Wild-type Parent Strain Research Articles

Contribution of Candida albicans cell wall components to recognition by and escape from murine macrophages.

The pathogenicity of the opportunistic human fungal pathogen Candida albicans depends on its ability to escape destruction by the host immune system. Using mutant strains that are defective in cell surface glycosylation, cell wall protein synthesis, and yeast-hypha morphogenesis, we have investigated three important aspects of C. albicans innate immune interactions: phagocytosis by primary macrophages and macrophage cell lines, hyphal formation within macrophage phagosomes, and the ability to escape from and kill macrophages. We show that cell wall glycosylation is critically important for the recognition and ingestion of C. albicans by macrophages. Phagocytosis was significantly reduced for mutants deficient in phosphomannan biosynthesis (mmn4Delta, pmr1Delta, and mnt3 mnt5Delta), whereas O- and N-linked mannan defects (mnt1Delta mnt2Delta and mns1Delta) were associated with increased ingestion, compared to the parent wild-type strains and genetically complemented controls. In contrast, macrophage uptake of mutants deficient in cell wall proteins such as adhesins (ece1Delta, hwp1Delta, and als3Delta) and yeast-locked mutants (clb2Delta, hgc1Delta, cph1Delta, efg1Delta, and efg1Delta cph1Delta), was similar to that observed for wild-type C. albicans. Killing of macrophages was abrogated in hypha-deficient strains, significantly reduced in all glycosylation mutants, and comparable to wild type in cell wall protein mutants. The diminished ability of glycosylation mutants to kill macrophages was not a consequence of impaired hyphal formation within macrophage phagosomes. Therefore, cell wall composition and the ability to undergo yeast-hypha morphogenesis are critical determinants of the macrophage's ability to ingest and process C. albicans.

Enhanced production of exopolysaccharide matrix and biofilm by a menadione-auxotrophic Staphylococcus aureus small-colony variant

The role of Staphylococcus aureus small-colony variants (SCVs) in the pathogenesis of biofilm-associated infections remains unclear. This study investigated the mechanism behind increased biofilm-forming potential of a menadione-auxotrophic Staphylococcus aureus SCV compared with the wild-type parental strain, as recently reported by our laboratory. SCVs displayed an autoaggregative phenotype, with a greater amount of polysaccharide intercellular adhesin (PIA), significantly reduced tricarboxylic acid cycle activity and a decreased susceptibility to aminoglycosides and cell-wall inhibitors compared with wild-type. The biofilms formed by the SCV were highly structured, consisting of large microcolonies separated by channels, and contained more biomass as well as significantly more PIA than wild-type biofilms. The surface hydrophobicity of the two phenotypes was similar. Thus, the autoaggregation and increased biofilm-forming capacity of menadione-auxotrophic Staphylococcus aureus SCVs in this study was related to the enhanced production of PIA in these variants.

RamA, a Member of the AraC/XylS Family, Influences Both Virulence and Efflux inSalmonella entericaSerovar Typhimurium

The transcriptomes of Salmonella enterica serovar Typhimurium SL1344 lacking a functional ramA or ramR or with plasmid-mediated high-level overexpression of ramA were compared to those of the wild-type parental strain. Inactivation of ramA led to increased expression of 14 SPI-1 genes and decreased expression of three SPI-2 genes, and it altered expression of ribosomal biosynthetic genes and several amino acid biosynthetic pathways. Furthermore, disruption of ramA led to decreased survival within RAW 264.7 mouse macrophages and attenuation within the BALB/c ByJ mouse model. Highly overexpressed ramA led to increased expression of genes encoding multidrug resistance (MDR) efflux pumps, including acrAB, acrEF, and tolC. Decreased expression of 34 Salmonella pathogenicity island (SPI) 1 and 2 genes, decreased SipC production, decreased adhesion to and survival within macrophages, and decreased colonization of Caenorhabditis elegans were also seen. Disruption of ramR led to the increased expression of ramA, acrAB, and tolC, but not to the same level as when ramA was overexpressed on a plasmid. Inactivation of ramR had a more limited effect on pathogenicity gene expression. In silico analysis of a suggested RamA-binding consensus sequence identified target genes, including ramR, acrA, tolC, sipABC, and ssrA. This study demonstrates that the regulation of a mechanism of MDR and expression of virulence genes show considerable overlap, and we postulate that such a mechanism is dependent on transcriptional activator concentration and promoter sensitivity. However, we have no evidence to support the hypothesis that increased MDR via RamA regulation of AcrAB-TolC gives rise to a hypervirulent strain.

The cation-uptake regulators AdcR and Fur are necessary for full virulence of Streptococcus suis

In streptococci, the pleiotropic regulators AdcR and Fur control the transport of, zinc and iron, respectively, which are essential components of many proteins. In this work, Δ adcR, Δ fur, and Δ adcR Δ fur mutants of Streptococcus suis, a serious pathogen in pigs and humans, were assayed in a mouse model to determine their involvement in the virulence of this bacterium. The results showed, for the first time, that the virulence of S. suis mutants carrying an inactivation of adcR, fur, or both genes is significantly attenuated compared to the wild-type parent strain. Furthermore, all mutants were found to be more sensitive to oxidative stress. Our data provide evidence that the adcR and fur genes play important roles in the oxidative stress response of S. suis as well as in the full virulence of this bacterium.

Involvement of the MyD88-independent pathway in controlling the intracellular fate ofBurkholderia pseudomalleiinfection in a mouse macrophage cell line (RAW 264.7)

Burkholderia pseudomallei is a facultative intracellular Gram-negative bacterium which is capable of surviving and multiplying inside macrophages. B. pseudomallei strain SRM117, a LPS mutant which lacks the O-antigenic polysaccharide moiety, is more susceptible to macrophage killing during the early phase of infection than is its parental wild type strain (1026b). In this study, it was shown that the wild type is able to induce expression of genes downstream of the MyD88-dependent (ikappabzeta, il-6 and tnf-alpha), but not of the MyD88-independent (inos, ifn-beta and irg-1), pathways in the mouse macrophage cell line RAW 264.7. In contrast, LPS mutant-infected macrophages were able to express genes downstream of both pathways. To elucidate the significance of activation of the MyD88-independent pathway in B. pseudomallei-infected macrophages, the expression of TBK1, an essential protein in the MyD88-independent pathway, was silenced prior to the infection. The results showed that silencing the tbk1 expression interferes with the gene expression profile in LPS mutant-infected macrophages and allows the bacteria to replicate intracellularly, thus suggesting that the MyD88-independent pathway plays an essential role in controlling intracellular survival of the LPS mutant. Moreover, exogenous IFN-gamma upregulated gene expression downstream of the MyD88-independent pathway, and interfered with intracellular survival in both wild type and tbk1-knockdown macrophages infected with either the wild type or the LPS mutant. These results suggest that gene expression downstream of the MyD88-independent pathway is essential in regulating the intracellular fate of B. pseudomallei, and that IFN-gamma regulates gene expression through the TBK1-independent pathway.

Metabolic profiling of Pseudomonas aeruginosa demonstrates that the anti-sigma factor MucA modulates osmotic stress tolerance

Metabolic footprinting has shown enormous potential as a phenotyping tool and we are interested in applying it to understand the physiology of the opportunistic pathogen Pseudomonas aeruginosa during its chronic infection of the lungs of cystic fibrosis patients. The selection pressures of surviving in the CF lung environment lead to genetic adaptations of the bacterium. A common adaptation is mutation of the mucA gene, resulting in a loss-of-function mutation to the anti-sigma factor MucA, which leads to a mucoid phenotype as a consequence of the overproduction of the extracellular polysaccharide alginate. However, apart from the mucoid phenotype little is known about the overall metabolic and physiological changes caused by mucA mutation. We investigated the pleiotropic metabolic effects of this mutation using time-resolved metabolic footprinting (extracellular metabolomics), and found changes in the levels of various metabolites associated with osmotic tolerance, including glycine-betaine, trehalose and glutamate. Physiological experiments confirmed that the isogenic mucA22 mutant is less resistant to osmotic stress than the parental PA01 wild-type strain, but only in the stationary phase of growth. Quantitative comparison of the endometabolome of the cells showed differences in the accumulation of osmoprotective metabolites by the wild-type and mucA22 mutant strains, suggesting a switch in osmo-protectant preference from glycine-betaine to trehalose.

Nuclease Expression by Staphylococcus aureus Facilitates Escape from Neutrophil Extracellular Traps

Neutrophils are key effectors of the host innate immune response against bacterial infection. Staphylococcus aureus is a preeminent human pathogen, with an ability to produce systemic infections even in previously healthy individuals, thereby reflecting a resistance to effective neutrophil clearance. The recent discovery of neutrophil extracellular traps (NETs) has opened a novel dimension in our understanding of how these specialized leukocytes kill pathogens. NETs consist of a nuclear DNA backbone associated with antimicrobial peptides, histones and proteases that provide a matrix to entrap and kill various microbes. Here, we used targeted mutagenesis to examine a potential role of S. aureus nuclease in NET degradation and virulence in a murine respiratory tract infection model. In vitro assays using fluorescence microscopy showed the isogenic nuclease-deficient (nuc-deficient) mutant to be significantly impaired in its ability to degrade NETs compared with the wild-type parent strain USA 300 LAC. Consequently, the nuc-deficient mutant strain was significantly more susceptible to extracellular killing by activated neutrophils. Moreover, S. aureus nuclease production was associated with delayed bacterial clearance in the lung and increased mortality after intranasal infection. In conclusion, this study shows that S. aureus nuclease promotes resistance against NET-mediated antimicrobial activity of neutrophils and contributes to disease pathogenesis in vivo.

The physiological and morphological phenotype of a yeast mutant resistant to the quaternary ammonium salt N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride.

We investigated the action of the quaternary ammonium salt (QAS) called IM (N-(dodecyloxycarboxymethyl)-N,N,N-trimethyl ammonium chloride) on Saccharomyces cerevisiae yeast cells. Changes in the yeast cell ultrastructure were confirmed by electron microscopy. We treated resistant mutant cells with QAS, and confirmed destruction of the mutant cytoplasm, an increase in the thickness of the cell wall, separation of the cell wall from the cytoplasm, and the accumulation of numerous lipid droplets. We also observed a relatively high production of lipids in the cells of the parental wild-type strain Σ1278b and in its IM-resistant (IMR) mutant in the presence of the QAS. The IMR mutant showed increased sensitivity to CaCl2 and SDS, and resistance to ethidium bromide, chloramphenicol, erythromycin and osmotic shock. It also tolerated growth at low pH. We suggest that the resistance to IM could be connected with the level of permeability of the cell membrane because the IMR mutant was sensitive to this compound in vivo in the presence of SDS and guanidine hydrochloride, which cause increased permeability of the cell plasma membrane.

Candida glabrata Persistence in Mice Does Not Depend on Host Immunosuppression and Is Unaffected by Fungal Amino Acid Auxotrophy

Candida glabrata has emerged as an important fungal pathogen of humans, causing life-threatening infections in immunocompromised patients. In contrast, mice do not develop disease upon systemic challenge, even with high infection doses. In this study we show that leukopenia, but not treatment with corticosteroids, leads to fungal burdens that are transiently increased over those in immunocompetent mice. However, even immunocompetent mice were not capable of clearing infections within 4 weeks. Tissue damage and immune responses to microabscesses were mild as monitored by clinical parameters, including blood enzyme levels, histology, myeloperoxidase, and cytokine levels. Furthermore, we investigated the suitability of amino acid auxotrophic C. glabrata strains for in vitro and in vivo studies of fitness and/or virulence. Histidine, leucine, or tryptophan auxotrophy, as well as a combination of these auxotrophies, did not influence in vitro growth in rich medium. The survival of all auxotrophic strains in immunocompetent mice was similar to that of the parental wild-type strain during the first week of infection and was only mildly reduced 4 weeks after infection, suggesting that C. glabrata is capable of utilizing a broad range of host-derived nutrients during infection. These data suggest that C. glabrata histidine, leucine, or tryptophan auxotrophic strains are suitable for the generation of knockout mutants for in vivo studies. Notably, our work indicates that C. glabrata has successfully developed immune evasion strategies enabling it to survive, disseminate, and persist within mammalian hosts.

The Murine Coronavirus Nucleocapsid Gene Is a Determinant of Virulence

The murine coronavirus, mouse hepatitis virus (MHV) strain A59, causes acute encephalitis and chronic demyelinating disease as well as hepatitis in mice. The JHM strain (also called MHV-4 or JHM.SD) causes fatal encephalitis and only minimal hepatitis. Previous analysis of chimeric recombinant MHVs in which the spike gene, encoding the protein that mediates viral entry and cell-to-cell fusion, was exchanged between JHM and A59 showed that the spike plays a major role in determining organ tropism and neurovirulence but that other genes also play important roles in pathogenic outcome. Here, we have investigated the role of the nucleocapsid protein in MHV-induced disease. The multifunctional nucleocapsid protein is complexed with the genomic RNA, interacts with the viral membrane protein during virion assembly, and plays an import role in enhancing the efficiency of transcription. A pair of chimeric recombinant viruses in which the nucleocapsid gene was exchanged between JHM and A59 was selected and compared to wild-type parental strains in terms of virulence. Importantly, expression of the JHM nucleocapsid in the context of the A59 genome conferred increased mortality and spread of viral antigen in the mouse central nervous system compared to the parental A59 strain, while having little effect on the induction of hepatitis. While the JHM nucleocapsid did not appear to enhance neuron-to-neuron spread in primary neuronal cultures, the increased neurovirulence it conferred may be due in part to the induction of a less robust T-cell response than that induced by strain A59.

FdTonB is involved in the photoregulation of cellular morphology during complementary chromatic adaptation in Fremyella diplosiphon

We have characterized a Fremyella diplosiphon TonB protein (FdTonB) and investigated its function during complementary chromatic adaptation. Sequence similarity analysis of FdTonB (571 aa) led to identification of several conserved domains characteristic of TonB proteins, including an N-terminal transmembrane domain, a central proline-rich spacer and a C-terminal TonB-related domain (TBRD). We identified a novel glycine-rich domain containing (Gly-X)(n) repeats. To assess FdTonB function, we constructed a DeltatonB mutant through homologous recombination based upon truncation of the central proline-rich spacer, glycine-rich domain and TBRD. Our DeltatonB mutant exhibited an aberrant cellular morphology under green light, with expanded cell width compared to the parental wild-type (WT) strain. The cellular morphology of the DeltatonB mutant recovered upon WT tonB expression. Interestingly, tonB expression was found to be independent of RcaE. As DeltatonB and WT strains respond in the same way when grown under iron-replete versus iron-limited conditions, our results suggest that FdTonB is not involved in the classic TonB function of mediating cellular adaptation to iron limitation, but exhibits a novel function related to the photoregulation of cellular morphology in F. diplosiphon.

Salmonella enterica serovar Typhimurium mutants completely lacking the F0F1 ATPase are novel live attenuated vaccine strains

The F0F1 ATPase plays a central role in both the generation of ATP and the utilisation of ATP for cellular processes such as rotation of bacterial flagella. We have deleted the entire operon encoding the F0F1 ATPase, as well as genes encoding individual F0 or F1 subunits, in Salmonella enteric serovar Typhimurium. These mutants were attenuated for virulence, as assessed by bacterial counts in the livers and spleens of intravenously infected mice. The attenuated in vivo growth of the entire atp operon mutant was complemented by the insertion of the atp operon into the malXY pseudogene region. Following clearance of the attenuated mutants from the organs, mice were protected against challenge with the virulent wild type parent strain. We have shown that the F0F1 ATPase is important for bacterial growth in vivo and that atp mutants are effective live attenuated vaccines against Salmonella infection.

Comparison of the metabolic activities of four wild-type Clostridium perfringens strains with their gatifloxacin-selected resistant mutants

The production of short-chain fatty acids, reductive enzymes, and hydrolytic enzymes by four gatifloxacin-selected, fluoroquinolone-resistant, mutant strains of C. perfringens, with stable mutations either in DNA gyrase or in both DNA gyrase and topoisomerase IV, was compared with that produced by the wild-type parent strains to investigate the effect of mutations associated with the selection of gatifloxacin resistance on bacterial metabolic activities. The mutants differed from their respective wild-type parent strains in the enzymatic activities of azoreductase, nitroreductase, and beta-glucosidase and in the ratio of butyric acid to acetic acid production. Microarray analysis of one wild type and the corresponding mutant revealed different levels of mRNA expression for the enzymes involved in short-chain fatty acid (SCFA) synthesis and for beta-glucosidase and oxidoreductases. In addition to mutations in the target genes, selection of resistance to gatifloxacin resulted in strain-specific physiological changes in the resistant mutants of C. perfringens that affected their metabolic activities.

Genome Shuffling in Clostridium diolis DSM 15410 for Improved 1,3-Propanediol Production

Several microorganisms are known for their efficient anaerobic conversion of glycerol to 1,3-propanediol, with Clostridium diolis DSM 15410 as one of the better performers in terms of molar yield and volumetric productivity. However, this performance is still insufficient to compete with established chemical processes. Previous studies have shown that high concentrations of 1,3-propanediol, glycerol, and fermentation side products can limit the productivity of C. diolis DSM 15410. Here, we describe the use of genome shuffling for improved 1,3-propanediol fermentation by the strict anaerobe C. diolis DSM 15410. By using chemical mutagenesis, strains with superior substrate and product tolerance levels were isolated and higher product yields were obtained. These superior strains were then used for genome shuffling and selection for 1,3-propanediol and organic acid tolerance. After four rounds of genome shuffling and selection, significant improvements were observed, with one strain attaining a 1,3-propanediol volumetric yield of 85 g/liter. This result represents an 80% improvement compared to the yield from the parental wild-type strain.

Escherichia coli Unsaturated Fatty Acid Synthesis: COMPLEX TRANSCRIPTION OF THE fabA GENE AND IN VIVO IDENTIFICATION OF THE ESSENTIAL REACTION CATALYZED BY FabB

Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the years. The key players are the fabA and fabB genes. Earlier studies of fabA transcription showed that the gene was transcribed from two promoters, with one being positively regulated by the FadR protein. The other weaker promoter (which could not be mapped with the technology then available) was considered constitutive because its function was independent of FadR. However, the FabR negative regulator was recently shown to represses fabA transcription. We report that the weak promoter overlaps the FadR-dependent promoter and is regulated by FabR. This promoter is strictly conserved in all E. coli and Salmonella enterica genomes sequenced to date and is thought to provide insurance against inappropriate regulation of fabA transcription by exogenous saturated fatty acids. Also, the fabAup promoter, a mutant promoter previously isolated by selection for increased FabA activity, was shown to be a promoter created de novo by a four-base deletion within the gene located immediately upstream of fabA. Demonstration of the key UFA synthetic reaction catalyzed by FabB has been elusive, although it was known to catalyze an elongation reaction. Strains lacking FabB are UFA auxotrophs indicating that the enzyme catalyzes an essential step in UFA synthesis. Using thioesterases specific for hydrolysis of short chain acyl-ACPs, the intermediates of the UFA synthetic pathway have been followed in vivo for the first time. These experiments showed that a fabB mutant strain accumulated less cis-5-dodecenoic acid than the parental wild-type strain. These data indicate that the key reaction in UFA synthesis catalyzed by FabB is elongation of the cis-3-decenoyl-ACP produced by FabA.

The Activities of the Yersinia Protein Kinase A (YpkA) and Outer Protein J (YopJ) Virulence Factors Converge on an eIF2α Kinase

The Yersinia protein kinase A (YpkA) and outer protein J (YopJ) are co-expressed from a single transcript and are injected directly into eukaryotic cells by the plague bacterium Yersinia pestis. When overexpressed in vertebrate or yeast cells, YpkA disrupts the actin-based cytoskeletal system by an unknown mechanism, whereas YopJ obstructs inductive chemokine expression by inhibiting MAPK and NF-kappaB signaling. Previously, we showed that the fission yeast Schizosaccharomyces pombe was sensitive to the kinase activity of YpkA. Here, we screened yeast for cellular processes important for YpkA activity and found that the eIF2alpha kinases mollify the toxicity imparted by the kinase activity of YpkA. Specifically, strains lacking the eIF2alpha kinase Hri2 were particularly sensitive to YpkA. Unexpectedly, the activity of YopJ, which conferred a phenotype consistent with its inhibitory effect on MAPK signaling, was also found to be dependent on Hri2. When expressed in S. pombe, YopJ sensitized cells to osmotic and oxidative stresses through a Hri2-dependent mechanism. However, when co-expressed with YpkA, YopJ protected cells from YpkA-mediated toxicity, and this protection was entirely dependent on Hri2. In contrast, YopJ did not confer protection against the toxic effects of the Yersinia virulence factor YopE. These findings are the first to functionally link YpkA and YopJ and suggest that eIF2alpha kinases, which are critically important in antiviral defenses and protection against environmental stresses, also play a role in bacterial virulence.

The Role of relA and spoT in Yersinia pestis KIM5+ Pathogenicity

The ppGpp molecule is part of a highly conserved regulatory system for mediating the growth response to various environmental conditions. This mechanism may represent a common strategy whereby pathogens such as Yersinia pestis, the causative agent of plague, regulate the virulence gene programs required for invasion, survival and persistence within host cells to match the capacity for growth. The products of the relA and spoT genes carry out ppGpp synthesis. To investigate the role of ppGpp on growth, protein synthesis, gene expression and virulence, we constructed a ΔrelA ΔspoT Y. pestis mutant. The mutant was no longer able to synthesize ppGpp in response to amino acid or carbon starvation, as expected. We also found that it exhibited several novel phenotypes, including a reduced growth rate and autoaggregation at 26°C. In addition, there was a reduction in the level of secretion of key virulence proteins and the mutant was>1,000-fold less virulent than its wild-type parent strain. Mice vaccinated subcutaneously (s.c.) with 2.5×104 CFU of the ΔrelA ΔspoT mutant developed high anti-Y. pestis serum IgG titers, were completely protected against s.c. challenge with 1.5×105 CFU of virulent Y. pestis and partially protected (60% survival) against pulmonary challenge with 2.0×104 CFU of virulent Y. pestis. Our results indicate that ppGpp represents an important virulence determinant in Y. pestis and the ΔrelA ΔspoT mutant strain is a promising vaccine candidate to provide protection against plague.

Characterization of aMycobacterium tuberculosisESX-3 Conditional Mutant: Essentiality and Rescue by Iron and Zinc

Recently, a novel type of secretory pathway, type VII secretion systems (T7SSs), has been characterized in mycobacteria. The chromosomes of Mycobacterium tuberculosis and Mycobacterium bovis encode five T7SSs (ESX-1 to ESX-5). The best characterized of them, ESX-1, is involved in host-pathogen interactions, and its deletion is one of the main causes of M. bovis BCG attenuation. Another T7SS, ESX-3, has been previously shown to be transcriptionally controlled by the zinc uptake repressor (Zur) and by the iron-dependent transcriptional repressor (IdeR), suggesting that it might be involved in zinc and iron homeostasis. In this study, we characterized an M. tuberculosis conditional mutant in which transcription of the ESX-3 gene cluster can be downregulated by anhydrotetracycline. We showed that this T7SS is essential for growth and that this phenotype can be complemented by zinc, iron, or supernatant from a wild-type parental strain culture, demonstrating that the ESX-3 secretion system is responsible for the secretion of some soluble factor(s) required for growth that is probably involved in optimal iron and zinc uptake.

Proteomic analysis of wild type and arsenite-resistant Leishmania donovani

Leishmania donovani, causative organism for visceral leishmaniasis, is responsible for considerable mortality and morbidity worldwide. Generation of drug-resistant variants continue to challenge the chemotherapy, the mainstay to fight the disease. The aim of current study was proteomic profiling of wild type (Ld-Wt) and arsenite-resistant (Ld-As20) L. donovani. Significant differences in protein profiles were observed between Ld-As20 and its parent Ld-Wt strain. Proteomic analysis of 158 spots from Ld-Wt and 144 spots from, Ld-As20 identified 77 and 74 protein entries, respectively, through MALDI-TOF/TOF based mass spectrometry and database search. A shift in the isoelectric point of few proteins was observed both in Ld-Wt and Ld-As20, which raises the possibility of continuous arsenite stress, resulting in the differences in the protein profiles of drug-resistant strain from its parent wild type strain. The comparative proteomic data holds the key for elucidation of the multifactorial and complex drug resistance mechanism, like arsenite resistance, in the parasite.

Deficiency in frataxin homologue YFH1 in the yeast Pichia guilliermondii leads to missregulation of iron acquisition and riboflavin biosynthesis and affects sulfate assimilation

Pichia guilliermondii is a representative of yeast species that overproduce riboflavin (vitamin B2) in response to iron deprivation. P. guilliermondii YFH1 gene coding for frataxin homologue, eukaryotic mitochondrial protein involved in iron trafficking and storage, was identified and deleted. Constructed P. guilliermondii Δyfh1 mutant grew very poorly in a sucrose-containing synthetic medium supplemented with sulfate or sulfite as a sole sulfur source. Addition of sodium sulfide, glutathione, cysteine, methionine, N-acetyl-L-cysteine partially restored growth rate of the mutant suggesting that it is impaired in sulfate assimilation. Cellular iron content in Δyfh1 mutant was ~3-3.5 times higher as compared to the parental strain. It produced 50-70 times more riboflavin in iron sufficient synthetic media relative to the parental wildtype strain. Biomass yield of the mutant in the synthetic glutathione containing medium supplemented with glycerol as a sole carbon source was 1.4- and 2.6-fold increased as compared to sucrose and succinate containing media, respectively. Oxygen uptake of the Δyfh1 mutant on sucrose, glycerol or succinate, when compared to the parental strain, was decreased 5.5-, 1.7- and 1.5-fold, respectively. Substitution of sucrose or glycerol in the synthetic iron sufficient medium with succinate completely abolished riboflavin overproduction by the mutants. Deletion of the YFH1 gene caused hypersensitivity to hydrogen peroxide and exogenously added riboflavin and led to alterations in superoxide dismutase activities. Thus, deletion of the gene coding for yeast frataxin homologue has pleiotropic effect on metabolism in P. guilliermondii.