Evaluation of functionality of type II toxin-antitoxin systems of Clostridioides difficile R20291.

Clostridioides difficile is a nosocomial, Gram-positive, strictly anaerobic, spore-forming pathogen capable of colonizing and proliferating in the human intestine. In bacteria, it has been shown that the Toxin-Antitoxin systems mediate the cellular response to external stress by initiating processes such as biofilm formation and programmed cell death. This work aims to evaluate the functionality of four type II TA modules of Clostridioides difficile R20291. We performed bioinformatic analysis to search for putative TA systems using the TADB platform. Then we performed a heterologous expression assay to evaluate the functionality of these systems. Our results showed that the MazEF and RelBE systems were functional, suggesting that their corresponding toxins possess an endoribonuclease activity. In conclusion, MazEF and RelBE systems of C. difficile R20291 are functional in a heterologous expression system.

Salmonella Typhimurium exhibits fluoroquinolone resistance mediated by the accumulation of the antioxidant molecule H2S in a CysK-dependent manner.

OBJECTIVES: To evaluate the contribution of cysK and cysM to the fluoroquinolone (ofloxacin) antibiotic resistance in Salmonella Typhimurium, and their impact on H2S and cysteine production through targeted mutagenesis. METHODS: Salmonella Typhimurium 14028s and its cysK and cysM mutants were tested for their susceptibility to ofloxacin, as determined by a broth microdilution test (to determine the MIC) and survival curves. H2S levels were measured by the Pb(AC)2 method and cysteine levels were determined using 5,5-dithio-bis-2-nitrobenzoic acid. DNA damage induced by antibiotic treatment was determined by PFGE. Finally, expression of cysK and cysM genes under antibiotic treatment was determined by real-time reverse transcription PCR. RESULTS: As determined by MIC, the ΔcysK strain was more resistant to ofloxacin, a reactive oxygen species (ROS)-producing fluoroquinolone, than the WT and ΔcysM strains, which correlates with survival curves. Moreover, the ΔcysK strain exhibited higher H2S levels and lower cysteine levels than the WT strain. Finally, the ΔcysK strain exhibited lower DNA damage upon challenge with ofloxacin than the WT and ΔcysM strains. These results are in accordance with lower expression of cysK under ofloxacin treatment in the WT strain. CONCLUSIONS: This work demonstrated that cysteine metabolism in Salmonella Typhimurium modulated H2S levels, conferring resistance to second-generation fluoroquinolones.

Lose to win: marT pseudogenization in Salmonella enterica serovar Typhi contributed to the surV-dependent survival to H2O2, and inside human macrophage-like cells.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and Salmonella enterica serovar Typhi (S. Typhi) can be partially attributed to the gain of functions, to the loss of functions (i.e. pseudogenization), or to a combination of both processes. As previously reported, the loss of functions by pseudogenization may play a role in bacterial evolution, especially in host-restricted pathogens such as S. Typhi. The marT-fidL operon, located at the SPI-3, encodes the MarT transcriptional regulator and a hypothetical protein (i.e. FidL) with no significant similarities to known proteins, respectively. Even though predicted S. Typhimurium FidL exhibit 99.4% identity with S. Typhi FidL, marT has been annotated as a pseudogene in S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium marT-fidL exhibited an increased accumulation of reactive oxygen species (ROS), leading to a decreased survival in presence of H2O2. Moreover, we found that that the presence of a functional copy of S. Typhimurium marT-fidL in S. Typhi resulted in a repression of surV (STY4039), an ORF found in the S. Typhi SPI-3 but absent from S. Typhimurium SPI-3, that contribute to the resistance to H2O2 by decreasing the accumulation of ROS. Finally, we observed that the presence of S. Typhimurium marT-fidL in S. Typhi negatively affected the survival inside macrophage-like cells, but not in epithelial cells, after 24h post infection. Therefore, this work provides evidence arguing that marT pseudogenization in Salmonella Typhi contributed to the surV-dependent survival against H2O2, and inside human macrophage-like cells. This is a good example of how the loss of functions (marT pseudogenization) and the gain of functions (presence of surV) might contribute to phenotypic changes improving virulence.

Pseudogenization of sopA and sopE2 is functionally linked and contributes to virulence of Salmonella enterica serovar Typhi.

The difference in host range between Salmonella enterica serovar Typhimurium (S. Typhimurium) and S. enterica serovar Typhi (S. Typhi) can be partially attributed to pseudogenes. Pseudogenes are genomic segments homologous to functional genes that do not encode functional products due to the presence of genetic defects. S. Typhi lacks several protein effectors implicated in invasion or other important processes necessary for full virulence of S. Typhimurium. SopA and SopE2, effectors that have been lost by pseudogenization in S. Typhi, correspond to an ubiquitin ligase involved in cytokine production by infected cells, and to a guanine exchange factor necessary for invasion of epithelial cells, respectively. We hypothesized that sopA and/or sopE pseudogenization contributed to the virulence of S. Typhi. In this work, we found that S. Typhi expressing S. Typhimurium sopE2 exhibited a decreased invasion in different epithelial cell lines compared with S. Typhi WT. S. Typhimurium sopA completely abolished the hypo-invasive phenotype observed in S. Typhi expressing S. Typhimurium sopE2, suggesting that functional SopA and SopE2 participate concertedly in the invasion process. Finally, the expression of S. Typhimurium sopA and/or sopE2 in S. Typhi, determined changes in the secretion of IL-8 and IL-18 in infected epithelial cells.

Salmonella enterica serovar Typhimurium BaeSR two-component system positively regulates sodA in response to ciprofloxacin.

In response to antibiotics, bacteria activate regulatory systems that control the expression of genes that participate in detoxifying these compounds, like multidrug efflux systems. We previously demonstrated that the BaeSR two-component system from Salmonella enterica serovar Typhimurium (S. Typhimurium) participates in the detection of ciprofloxacin, a bactericidal antibiotic, and in the positive regulation of mdtA, an efflux pump implicated in antibiotic resistance. In the present work, we provide further evidence for a role of the S. Typhimurium BaeSR two-component system in response to ciprofloxacin treatment and show that it regulates sodA expression. We demonstrate that, in the absence of BaeSR, the transcript levels of sodA and the activity of its gene product are lower. Using electrophoretic mobility shift assays and transcriptional fusions, we demonstrate that BaeR regulates sodA by a direct interaction with the promoter region.

ompW is cooperatively upregulated by MarA and SoxS in response to menadione.

OmpW is a minor porin whose biological function has not been clearly defined. Evidence obtained in our laboratory indicates that in Salmonella enterica serovar Typhimurium the expression of OmpW is activated by SoxS upon exposure to paraquat and it is required for resistance. SoxS belongs to the AraC family of transcriptional regulators, like MarA and Rob. Due to their high structural similarity, the genes under their control have been grouped in the mar/sox/rob regulon, which presents a DNA-binding consensus sequence denominated the marsox box. In this work, we evaluated the role of the transcription factors MarA, SoxS and Rob of S. enterica serovar Typhimurium in regulating ompW expression in response to menadione. We determined the transcript and protein levels of OmpW in different genetic backgrounds; in the wild-type and Δrob strains ompW was upregulated in response to menadione, while in the ΔmarA and ΔsoxS strains the induction was abolished. In a double marA soxS mutant, ompW transcript levels were lowered after exposure to menadione, and only complementation in trans with both genes restored the positive regulation. Using transcriptional fusions and electrophoretic mobility shift assays with mutant versions of the promoter region we demonstrated that two of the predicted sites were functional. Additionally, we demonstrated that MarA increases the affinity of SoxS for the ompW promoter region. In conclusion, our study shows that ompW is upregulated in response to menadione in a cooperative manner by MarA and SoxS through a direct interaction with the promoter region.

Differential expression of the transcription factors MarA, Rob, and SoxS of Salmonella Typhimurium in response to sodium hypochlorite: down-regulation of rob by MarA and SoxS.

To survive, Salmonella enterica serovar Typhimurium (S. Typhimurium) must sense signals found in phagocytic cells and modulate gene expression. In the present work, we evaluated the expression and cross-regulation of the transcription factors MarA, Rob, and SoxS in response to NaOCl. We generated strains ΔsoxS and ΔmarA, which were 20 times more sensitive to NaOCl as compared to the wild-type strain; while Δrob only 5 times. Subsequently, we determined that marA and soxS transcript and protein levels were increased while those of rob decreased in a wild-type strain treated with NaOCl. To assess if changes in S. Typhimurium after exposure to NaOCl were due to a cross-regulation, as in Escherichia coli, we evaluated the expression of marA, soxS, and rob in the different genetic backgrounds. The positive regulation observed in the wild-type strain of marA and soxS was retained in the Δrob strain. As in the wild-type strain, rob was down-regulated in the ΔmarA and ΔsoxS treated with NaOCl; however, this effect was decreased. Since rob was down-regulated by both factors, we generated a ΔmarA ΔsoxS strain finding that the negative regulation was abolished, confirming our hypothesis. Electrophoretic mobility shift assays using MarA and SoxS confirmed an interaction with the promoter of rob.

Characterization of the BaeSR two-component system from Salmonella Typhimurium and its role in ciprofloxacin-induced mdtA expression.

Two-component systems are one of the most prevalent mechanisms by which bacteria sense, respond and adapt to changes in their environment. The activation of a sensor histidine kinase leads to autophosphorylation of a conserved histidine residue followed by transfer of the phosphoryl group to a cognate response regulator in an aspartate residue. The search for antibiotics that inhibit molecular targets has led to study prokaryotic two-component systems. In this study, we characterized in vitro and in vivo the BaeSR two-component system from Salmonella Typhimurium and evaluated its role in mdtA regulation in response to ciprofloxacin treatment. We demonstrated in vitro that residue histidine 250 is essential for BaeS autophosphorylation and aspartic acid 61 for BaeR transphosphorylation. By real-time PCR, we showed that mdtA activation in the presence of ciprofloxacin depends on both members of this system and that histidine 250 of BaeS and aspartic acid 61 of BaeR are needed for this. Moreover, the mdtA expression is directly regulated by binding of BaeR at the promoter region, and this interaction is enhanced when the protein is phosphorylated. In agreement, a BaeR mutant unable to phosphorylate at aspartic acid 61 presents a lower affinity with the mdtA promoter.

cAMP receptor protein (CRP) positively regulates the yihU-yshA operon in Salmonella enterica serovar Typhi.

Salmonella enterica serovar Typhi (S. Typhi) is the aetiological agent of typhoid fever in humans. This bacterium is also able to persist in its host, causing a chronic disease by colonizing the spleen, liver and gallbladder, in the last of which the pathogen forms biofilms in order to survive the bile. Several genetic components, including the yihU-yshA genes, have been suggested to be involved in the survival of Salmonella in the gallbladder. In this work we describe how the yihU-yshA gene cluster forms a transcriptional unit regulated positively by the cAMP receptor global regulator CRP (cAMP receptor protein). The results obtained show that two CRP-binding sites on the regulatory region of the yihU-yshA operon are required to promote transcriptional activation. In this work we also demonstrate that the yihU-yshA transcriptional unit is carbon catabolite-repressed in Salmonella, indicating that it forms part of the CRP regulon in enteric bacteria.

A new mutation in the yeast aspartate kinase induces threonine accumulation in a temperature-regulated way.

In Saccharomyces cerevisiae, aspartate kinase (the HOM3 product) regulates the metabolic flux through the threonine biosynthetic pathway through feedback inhibition by the end product. In order to obtain a strain able to produce threonine in a controlled way, we have isolated a mutant allele (HOM3-ts31d) that gives rise to a deregulated aspartate kinase. This allele has been isolated as an extragenic suppressor of ilv1, which confers an Ilv+ phenotype at 37 degrees C but not at 22 degrees C. We have stated that at high temperature the mutant aspartate kinase is slightly more deregulated and shows a higher specific activity, inducing threonine accumulation. The HOM3-ts31d allele carries a mutation that leads to a Ser399 --> Phe substitution in the postulated regulatory region of the enzyme. We have detected other changes in the nucleotide sequence but they are also present in the parental strain, reflecting the genetic differences between different wild-type strains. A sequence comparison among all the reported mutant aspartate kinases suggests that not all residues involved in regulation of the activity are clustered in the so-called regulatory domain, as is the case of that mutated in AK-R7, another deregulated aspartate kinase obtained with the same strategy of ilv1 suppression.

Biochemical characterization of a thermostable cysteine synthase from Geobacillus stearothermophilus V.

The cysK gene encoding a cysteine synthase of Geobacillus stearothermophilus V was overexpressed in E. coli and the recombinant protein was purified and characterized. The enzyme is a thermostable homodimer (32 kDa/monomer) belonging to the beta family of pyridoxal phosphate (PLP)-dependent enzymes. UV-visible spectra showed absorption bands at 279 and 410 nm. The band at 279 nm is due to tyrosine residues as the enzyme lacks tryptophan. The 410 nm band represents absorption of the coenzyme bound as a Schiff base to a lysine residue of the protein. Fluorescence characteristics of CysK's Schiff base were influenced by temperature changes suggesting different local structures at the cofactor binding site. The emission of the Schiff base allowed the determination of binding constants for products at both 20 degrees C and 50 degrees C. At 50 degrees C and in the absence of sulphide the enzyme catalyzes the decomposition of O-acetyl-l-serine to pyruvate and ammonia. At 20 degrees C, however, a stable alpha-aminoacrylate intermediate is formed.

Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae.

The HOM3 gene of Saccharomyces cerevisiae encodes aspartate kinase, which catalyses the first step in the branched pathway leading to the synthesis of threonine and methionine from aspartate. Regulation of the carbon flow into this pathway takes place mainly by feedback inhibition of this enzyme by threonine. We have isolated and characterized three HOM3 mutants that show growth inhibition by threonine due to a severe, threonine-induced reduction of the carbon flow into the aspartate pathway, leading to methionine limitation. One of the mutants has an aspartate kinase which is 30-fold more strongly inhibited by threonine than the wild-type enzyme. The predicted amino acid substitution in this mutant, A406T, is located in a region associated with the modulation of the enzymatic activity. The other two mutants carry an aspartate kinase with reduced affinity for its substrates, aspartate and ATP. The corresponding amino acid substitutions, K26I and G25D, affect residues located in the vicinity of a highly conserved lysine-phenylalanine-glycine-glycine (KFGG) stretch present in the N-terminal part of the aspartate kinase, to which no function has so far been assigned. We suggest that this region is involved in substrate binding. Mutagenesis of a HOM3 region centred in the KFGG-coding triplets generated alleles that determine threonine sensitivity or auxotrophy for threonine and methionine, but not a phenotype associated with a feedback-resistant aspartate kinase, indicating that this region is not involved in the allosteric response of the enzyme.

Threonine overproduction in yeast strains carrying the HOM3-R2 mutant allele under the control of different inducible promoters.

The HOM3 gene of Saccharomyces cerevisiae codes for aspartate kinase, which plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis. With the aim of obtaining yeast strains able to overproduce threonine in a controlled way, we have placed the HOM3-R2 mutant allele, which causes expression of a feedback-insensitive enzyme, under the control of four distinctive regulatable yeast promoters, namely, PGAL1, PCHA1, PCYC1-HSE2, and PGPH1. The amino acid contents of strains bearing the different constructs were analyzed both under repression and induction conditions. Although some differences in overall threonine production were found, a maximum of around 400 nmol/mg (dry weight) was observed. Other factors, such as excretion to the medium and activity of the catabolic threonine/serine deaminase, also affect threonine accumulation. Thus, improvement of threonine productivity by yeast cells would probably require manipulation of these and other factors.

Locus-specific suppression of ilv1 in Saccharomyces cerevisiae by deregulation of CHA1 transcription.

The ILV1 gene of Saccharomyces cerevisiae encodes the anabolic threonine deaminase, which catalyzes the first committed step in isoleucine biosynthesis. Strains devoid of a functional Ilv1p have a requirement for isoleucine. Threonine can also be deaminated by a second serine/threonine deaminase encoded by the CHA1 gene. CHA1 is regulated by transcriptional induction by serine and threonine, and enables yeast to utilize the hydroxyamino acids as sole nitrogen source. Phenotypic suppression of ilv1 can occur by inducer-mediated transcriptional activation of the CHA1 gene. To identify mutations in putative trnas-acting factors regulating CHA1 expression, we have isolated and characterized three extragenic suppressors of ilv1. A dominant mutation, SIL4 (suppressor of ilv1), is allelic to HOM3. It increases the size of the threonine pool, by 15- to 20-fold, which is sufficient to induce CHA1 transcription, thereby creating a metabolic bypass of ilv1. A second dominant mutation, SIL3, and a recessive mutation, sil2, both suppress ilv1 by causing inducer-independent, constitutive transcription of CHA1. Importantly, sil2 and SIL3 increase the expression of a CHA1p-lacZ translational gene fusion, demonstrating that they exert their action through the CHA1 promoter. Genetic analysis showed that both SIL3 and sil2 are alleles of CHA4, a positive regulator of CHA1, i.e., they convert Cha4p to a constitutive activator.

Effect of gene amplification on threonine production by yeast.

In this work, we have studied the effect of amplifying different alleles involved in the threonine biosynthesis on the amino acid production by Saccharomyces cerevisiae. The genes used were wild-type HOM3, HOM2, HOM6, THR1, and THR4, and two mutant alleles of HOM3 (namely HOM3-R2 and HOM3-R6), that code for feedback-insensitive aspartate kinases. The results show that only the amplification of the HOM3 alleles leads to threonine and, in some instances, to homoserine overproduction. In terms of the regulation of the pathway, the data indicate that the main control is exerted by inhibition of the aspartate kinase and that, probably, a second and less important regulation takes place at the level of the homoserine kinase, the THR1 gene product. However, amplification of THR1 in two related Hom3-R2 strains does not increase the amount of threonine but, in one of them, it does induce accumulation of more homoserine. This result probably reflects differences between these strains in some undetermined genetic factor/s related with threonine metabolism. In general, the data indicate that the common laboratory yeast strains are genetically rather heterogeneous and, thus, extrapolation of conclusions must be done carefully. (c) 1996 John Wiley & Sons, Inc.

Biochemical evidence that the Saccharomyces cerevisiae THR4 gene encodes threonine synthetase.

In yeast, the assignment of the threonine synthetase activity to the THR4 gene has been inferred from different data, but never really proved enzymatically. In this work, an assay system for threonine synthetase activity in yeast crude extract is reported. The method is based on the quantification by reverse-phase high-performance liquid chromatography, of the threonine formed from O-phosphohomoserine. Using this method we have determined that this activity depends on the presence in the cell of an active form of the THR4 gene, thus demonstrating the univocal relationship between them.

Isolation of a mutant allele that deregulates the threonine biosynthesis in Saccharomyces cerevisiae.

We have cloned the yeast allele HOM3-R2, that codes for a mutant aspartate kinase which is insensitive to feedback inhibition by threonine, by gap-repair. A strain carrying this allele in a multicopy plasmid, or integrated into the genome, accumulates 14-times and 8-times more threonine than the wild-type, respectively. The sequence of the mutant allele differs from that of the wild-type in a single base pair change, namely a G by an A, at position 1355 in the open reading frame. The fact that the presence of this mutant allele in a cell induces threonine overproduction points to aspartate kinase as the key enzyme in the regulation of threonine biosynthesis in yeast.

Identification of yeast cloned genes by genetic analysis.

Gene cloning in yeast is usually carried out by complementation of recessive mutations. However, the fact that a DNA fragment is able to complement a mutation in a certain gene does not necessarily mean that it contains that gene. The identification of a cloned gene can involve the use of Molecular and/or Classical Genetics techniques. In this paper we describe the strategy to be followed in order to establish the identity of a cloned gene, by using genetic crosses and tetrad analysis. As a practical example of the use of this strategy, we describe the cloning of the THR1 gene which codes for the homoserine kinase in S. cerevisiae. This gene has been isolated from a yeast genomic library by complementation of a thr1 mutation. The complementing DNA fragment has been subcloned and integrated into the yeast genome. By genetic crosses and tetrad analysis it has been demonstrated that integration has occurred at the THR1 locus. Since in this organism integration takes place mainly by homologous recombination, it can be inferred that we have, in fact, cloned the THR1 gene. Biochemical analysis of the transformant that carries multiple copies of the cloned gene confirms this result. It shows that this strain presents a homoserine kinase activity about 60 times higher than that of the wild type.

Overproduction of threonine by Saccharomyces cerevisiae mutants resistant to hydroxynorvaline.

In this work, we isolated and characterized mutants that overproduce threonine from Saccharomyces cerevisiae. The mutants were selected for resistance to the threonine analog alpha-amino-beta-hydroxynorvalerate (hydroxynorvaline), and, of these, the ones able to excrete threonine to the medium were chosen. The mutant strains produce between 15 and 30 times more threonine than the wild type does, and, to a lesser degree, they also accumulate isoleucine. Genetic and biochemical studies have revealed that the threonine overproduction is, in all cases studied, associated with the presence in the strain of a HOM3 allele coding for a mutant aspartate kinase that is totally or partially insensitive to feedback inhibition by threonine. This enzyme seems, therefore, to be crucial in the regulation of threonine biosynthesis in S. cerevisiae. The results obtained suggest that this strategy could be efficiently applied to the isolation of threonine-overproducing strains of yeasts other than S. cerevisiae, even those used industrially.

Inhibition by different amino acids of the aspartate kinase and the homoserine kinase of the yeast Saccharomyces cerevisiae.

In this paper, we describe a simple method to measure the yeast homoserine kinase and aspartate kinase activities, independently but in the same extract. With this method, we have determined some kinetic parameters for the physiological substrates of both enzymes, and investigated the inhibition exerted by different amino acids on these activities. Of all natural amino acids tested, only threonine inhibits effectively both enzymatic activities, although to a different degree. We did not find the reported inhibition by L-homoserine over the aspartate kinase. Altogether the data point to the aspartate kinase and to the threonine as the key factors in the regulation of this route.