant values of correlation were found for exoglucanase and endoglucanase activities, suggesting that these enzymes may be co-regulated in T. pseudokoningii.

L-phenylalanine production by double auxotrophic multianalogue-resistant mutant ofArthrobacter globiformis

Tryptophan-plus-tyrosine double auxotrophic mutants resistant to fluorophenylalanine (PFP) and β-2-thienylalanine (TA) were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in mineral salts medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to 5-methyltryptophan (MT) and 3-nitrotyrosine (NT) from a double auxotrophic PFPr and TAr mutant. Under optimal cultural condition one mutant yielded 9.6g phenylalanine per L medium in flask culture. Enzymic activity of regulatory enzymes (deoxy-d-arabino-heptulosonate-7-phosphate synthase, chorismate mutase and prephenate dehydratase) were observed in the wild type, double auxotroph and double-auxotrophic multianalogue-resistant mutant.

L‐Phenylalanine production by double auxotrophic analogue resistant mutants of Arthrobacter globiformis

AbstractA number of tryptophan plus tyrosine double auxotrophic mutants isolated by the NTG treatment of a glutamate producing strain of Arthrobacter globiformis were found to excrete phenylalanine in a mineral salt medium. By controlling the pH of the medium to near neutrality, the active growth period could be extended up to 72 h and more phenylalanine was accumulated compared to the unregulated culture where the growth period took up to 48 h. Under optimum culture conditions, the best double auxotroph (TT‐39) produced 3 g phenylalanine/l. Further improvement of phenylalanine production has been achieved by the step‐by‐step isolation of a mutant resistant to the phenylalanine analogues p‐fluorophenylalanine (PFP) and β‐2‐thienylalanine (TA) from the TT‐39 strain. Under optimum culture conditions, the best double auxotrophic analogue resistant mutant TT‐39 PTr‐21 yielded 8.7 g/l phenylalanine.

L‐Methionine production by double auxotrophic mutants of a ethionine resistant strain of Brevibacterium heali

AbstractA number of lysine plus threonine double auxotrophs have been isolated from a ethionine resistant methionine producing strain of Brevibacterium heali previously isolated from soil by mutagenesis with N‐methyl N′‐nitro‐N‐nitrosoguanidine in two steps. This strain excreted L‐methionine in sufficient amounts. For the three potent mutants tested, the medium of ALFOLDI was judged to be the best. Biotin and ammonium nitrate were found to be optimal at 5 μg/l and at a 40 mM level, respectively. With such an optimal dose, the strain BhLT 27 yielded 25.5 g/l methionine in a flask culture containing methionine‐analogue ethionine at a minimal inhibitory concentration.

Production ofl-phenylalanine by double auxotrophic mutants ofArthrobacter globiformis: Effect of temperature, trace salts and inoculum dose

Three tryptophan-plus-tyrosine double auxotrophic mutants were isolated from a biotin-requiring glutamate-producingArthrobacter globiformis. The mutants were found to producel-phenylalanine in a mineral salt medium. Further improvement ofl-phenylalanine production was achieved by isolation of mutants resistant to β-2-thienylalanine from these double auxotrophs. Temperature of 30 °C and a 4% inoculum dose were found to be optimum for phenylalanine production. Addition of some trace salts does not enhance phenylalanine yield. Under optimal cultural conditions one mutant yielded 6.8 g phenylalanine per L medium in flask culture.

Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522.

Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine-methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10(-6) to 10(-8) for each marker.

Intraspecific hybridisation of Trichoderma pseudokoningii by anastomosis and by protoplast fusion

Double auxotrophic and morphological mutants of Trichoderma pseudokoningii Rifai were fused by anastomosis and by protoplast fusion. The recovery of recombinants from heterokaryons on different selective media and from heterokaryotic colonies indicated the occurrence of parasexual events. Prototrophic colonies growing on minimal medium produced binucleate spores, green in colour, revealing a non-autonomous system for conidial pigmentation. Recombinants were obtained from these dikaryotic colonies suggesting the occurrence of a highly unstable diploid phase.

L-Phenylalanine production by double auxotrophic mutants ofArthrobacter globiformis

A number of tryptophan-plus-tyrosine double auxotrophs have been isolated from a glutamate producing Arthrobacter globiformis excreting L-phenylalanine by two-step mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. For the three potent mutants tested the medium of Alföldi was found to be the best. The optimum tryptophan, tyrosine and biotin concentrations for phenylalanine production of these mutants were 0.5 mmol/L, 0.1 mmol/L and 5 micrograms/L, respectively. At these levels strain TT-39 yielded 2.6 g phenylalanine per L of medium in flask culture with glucose (350 mmol/L) and NH4Cl (60 mmol/L).

Production of l‐phenylalanine by double auxotrophic mutants of arthrobacter globiformis: Optimization of c and n‐source

AbstractA number of tryptophan plus tyrosine double auxotrophic mutants isolated by NTG treatment of a glutamic acid prodcing strain of A. globiformis were found to excrete phenylalanine alone in glucose, ammonium chloride, mineral salt medium. Among 24 different carbon sources tested glucose was found to be the best and optimum for ‐phenylalanine production was at 350 mM level. Among 14 different inorganic and organic nitrogen sources tested ammonium chloride was the best and was optimal at 60 mM level for phenylalanine production. On the optimum level of C and N sources a yield of 1.5 g/l phenylalanine was achieved.

4812405 Double auxotrophic mutants of pichia pastoris and methods for preparation: Stephen V Lair, Mary Digan assigned to Phillips Petroleum Company

4812405 Double auxotrophic mutants of pichia pastoris and methods for preparation: Stephen V Lair, Mary Digan assigned to Phillips Petroleum Company

Auswirkungen von Mutanteninduktion und Protoplastenfusion auf ausgewählte Dehydrogenase‐Muster von Pichia guilliermondii

AbstractWe investigated the enzyme patterns of two wild‐type strains, two auxotrophic mutants and some fusion hybrids of Pichia guilliermondii after the electrophoretic separation of their crude cell extracts in dependence on the cultivation on mineral salt medium with different carbon sources. The wild‐type strains showed a constitutive synthesis of one NAD‐specific and some NADP‐specific enzymes and their ability to oxidize primary alcohols with a chain length from C2 to C8. After the induction of mutants auxotroph for amino acids, nicotinic acid or adenine we found different enzyme patterns, too. Prototrophic fusion hybrids obtained via protoplast fusion of the two double auxotrophic mutants showed behaviour either characteristic for the wild‐type strains or characteristics of their parental strains and in some cases also combination of parental enzyme patterns. The behaviour of the hybrids seems to be dependent on the quantity of DNA transferred into each fusion hybrid from the parental mutant strains.So the method of protoplast fusion allows to get qualitatively different hybrids.

Interspecific protoplast fusion of Trichoderma

Summary Protoplasts obtained from immature conidia of double auxotrophic mutant strains of Trichoderma reesei were fused with those of T. viride or T. koningii in the presence of polyethyleneglycol 6000. Fusant colonies from T. reesei × T. viride and T. reesei × T. koningii were isolated as colonies requiring only the amino acid required by both parents. The auxotrophic phenoypes of the conidia generated from each fusant showed two types: one recombinant and the other parental. Colonies of the recombinant type in the presence of Triton X-100 in an agar medium contained specific spots having a higher mycelial density, termed ‘knobs’, of which the phenotype again varied between the prototroph, recombinant type and one of the parental types, i.e. T. viride or T. koningii type, but no T. reesei type was detected. Their traits were inherited stably as was observed in the intraspecific fusion of T. reesi strains (Toyama et al. , paper submitted). The conidial DNA content of the knob clones was almost the same as that of the parents, independent of their phenotypes, but that of the fusants was twice that of the parents. The activities of carboxymethylcellulose-hydrolysing enzymes and β -glucosidases produced by the knob clones showed a wide distribution.