Auxotrophic Research Articles

Plant hormone functions and interactions in biological systems

Plant hormone functions and interactions in biological systems

An efficient transformation system for Trichoderma atroviride using the pyr4 gene as a selectable marker.

The development of an efficient transformation system is essential to enrich the genetic understanding of Trichoderma atroviride. To acquire an additional homologous selectable marker, uracil auxotrophic mutants were generated. First, the pyr4 gene encoding OMP decarboxylase was replaced by the hph marker gene, encoding a hygromycin phosphotransferase. Then, uracil auxotrophs were employed to determine that 5mM uracil restores their growth and conidia production, and 1mgml-1 is the lethal dose of 5-fluoroorotic acid in T. atroviride. Subsequently, uracil auxotrophic strains, free of a drug-selectable marker, were selected by 5-fluoroorotic acid resistance. Two different deletions in pyr4 were mapped in four auxotrophs, encoding a protein with frameshifts at the 310 and 335 amino acids in their COOH-terminal. Six auxotrophs did not have changes in the pyr4 ORF even though a specific cassette to delete the pyr4 was used, suggesting that 5-FOA could have mutagenic activity. The Ura-1 strain was selected as a genetic background to knock out the MAPKK Pbs2, MAPK Tmk3, and the blue light receptors Blr1/Blr2, using a short version of pyr4 as a homologous marker. The ∆tmk3 and ∆pbs2 mutants selected with pyr4 or hph marker were phenotypically identical, highly sensitive to different stressors, and affected in photoconidiation. The ∆blr1 and ∆blr2 mutants were not responsive to light, and complementation of uracil biosynthesis did not interfere in the expression of blu1, grg2, phr1, and env1 genes upregulated by blue light. Overall, uracil metabolism can be used as a tool for genetic manipulation in T. atroviride.

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.

Construction of wild-type Yarrowia lipolytica IMUFRJ 50682 auxotrophic mutants using dual CRISPR/Cas9 strategy for novel biotechnological approaches

Yarrowia lipolytica IMUFRJ 50682 is a Brazilian wild-type strain with potential application in bioconversion processes which can be improved through synthetic biology. In this study, we focused on a combinatorial dual cleavage CRISPR/Cas9-mediated for construction of irreversible auxotrophic mutants IMUFRJ 50682, which genomic information is not available, thought paired sgRNAs targeting upstream and downstream sites of URA3 gene. The disruption efficiency ranged from 5 to 28 % for sgRNAs combinations closer to URA3’s start and stop codon and the auxotrophic mutants lost about 970 bp containing all coding sequence, validating this method for genomic edition of wild-type strains. In addition, we introduced a fluorescent phenotype and achieved cloning rates varying from 80 to 100 %. The ura3Δ strains IMUFRJ 50682 were also engineered for β-carotene synthesis as proof of concept. Carotenoid-producing strains exhibited a similar growth profile compared to the wild-type strain and were able to synthesized 30.54–50.06 mg/L (up to 4.8 mg/g DCW) of β-carotene in YPD and YNB flask cultures, indicating a promisor future of the auxotrophic mutants IMUFRJ 50682 as a chassis for production of novel value-added chemicals.

Phenotypic Characterization of Auxotroph Mutants Obtained by Transposon Mutagenesis

Phenotypic Characterization of Auxotroph Mutants Obtained by Transposon Mutagenesis

Characterization of the Transcription Termination Factor Responsible for Polarity in Archaea

The absence of a nuclear compartment permits prokaryotic ribosomes to bind nascent RNAs to initiate translation before a transcript is completely synthesized. Transcription and translation are normally tightly coupled in prokaryotes (Archaea and Bacteria) and uncoupling translation from transcription serves as a regulatory signal, permitting premature transcription termination when translation fails or is inhibited. The resultant decrease in downstream gene expression, termed polarity, is a major regulatory pathway to ensure coordinated gene expression. While the importance and widespread use of polar repression is well documented, to date, the factor responsible for polarity in archaea cells has not been explicitly identified.In bacteria, the transcription termination factor rho is responsible for polarity, but there are no rho homologues encoded in archaeal genomes. FttA (Factor that terminates transcription in Archaea), a recently identified archaeal termination factor, appears to potentially fulfill the requirements to direct polarity in archaea, but other factors may be responsible. Our primary objective, employing an unbiased genetic selection, is to identify the transcription termination factor(s) responsible for polar repression in Archaea.We have established and will present our findings resultant from a three‐tiered genetic selection for spontaneous mutations that impact the efficiency of polar repression in archaeal cells. The selections were carried out in specialized strains of Thermococcus kodakarensis, a marine, hyperthermophilic archaeon with a facile genetic system and robust laboratory growth.Briefly, genomic modifications were introduced to place a non‐essential gene with a premature translation stop codon directly upstream and within the same operon of a reporter or essential (selection) gene. In wide‐type strains, the early stop codon permits a polarity factor to recognize uncoupling of transcription and translation and terminate transcription before productive expression of the downstream essential gene. However, when the activity of a polarity factor(s) is hindered, these selective genes can be expressed at high levels. For selection, the final genes of the tryptophan and histidine synthesis pathway were chosen, constructing auxotrophic mutant strains which can only develop in the absence of tryptophan and histidine if there is some disruption in polarity‐ allowing them to become prototrophic. The spontaneous mutations resultant in reduced polar repression are easily identified through whole‐genome sequencing and we present evidence for roles of multiple factors in mediating polarity in archaeal cells.

Mutational Analysis of N-Ethyl-N-Nitrosourea (ENU) in the Fission Yeast Schizosaccharomyces pombe.

Forward genetics in model organisms has boosted our knowledge of the genetic bases of development, aging, and human diseases. In this experimental pipeline, it is crucial to start by inducing a large number of random mutations in the genome of the model organism to search for phenotypes of interest. Many chemical mutagens are used to this end because most of them display particular reactivity properties and act differently over DNA. Here we report the use of N-ethyl-N-nitrosourea (ENU) as a mutagen in the fission yeast Schizosaccharomyces pombe. As opposed to many other alkylating agents, ENU only induces an SN1-type reaction with a low s constant (s = 0.26), attacking preferentially O and O in thymine and O deoxyguanosine, leading to base substitutions rather than indels, which are extremely rare in its resulting mutagenic repertoire. Using ENU, we gathered a collection of 13 temperature-sensitive mutants and 80 auxotrophic mutants including two deleterious alleles of the human ortholog ATIC. Defective alleles of this gene cause AICA-ribosiduria, a severe genetic disease. In this screen, we also identified 13 aminoglycoside-resistance inactivating mutations in APH genes. Mutations reported here may be of interest for metabolism related diseases and antibiotic resistance research fields.

PtsI gene in the phosphotransfer system is a potential target for developing a live attenuated Salmonella vaccine.

Salmonella enterica serovar Typhimurium causes invasive non-typhoidal Salmonella diseases in animals and humans, resulting in a high mortality rate and huge economic losses globally. As the prevalence of antibiotic-resistant Salmonella has been increasing, vaccination is thought to be the most effective and economical strategy to manage salmonellosis. The present study aimed to investigate whether dysfunction in the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), which is critical for carbon uptake and survival in macrophages, may be adequate to generate Salmonella-attenuated vaccine strains. A Salmonella strain (KST0555) was generated by deleting the ptsI gene from the PTS and it was revealed that this auxotrophic mutant was unable to efficiently utilize predominant carbon sources during infection (glucose and glycerol), reduced its invasion and replication capacity in macrophages, and significantly (P=0.0065) lowered its virulence in the setting of a mouse colitis model, along with a substantially decreased intestinal colonization and invasiveness compared with its parent strain. The reverse transcription-quantitative PCR results demonstrated that the virulence genes in Salmonella pathogenicity island-1 (SPI-1) and -2 (SPI-2) and the motility of KST0555 were all downregulated compared with its parent strain. Finally, it was revealed that when mice were immunized orally with live KST0555, Salmonella-specific humoral and cellular immune responses were effectively elicited, providing protection against Salmonella infection. Thus, the present promising data provides a strong rationale for the advancement of KST0555 as a live Salmonella vaccine candidate and ptsI as a potential target for developing a live attenuated bacterial vaccine strain.

Auxotrophic mutations of Trichophyton rubrum created by in vitro synthesized Cas9 ribonucleoprotein

BackgroundTrichophyton rubrum is an obligate human parasitic fungus and responsible for approximately 80–90% of dermatomycosis in human. Molecular genetic manipulations of this pathogen are challenging and available tools and protocols are only rudimentary. We adapt molecular genetics methods of well established fungal model organism, to knock out genes in T. rubrum. For the adaptation, crucial modifications are necessary. With the implementation of in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, it is possible to adapt molecular genetic methods, to knock out genes in T. rubrum.ResultsThe gene knock-out method is based on integration of a selection marker into the target site, to interrupt the gene translation. The target gene gets preassigned by the homologous sequence of the in vitro synthesized Cas9-sgRNA ribonucleoprotein complex. To develop the method, we first isolated and characterized a T. rubrum strain with a high amount of microconidia. Next, we developed a transformation protocol, whereby the Cas9-sgRNA ribonucleoprotein gets delivered into the fungal protoplast by the PEG method.We knocked out the URA3 gene and resulted, as predicted, uracil auxotrophic strains. These strains can be used for specific gene knock-outs by reintegrating the URA3 fragment and selection on uracil lacking cultivation media. Exemplary, we knocked out the TRP3 gene and got the predicted phenotype, tryptophan auxotrophic strains. The mutation had been verified by sequencing.ConclusionsWe developed a method, based on in vitro synthesized Cas9-sgRNA ribonucleoprotein complex, for target specific gene knock-outs in T. rubrum. We knocked out the Ura3 gene and resulted uracil auxotrophic strains. These strains were used for target specific gene knock-outs by reintegrating the Ura3 fragment into the target gene site to interrupt the gene transcription. The developed method allows to adapt sophisticate gene manipulation methods of model fungal species to non-model species.

Draft genome sequences of Salmonella Oslo isolated from seafood and its laboratory generated auxotrophic mutant.

In recent years, the concept of bacteria-mediated cancer therapy has gained significant attention as an alternative to conventional therapy. The focus has been on non-typhoidal Salmonella (NTS), particularly S. Typhimurium, for its anti-cancer properties, however, other NTS serovars such as Salmonella Oslo, which are associated with foodborne illnesses could potentially be effective anti-cancer agents. Here, we report the draft genome sequence of Salmonella Oslo isolated from seafood and its laboratory generated auxotrophic mutant.

Construction of an Auxotrophic Mutant from an Industrial Saccharomyces cerevisiae Strain by CRISPR-Cas9 System

Construction of an Auxotrophic Mutant from an Industrial Saccharomyces cerevisiae Strain by CRISPR-Cas9 System

Self-fusion and fusion cell isolation of transformants derived from white rot fungus Phanerochaete sordida YK-624 by simple visual method

In order to develop a simple method for crossing two transformants, we first attempted to elucidate the fusion type (self-compatibility or -incompatible) of Phanerochaete sordida YK-624. Two transformants expressing green or red fluorescent protein derived from an auxotrophic mutant were constructed. Each recombinant protein fluoresced by expression as a fused protein with glyceraldehyde-3-phosphate dehydrogenase. On co-culture of both transformants, a number of sequential hyphal cells emitting dual fluorescence were formed at the contact areas of both hyphae. Some of the single cells isolated as protoplasts and chlamydospore from the co-cultures also expressed these fluorescent proteins. These results suggest that P.sordida YK-624 possesses a self-compatible fusion system. In addition, transformant strains with different fluorescence derived from this fungus can readily undergo self-fusion and nuclear interchange events by confrontational and mixed cultivation, and we developed a simple method that allows fused cells to be isolated as chlamydospores.

Involvement of HisF in the Persistence of Acinetobacter baumannii During a Pneumonia Infection.

Acinetobacter baumannii is currently considered one of the most problematic nosocomial microorganisms. In the present work the hisF gene from the ATCC 17978 strain and the AbH12O-A2 clinical isolate of A. baumannii was found over-expressed during the course of murine pneumonia infections. The study demonstrated that the A. baumannii ATCC 17978 mutant strain lacking the hisF gene induces a sub-lethal pneumonia infection in mice, while the complemented mutant strain increased its virulence. This histidine auxotroph mutant showed an increase on IL-6 secretion and leukocytes recruitment during infections. Furthermore, data revealed that the hisF gene, implicated in the innate immunity and inflammation, is involved in virulence during a pneumonia infection, which may partly explain the ability of this strain to persist in the lung. We suggest that HisF, essential for full virulence in this pathogen, should be considered a potential target for developing new antimicrobial therapies against A. baumannii.Importance Nosocomial pathogens such as A. baumannii are able to acquire and develop multi-drug resistance and represent an important clinical and economic problem. There is therefore an urgent need to find new therapeutic targets to fight against A. baumannii. In the present work, the potential of HisF from A. baumannii as a therapeutic target has been addressed since this protein is involved in the innate inmunity and the inflamatory response and seems essential to develop a pneumonia in mice. This work lays the groundwork for designing antimicrobial therapies that block the activity of HisF.

Amino acids as nutritional factors and (p)ppGpp as an alarmone of the stringent response regulate natural transformation in Micrococcus luteus

Natural competence for genetic transformation refers to the natural ability of various bacteria to take up exogenous DNA from their surroundings and to incorporate internalized genetic information into their genomes. By promoting bacterial diversification and adaptability, this process represents a major driving force in bacterial evolution. Micrococcus luteus was one of the first organisms used to study natural transformation in bacteria. Since then, however, only very little information about this phenomenon has been reported in M. luteus or in any member of the Actinobacteria phylum (low-GC Gram-positive bacteria). Previous work in our group indicated major differences between the transformation apparatus of M. luteus and the transformation machinery described for various Gram-negative and Gram-positive model bacteria belonging to the phyla Proteobacteria and Firmicutes (high-GC Gram-positive bacteria). This prompted us to initiate a study concerning the regulation mechanism of competence development in M. luteus. In this report, we identify amino acids as a nutritional factor that influences competence in a concentration-dependent manner. By using a transcriptional reporter strain for one of the late competence genes, we demonstrate how increasing concentrations of both amino acids mixtures and single amino acids supplemented to the growth medium affect transformability on transcriptional and post-transcriptional level. Furthermore, we revisit previously generated auxotrophic mutants to show that the transformation machinery is turned down during a state of extreme hunger for amino acids presumably as a part of a general response to auxotrophy. Finally, by generating and analysing knockout mutants for two predicted stringent response enzymes, we provide evidence for the involvement of the alarmone (p)ppGpp as a putative mediator of the effects on transformation development caused by amino acids. As a member of the Actinobacteria phylum, M. luteus could serve as a model for other representatives of the phylum, including a number of important human pathogens.

Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples.

The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents.IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

Data for: Enabling HEK293 cells for antibiotic-free media bioprocessing through CRISPR/Cas9 gene editing

Abstract Unlike other cell lines used in industry, HEK293 have not well-established metabolic markers yet for the selection of positive transfected cells expressing recombinant proteins. Three different metabolic markers were explored and assessed in HEK293 to avoid antibiotic-based selection methods. Firstly, PAH gene (Phenylalanine Hydroxylase) was tested as selection marker in tyrosine free medium exploring the potential of the natural tyrosine auxotrophy found in HEK cells. Secondly, two HEK293 mutant cell lines defectives in Thymidylate Synthase (TYMS) and Glutamine Synthetase (GS) were generated using CRISPR/Cas9, and the knocked-out genes were used as selectable markers. Efficiency of selection and eGFP production were evaluated for each selection marker by FACS and compared with the performance based on puromycin selection. Selection efficiencies of 98.60% and 99.95% eGFP + cells were found for PAH and GS selection markers respectively, comparable to puromycin-based selection (99.86% eGFP+). TYMS-based selection system achieved 87.40% eGFP + cells in transfected cultures after one week. Regarding eGFP expression levels, PAH system showed the highest specific production (4,7 × 10 -4 rfu/cell) followed by GS and puromycin (about 2.0 × 10 -4 rfu/cell and 2.7 × 10 -4 rfu/cell, respectively). Inhibitors were then used to increase selection astringency to enhance productivity in selected pools. Eventually, the GS system was used to produce human interferon gamma (hINFγ) as an additional proof of concept. Growth kinetics, advantages and intrinsic limitations of each strategy are assessed and discussed.

Construction and in vitro characterisation of aroA defective (aroAΔ) mutant Salmonella Infantis.

Poultry vaccine programs are important for control of Salmonella infections. Although there are vaccines for Salmonella Enteritidis, Salmonella Typhimurium and Salmonella Typhi, there are no vaccines for Salmonella Infantis which has an increased rate in the world. In this study, it was aimed to generate aroA gene deleted mutant bacteria for the constitution of S. Infantis vaccine prototype and the in vitro characterisation of this bacterium. S. Infantis auxotrophic mutant which has a block at any step of chorismate pathway has been constituted for the first time in the world and it was determined that this bacterium gets susceptibility against some antibiotics and antimicrobial substances. It was also observed that the adhesion and invasion rate of mutant strain tenfold decreased in comparison with the field strain in cell culture assay. It is understood from the in vitro evaluation of this mutant strain that it can be used as a vaccine candidate in further vaccine development studies.

Genotoxicity evaluation of a Phragmitis rhizoma extract using a standard battery of in vitro and in vivo assays

Ethnopharmacological relevanceA rhizome of Phragmites communis Trinius has been used in traditional medicine to remove a heat, relieve vomiting and fever, nourish body fluids, and treat diseases like cancers. However, the safety of Phragmitis rhizoma has not yet been fully assessed.Aim of the study: The present study evaluated the genotoxicity of an aqueous extract of Phragmitis rhizoma (AEPR). Materials and methodsThe genotoxic potential of AEPR was evaluated using both in vitro and in vivo assay systems: a bacterial reverse mutation (AMES) test using auxotrophic mutant strains of Salmonella typhimurium (TA100, TA1535, TA98, TA1537) and Escherichia coli (WP2 uvrA), a chromosomal aberration test using Chinese hamster lung cells, and a micronucleus test using bone marrow cells from male ICR mice subjected to an oral administration of AEPR. All tests were completed in compliance with the OECD guidelines or regional regulatory standards for toxicity study, and Good Laboratory Practice. ResultsWhen compared with the negative control, no genotoxic signs related to the AEPR treatment were observed in the AMES test up to 5000 μg/plate of AEPR and in the chromosomal aberration test up to 500 μg/ml of AEPR regardless of metabolic activation. Repeated oral administration of AEPR up to 5000 mg/kg/day for 2 days did not affect the body weight gains or mortalities of the experimental mice and did not induce any significant changes in the frequency of micronucleated polychromatic erythrocytes. ConclusionsThe present study demonstrated that aqueous extract of Phragmitis rhizoma is safe regarding genotoxicity in an experimental model at least under the conditions tested. Further toxicity assessment in a human clinical study should be done to support the safe use of Phragmitis rhizoma by patients and healthcare providers.

Cysteine auxotrophy drives reduced susceptibility to quinolones and paraquat by inducing the expression of efflux-pump systems and detoxifying enzymes in S. Typhimurium

Currently, Salmonella enterica serovar Typhimurium (S. Typhimurium), is a major global public health problem, which has caused food-borne illnesses in many countries. Today, with the extensive use of antimicrobials, antimicrobial resistance is increasing at a serious rate in S. Typhimurium isolates. The present study sought the role of cysteine (Cys) auxotrophy on the resistance to quinolones and paraquat in S. Typhimurium. Cys auxotrophy was achieved by deleting either the cysDNC, cysJIH or cysQ loci. Deletion of these loci resulted in loss of susceptibility against nalidixic acid, levofloxacin, ciprofloxacin (CIP) and paraquat. Further studies with cysJIH mutant indicated increased expression of multi-antibiotic resistance genes marA and ramA, and consequently increased expression of efflux-pump systems. The cysJIH mutant presented a smaller increase of reactive oxygen species (ROS) in presence of paraquat or CIP. Expression of katG and sodA (expressing for a catalase and a superoxide dismutase, respectively) genes was increased in presence of paraquat in the cysJIH mutant; while expression of the superoxide dismutase gene sodB was decreased. These results indicate that deletion of cysDNC, cysJIH or cysQ genes of S. Typhimurium renders Cys auxotrophy along with decreased susceptibility in response to quinolone and paraquat. Overexpression of efflux-pump systems AcrB-TolC and SmvA-OmpD and antioxidant enzymes KatG and SodA could explain the mechanisms of antimicrobial resistance in the Cys auxotrophic mutants.

Methionine Limitation Impairs Pathogen Expansion and Biofilm Formation Capacity.

Multidrug-resistant bacterial pathogens are becoming increasingly prevalent, and novel strategies to treat bacterial infections caused by these organisms are desperately needed. Bacterial central metabolism is crucial for catabolic processes and provides precursors for anabolic pathways, such as the biosynthesis of essential biomolecules like amino acids or vitamins. However, most essential pathways are not regarded as good targets for antibiotic therapy since their products might be acquired from the environment. This issue raises doubts about the essentiality of such targets during infection. A putative target in bacterial anabolism is the methionine biosynthesis pathway. In contrast to humans, almost all bacteria carry methionine biosynthesis pathways which have often been suggested as putative targets for novel anti-infectives. While the growth of methionine auxotrophic strains can be stimulated by exogenous methionine, the extracellular concentrations required by most bacterial species are unknown. Furthermore, several phenotypic characteristics of methionine auxotrophs are only partly reversed by exogenous methionine. We investigated methionine auxotrophic mutants of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli (all differing in methionine biosynthesis enzymes) and found that each needed concentrations of exogenous methionine far exceeding that reported for human serum (∼30 µM). Accordingly, these methionine auxotrophs showed a reduced ability to proliferate in human serum. Additionally, S. aureus and P. aeruginosa methionine auxotrophs were significantly impaired in their ability to form and maintain biofilms. Altogether, our data show intrinsic defects of methionine auxotrophs. This result suggests that the pathway should be considered for further studies validating the therapeutic potential of inhibitors.IMPORTANCE New antibiotics that attack novel targets are needed to circumvent widespread resistance to conventional drugs. Bacterial anabolic pathways, such as the enzymes for biosynthesis of the essential amino acid methionine, have been proposed as potential targets. However, the eligibility of enzymes in these pathways as drug targets is unclear because metabolites might be acquired from the environment to overcome inhibition. We investigated the nutritional needs of methionine auxotrophs of the pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli We found that each auxotrophic strain retained a growth disadvantage at methionine concentrations mimicking those available in vivo and showed that biofilm biomass was strongly influenced by endogenous methionine biosynthesis. Our experiments suggest that inhibition of the methionine biosynthesis pathway has deleterious effects even in the presence of external methionine. Therefore, additional efforts to validate the effects of methionine biosynthesis inhibitors in vivo are warranted.