Insights into the Evolution of IncR Plasmids Found in the Southern European Clone of the Monophasic Variant of Salmonella enterica Serovar Typhimurium.

Salmonella enterica subspecies enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium which has emerged as a world-wide distributed pathogen in the last decades. Several clones have been identified within this variant, the European clone, the Spanish clone, the Southern European clone and the U.S./American clone. The present study focused on isolates of the Southern European clone that were obtained from clinical samples at Spanish hospitals. The selected isolates were multidrug resistant, with most resistance genes residing on IncR plasmids that also carried virulence genes. These plasmids had a mosaic structure, comprising a highly reduced IncR backbone, which has acquired a large amount of exogenous DNA mostly derived from pSLT and IncI1-I(alfa) plasmids. Although composed of approximately the same elements, the investigated plasmids displayed a high diversity, consistent with active evolution driven by a wealth of mobile genetic elements. They comprise multiple intact or truncated insertion sequences, transposons, pseudo-compound transposons and integrons. Particularly relevant was the role of IS26 (with six to nine copies per plasmid) in generating insertions, deletions and inversions, with many of the rearrangements uncovered by tracking the patterns of eight bp target site duplications. Most of the resistance genes detected in the analyzed isolates have been previously associated with the Southern European clone. However, erm(B), lnu(G) and blaTEM-1B are novel, with the last two carried by a second resistance plasmid found in one of the IncR-positive isolates. Thus, evolution of resistance in the Southern European clone is not only mediated by diversification of the IncR plasmids, but also through acquisition of additional plasmids. All isolates investigated in the present study have the large deletion affecting the fljBA region previously found to justify the monophasic phenotype in the Southern European and U.S./American clones. An SNP-based phylogenetic analysis revealed the close relationship amongst our isolates, and support that those sharing the large fljBA deletion could be more heterogeneous than previously anticipated.

Functional Conservation of the Small GTPase Rho5/Rac1-A Tale of Yeast and Men.

Small GTPases are molecular switches that participate in many essential cellular processes. Amongst them, human Rac1 was first described for its role in regulating actin cytoskeleton dynamics and cell migration, with a close relation to carcinogenesis. More recently, the role of Rac1 in regulating the production of reactive oxygen species (ROS), both as a subunit of NADPH oxidase complexes and through its association with mitochondrial functions, has drawn attention. Malfunctions in this context affect cellular plasticity and apoptosis, related to neurodegenerative diseases and diabetes. Some of these features of Rac1 are conserved in its yeast homologue Rho5. Here, we review the structural and functional similarities and differences between these two evolutionary distant proteins and propose yeast as a useful model and a device for high-throughput screens for specific drugs.

The small yeast GTPase Rho5 requires specific mitochondrial outer membrane proteins for translocation under oxidative stress and interacts with the VDAC Por1.

Yeast Rho5 is a small GTPase which mediates the response to nutrient and oxidative stress, and triggers mitophagy and apoptosis. We here studied the rapid translocation of a GFP-tagged Rho5 to mitochondria under such stress conditions by live-cell fluorescence microscopy in the background of strains lacking different mitochondrial outer membrane proteins (MOMP). Fun14, Msp1 and Alo1 were found to be required for efficient recruitment of the GTPase, whereas translocation of Dck1 and Lmo1, the subunits of its dimeric GDP/GTP exchange factor (GEF), remained unaffected. An influence of the voltage-dependent anion channel (VDAC) Por1 on the association of GFP-Rho5 with mitochondria under oxidative stress conditions appeared to be strain-dependent. However, epistasis analyses and bimolecular fluorescence complementation (BiFC) studies indicate a genetic and physical interaction. All four strains lacking a single MOMP were investigated for their effect on mitophagy.

The Role of Glucose-6-phosphate Dehydrogenase in the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is the predominant yeast species in the majority of wine fermentations, which has only recently become amenable to directed genetic manipulation. The genetics and metabolism of H. uvarum have been poorly studied as compared to other yeasts of biotechnological importance. This work describes the construction and characterization of homozygous deletion mutants in the HuZWF1 gene, encoding glucose-6-phosphate dehydrogenase (G6PDH), which provides the entrance into the oxidative part of the pentose phosphate pathway (PPP) and serves as a major source of NADPH for anabolic reactions and oxidative stress response. Huzwf1 deletion mutants grow more slowly on glucose medium than wild-type and are hypersensitive both to hydrogen peroxide and potassium bisulfite, indicating that G6PDH activity is required to cope with these stresses. The mutant also requires methionine for growth. Enzyme activity can be restored by the expression of heterologous G6PDH genes from other yeasts and humans under the control of a strong endogenous promoter. These findings provide the basis for a better adaptation of H. uvarum to conditions used in wine fermentations, as well as its use for other biotechnological purposes and as an expression organism for studying G6PDH functions in patients with hemolytic anemia.

A Versatile Toolset for Genetic Manipulation of the Wine Yeast Hanseniaspora uvarum.

Hanseniaspora uvarum is an ascomycetous yeast that frequently dominates the population in the first two days of wine fermentations. It contributes to the production of many beneficial as well as detrimental aroma compounds. While the genome sequence of the diploid type strain DSM 2768 has been largely elucidated, transformation by electroporation was only recently achieved. We here provide an elaborate toolset for the genetic manipulation of this yeast. A chromosomal replication origin was isolated and used for the construction of episomal, self-replicating cloning vectors. Moreover, homozygous auxotrophic deletion markers (Huura3, Huhis3, Huleu2, Huade2) have been obtained in the diploid genome as future recipients and a proof of principle for the application of PCR-based one-step gene deletion strategies. Besides a hygromycin resistance cassette, a kanamycin resistance gene was established as a dominant marker for selection on G418. Recyclable deletion cassettes flanked by loxP-sites and the corresponding Cre-recombinase expression vectors were tailored. Moreover, we report on a chemical transformation procedure with the use of freeze-competent cells. Together, these techniques and constructs pave the way for efficient and targeted manipulations of H. uvarum.

The Intracellular Distribution of the Small GTPase Rho5 and Its Dimeric Guanidine Nucleotide Exchange Factor Dck1/Lmo1 Determine Their Function in Oxidative Stress Response.

Rho5, the yeast homolog of human Rac1, is a small GTPase which regulates the cell response to nutrient and oxidative stress by inducing mitophagy and apoptosis. It is activated by a dimeric GEF composed of the subunits Dck1 and Lmo1. Upon stress, all three proteins rapidly translocate from the cell surface (Rho5) and a diffuse cytosolic distribution (Dck1 and Lmo1) to mitochondria, with translocation of the GTPase depending on both GEF subunits. We here show that the latter associate with mitochondria independent from each other and from Rho5. The trapping of Dck1-GFP or GFP-Lmo1 to the mitochondrial surface by a specific nanobody fused to the transmembrane domain (TMD) of Fis1 results in a loss of function, mimicking the phenotypes of the respective gene deletions, dck1 or lmo1. Direct fusion of Rho5 to Fis1TMD, i.e., permanent attachment to the mitochondria, also mimics the phenotypes of an rho5 deletion. Together, these data suggest that the GTPase needs to be activated at the plasma membrane prior to its translocation in order to fulfill its function in the oxidative stress response. This notion is substantiated by the observation that strains carrying fusions of Rho5 to the cell wall integrity sensor Mid2, confining the GTPase to the plasma membrane, retained their function. We propose a model in which Rho5 activated at the plasma membrane represses the oxidative stress response under standard growth conditions. This repression is relieved upon its GEF-mediated translocation to mitochondria, thus triggering mitophagy and apoptosis.

Neprilysins regulate muscle contraction and heart function via cleavage of SERCA-inhibitory micropeptides.

Muscle contraction depends on strictly controlled Ca2+ transients within myocytes. A major player maintaining these transients is the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase, SERCA. Activity of SERCA is regulated by binding of micropeptides and impaired expression or function of these peptides results in cardiomyopathy. To date, it is not known how homeostasis or turnover of the micropeptides is regulated. Herein, we find that the Drosophila endopeptidase Neprilysin 4 hydrolyzes SERCA-inhibitory Sarcolamban peptides in membranes of the sarcoplasmic reticulum, thereby ensuring proper regulation of SERCA. Cleavage is necessary and sufficient to maintain homeostasis and function of the micropeptides. Analyses on human Neprilysin, sarcolipin, and ventricular cardiomyocytes indicates that the regulatory mechanism is evolutionarily conserved. By identifying a neprilysin as essential regulator of SERCA activity and Ca2+ homeostasis in cardiomyocytes, these data contribute to a more comprehensive understanding of the complex mechanisms that control muscle contraction and heart function in health and disease.

A Drosophila melanogaster model for TMEM43-related arrhythmogenic right ventricular cardiomyopathy type 5.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a severe cardiac disease that leads to heart failure or sudden cardiac death (SCD). For the pathogenesis of ARVC, various mutations in at least eight different genes have been identified. A rare form of ARVC is associated with the mutation TMEM43 p.S358L, which is a fully penetrant variant in male carriers. TMEM43 p.S358 is homologous to CG8111 p.S333 in Drosophila melanogaster. We established CRISPR/Cas9-mediated CG8111 knock-out mutants in Drosophila, as well as transgenic fly lines carrying an overexpression construct of the CG8111 p.S333L substitution. Knock-out flies developed normally, whereas the overexpression of CG8111 p.S333L caused growth defects, loss of body weight, cardiac arrhythmias, and premature death. An evaluation of a series of model mutants that replaced S333 by selected amino acids proved that the conserved serine is critical for the physiological function of CG8111. Metabolomic and proteomic analyses revealed that the S333 in CG8111 is essential to proper energy homeostasis and lipid metabolism in the fly. Of note, metabolic impairments were also found in the murine Tmem43 disease model, and fibrofatty replacement is a hallmark of human ARVC5. These findings contribute to a more comprehensive understanding of the molecular functions of CG8111 in Drosophila, and can represent a valuable basis to assess the aetiology of the human TMEM43 p.S358L variant in more detail.

Caspase-cleaved tau is senescence-associated and induces a toxic gain of function by putting a brake on axonal transport.

The microtubule-associated protein tau plays a central role in tauopathies such as Alzheimer's disease (AD). The exact molecular mechanisms underlying tau toxicity are unclear, but aging is irrefutably the biggest risk factor. This raises the question of how cellular senescence affects the function of tau as a microtubule regulator. Here we report that the proportion of tau that is proteolytically cleaved at the caspase-3 site (TauC3) doubles in the hippocampus of senescent mice. TauC3 is also elevated in AD patients. Through quantitative live-cell imaging, we show that TauC3 has a drastically reduced dynamics of its microtubule interaction. Single-molecule tracking of tau confirmed that TauC3 has a longer residence time on axonal microtubules. The reduced dynamics of the TauC3-microtubule interaction correlated with a decreased transport of mitochondria, a reduced processivity of APP-vesicle transport and an induction of region-specific dendritic atrophy in CA1 neurons of the hippocampus. The microtubule-targeting drug Epothilone D normalized the interaction of TauC3 with microtubules and modulated the transport of APP-vesicles dependent on the presence of overexpressed human tau. The results indicate a novel toxic gain of function, in which a post-translational modification of tau changes the dynamics of the tau-microtubule interaction and thus leads to axonal transport defects and neuronal degeneration. The data also introduce microtubule-targeting drugs as pharmacological modifiers of the tau-microtubule interaction with the potential to restore the physiological interaction of pathologically altered tau with microtubules.

Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis.

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

The small GTPase KlRho5 responds to oxidative stress and affects cytokinesis.

Rho5 is the yeast homolog of the human small GTPase Rac1. We characterized the genes encoding Rho5 and the subunits of its dimeric activating guanine-nucleotide-exchange factor (GEF), Dck1 and Lmo1, in the yeast Kluyveromyces lactis. Rapid translocation of the three GFP-tagged components to mitochondria upon oxidative stress and carbon starvation indicate a similar function of KlRho5 in energy metabolism and mitochondrial dynamics as described for its Saccharomyces cerevisiae homolog. Accordingly, Klrho5 deletion mutants are hyper-resistant towards hydrogen peroxide. Moreover, synthetic lethalities of rho5 deletions with key components in nutrient sensing, such as sch9 and gpr1, are not conserved in K. lactis. Instead, Klrho5 deletion mutants display morphological defects with strengthened lateral cell walls and protruding bud scars. The latter result from aberrant cytokinesis, as observed by following the budding process in vivo and by transmission electron microscopy of the bud neck region. This phenotype can be suppressed by KlCDC42G12V, which encodes a hyper-active variant. Data from live-cell fluorescence microscopy support the notion that KlRho5 interferes with the actin moiety of the contractile actomyosin ring, with consequences different from those previously reported for mutants lacking myosin.

The Pentose Phosphate Pathway in Yeasts-More Than a Poor Cousin of Glycolysis.

The pentose phosphate pathway (PPP) is a route that can work in parallel to glycolysis in glucose degradation in most living cells. It has a unidirectional oxidative part with glucose-6-phosphate dehydrogenase as a key enzyme generating NADPH, and a non-oxidative part involving the reversible transketolase and transaldolase reactions, which interchange PPP metabolites with glycolysis. While the oxidative branch is vital to cope with oxidative stress, the non-oxidative branch provides precursors for the synthesis of nucleic, fatty and aromatic amino acids. For glucose catabolism in the baker's yeast Saccharomyces cerevisiae, where its components were first discovered and extensively studied, the PPP plays only a minor role. In contrast, PPP and glycolysis contribute almost equally to glucose degradation in other yeasts. We here summarize the data available for the PPP enzymes focusing on S. cerevisiae and Kluyveromyces lactis, and describe the phenotypes of gene deletions and the benefits of their overproduction and modification. Reference to other yeasts and to the importance of the PPP in their biotechnological and medical applications is briefly being included. We propose future studies on the PPP in K. lactis to be of special interest for basic science and as a host for the expression of human disease genes.

Genomic analysis and phylogenetic position of the complex IncC plasmid found in the Spanish monophasic clone of Salmonella enterica serovar Typhimurium.

pUO-STmRV1 is an IncC plasmid discovered in the Spanish clone of the emergent monophasic variant of Salmonella enterica serovar Typhimurium, which has probably contributed to its epidemiological success. The sequence of the entire plasmid determined herein revealed a largely degenerated backbone with accessory DNA incorporated at four different locations. The acquired DNA constitutes more than two-thirds of the pUO-STmRV1 genome and originates from plasmids of different incompatibility groups, including IncF (such as R100 and pSLT, the virulence plasmid specific of S. Typhimurium), IncN and IncI, from the integrative element GIsul2, or from yet unknown sources. In addition to pSLT virulence genes, the plasmid carries genes conferring resistance to widely-used antibiotics and heavy metals, together with a wealth of genetic elements involved in DNA mobility. The latter comprise class 1 integrons, transposons, pseudo-transposons, and insertion sequences, strikingly with 14 copies of IS26, which could have played a crucial role in the assembly of the complex plasmid. Typing of pUO-STmRV1 revealed backbone features characteristically associated with type 1 and type 2 IncC plasmids and could therefore be regarded as a hybrid plasmid. However, a rooted phylogenetic tree based on core genes indicates that it rather belongs to an ancient lineage which diverged at an early stage from the branch leading to most extant IncC plasmids detected so far. pUO-STmRV1 may have evolved at a time when uncontrolled use of antibiotics and biocides favored the accumulation of multiple resistance genes within an IncC backbone. The resulting plasmid thus allowed the Spanish clone to withstand a wide variety of adverse conditions, while simultaneously promoting its own propagation through vertical transmission.

A Three-Dimensional Model of the Yeast Transmembrane Sensor Wsc1 Obtained by SMA-Based Detergent-Free Purification and Transmission Electron Microscopy.

The cell wall sensor Wsc1 belongs to a small family of transmembrane proteins, which are crucial to sustain cell integrity in yeast and other fungi. Wsc1 acts as a mechanosensor of the cell wall integrity (CWI) signal transduction pathway which responds to external stresses. Here we report on the purification of Wsc1 by its trapping in water-soluble polymer-stabilized lipid nanoparticles, obtained with an amphipathic styrene-maleic acid (SMA) copolymer. The latter was employed to transfer tagged sensors from their native yeast membranes into SMA/lipid particles (SMALPs), which allows their purification in a functional state, i.e., avoiding denaturation. The SMALPs composition was characterized by fluorescence correlation spectroscopy, followed by two-dimensional image acquisition from single particle transmission electron microscopy to build a three-dimensional model of the sensor. The latter confirms that Wsc1 consists of a large extracellular domain connected to a smaller intracellular part by a single transmembrane domain, which is embedded within the hydrophobic moiety of the lipid bilayer. The successful extraction of a sensor from the yeast plasma membrane by a detergent-free procedure into a native-like membrane environment provides new prospects for in vitro structural and functional studies of yeast plasma proteins which are likely to be applicable to other fungi, including plant and human pathogens.

Uptake of exogenous serine is important to maintain sphingolipid homeostasis in Saccharomyces cerevisiae.

Sphingolipids are abundant and essential molecules in eukaryotes that have crucial functions as signaling molecules and as membrane components. Sphingolipid biosynthesis starts in the endoplasmic reticulum with the condensation of serine and palmitoyl-CoA. Sphingolipid biosynthesis is highly regulated to maintain sphingolipid homeostasis. Even though, serine is an essential component of the sphingolipid biosynthesis pathway, its role in maintaining sphingolipid homeostasis has not been precisely studied. Here we show that serine uptake is an important factor for the regulation of sphingolipid biosynthesis in Saccharomyces cerevisiae. Using genetic experiments, we find the broad-specificity amino acid permease Gnp1 to be important for serine uptake. We confirm these results with serine uptake assays in gnp1Δ cells. We further show that uptake of exogenous serine by Gnp1 is important to maintain cellular serine levels and observe a specific connection between serine uptake and the first step of sphingolipid biosynthesis. Using mass spectrometry-based flux analysis, we further observed imported serine as the main source for de novo sphingolipid biosynthesis. Our results demonstrate that yeast cells preferentially use the uptake of exogenous serine to regulate sphingolipid biosynthesis. Our study can also be a starting point to analyze the role of serine uptake in mammalian sphingolipid metabolism.

A trimeric metazoan Rab7 GEF complex is crucial for endocytosis and scavenger function.

Endosome biogenesis in eukaryotic cells is critical for nutrient uptake and plasma membrane integrity. Early endosomes initially contain Rab5, which is replaced by Rab7 on late endosomes prior to their fusion with lysosomes. Recruitment of Rab7 to endosomes requires the Mon1-Ccz1 guanine-nucleotide-exchange factor (GEF). Here, we show that full function of the Drosophila Mon1-Ccz1 complex requires a third stoichiometric subunit, termed Bulli (encoded by CG8270). Bulli localises to Rab7-positive endosomes, in agreement with its function in the GEF complex. Using Drosophila nephrocytes as a model system, we observe that absence of Bulli results in (i) reduced endocytosis, (ii) Rab5 accumulation within non-acidified enlarged endosomes, (iii) defective Rab7 localisation and (iv) impaired endosomal maturation. Moreover, longevity of animals lacking bulli is affected. Both the Mon1-Ccz1 dimer and a Bulli-containing trimer display Rab7 GEF activity. In summary, this suggests a key role for Bulli in the Rab5 to Rab7 transition during endosomal maturation rather than a direct influence on the GEF activity of Mon1-Ccz1.

Investigation of Heterologously Expressed Glucose-6-Phosphate Dehydrogenase Genes in a Yeast zwf1 Deletion.

Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme of the oxidative part of the pentose phosphate pathway and serves as the major source of NADPH for metabolic reactions and oxidative stress response in pro- and eukaryotic cells. We here report on a strain of the model yeast Saccharomyces cerevisiae which lacks the G6PD-encoding ZWF1 gene and displays distinct growth retardation on rich and synthetic media, as well as a strongly reduced chronological lifespan. This strain was used as a recipient to introduce plasmid-encoded heterologous G6PD genes, synthesized in the yeast codon usage and expressed under the control of the native PFK2 promotor. Complementation of the hypersensitivity of the zwf1 mutant towards hydrogen peroxide to different degrees was observed for the genes from humans (HsG6PD1), the milk yeast Kluyveromyces lactis (KlZWF1), the bacteria Escherichia coli (EcZWF1) and Leuconostoc mesenteroides (LmZWF1), as well as the genes encoding three different plant G6PD isoforms from Arabidopsis thaliana (AtG6PD1, AtG6PD5, AtG6PD6). The plastidic AtG6PD1 isoform retained its redox-sensitive activity when produced in the yeast as a cytosolic enzyme, demonstrating the suitability of this host for determination of its physiological properties. Mutations precluding the formation of a disulfide bridge in AtG6PD1 abolished its redox-sensitivity but improved its capacity to complement the yeast zwf1 deletion. Given the importance of G6PD in human diseases and plant growth, this heterologous expression system offers a broad range of applications.

How to study intertwined and autoregulated eukaryotic signal transduction pathways.

Mitogen-activated protein kinase (MAPK) pathways are a major means of eukaryotic cells to adapt to environmental changes, in the case of microorganisms, and to nutritional and hormonal signals, in the case of multicellular organisms. Numerous defects in such architecturally conserved pathways have been associated with different human cancers. These signaling cascades usually commence with sensors located in the plasma membrane, which through specific protein kinases activate a conserved tripartite MAPK module. Phosphorylation of their targets, that is, cytosolic proteins and/or transcription factors, then triggers the proper cellular response. In the model yeast Saccharomyces cerevisiae and other fungi, the cell wall integrity pathway (CWI) has been extensively studied and its components may serve as targets for antifungal drugs of clinical and agricultural importance. Another well-known MAPK cascade, the high osmolarity glycerol (HOG) pathway, is required to cope with osmotic stress. In the past decade, it has become increasingly evident that such pathways do not act in a linear top-down fashion, but are highly regulated by internal feedback mechanisms as well as by cross-pathway interactions. The work of Jiménez-Gutiérrez et al. in this issue provides an elegant way to identify new players in these complex networks. Comment on: https://doi.org/10.1111/febs.15288.

Fungal homologues of human Rac1 as emerging players in signal transduction and morphogenesis.

A wealth of data is accumulating on the physiological functions of human Rac1, a member of the Rho GTPase family of molecular switches and substrate of botulinum toxin, which was first identified as a regulator of cell motility through its effect on the actin cytoskeleton. Later on, it was found to be involved in different diseases like cancers, cardiac function, neuronal disorders, and apoptotic cell death. Despite the presence of Rac1 homologues in most fungi investigated so far, including Rho5 in the genetically tractable model yeast Saccharomyces cerevisiae, knowledge on their physiological functions is still scarce, let alone the details of the molecular mechanisms of their actions and interactions. Nevertheless, all functions proposed for human Rac1 seem to be conserved in one or the other fungus. This includes the regulation of MAPK cascades, polarized growth, and actin dynamics. Moreover, both the production and response to reactive oxygen species, as well as the reaction to nutrient availability, can be affected. We here summarize the studies performed on fungal Rac1 homologues, with a special focus on S. cerevisiae Rho5, which may be of use in drug development in medicine and agriculture.

Analysis of the Degradation of Broad-Spectrum Cephalosporins by OXA-48-Producing Enterobacteriaceae Using MALDI-TOF MS.

The objective of the study was to evaluate the activity of OXA-48 against different broad-spectrum cephalosporins and to identify the reaction products by MALDI-TOF MS. The action of OXA-48 on cefotaxime, ceftazidime, and ceftriaxone was assessed by this method, using an Escherichia coli J53 transconjugant carrying only the ~62 Kb IncL plasmid containing the blaOXA-48 gene, and the same strain without any plasmid was included as a negative control. In addition, a collection of 17 clinical OXA-48-producing Enterobacteriaceae, which were susceptible to broad-spectrum cephalosporins, was evaluated. MALDI-TOF MS-based analysis of the E. coli transconjugant carrying the blaOXA-48-harboring plasmid, and also the clinical isolates, showed degradation of cefotaxime into two inactive compounds-decarboxylated and deacetylated cefotaxime (~370 Da) and deacetyl cefotaxime (~414 Da), both with the hydrolyzed beta-lactam ring. Reaction products were not obtained when the experiment was performed with ceftriaxone or ceftazidime. From a clinical point of view, our study supports the idea that the efficacy of cefotaxime against OXA-48-producing Enterobacteriaceae is doubtful, in contrast to ceftazidime and ceftriaxone which could be valid choices for treating infections caused by these bacteria. However, further clinical studies confirming this hypothesis are required.