Multidrug- and colistin-resistant Salmonella enterica 4,[5],12:i:- sequence type 34 carrying the mcr-3.1 gene on the IncHI2 plasmid recovered from a human.

A colistin-resistant Salmonella enterica 4, [5],12:i:- sequence type (ST) 34 harbouring mcr-3.1 was recovered from a patient who travelled to China 2 weeks prior to diarrhoea onset. Genomic analysis revealed the presence of the mcr-3.1 gene located in the globally disseminated IncHI2 plasmid, highlighting the intercontinental dissemination of the colistin-resistant S. enterica 4, [5],12:i:- ST34 pandemic clone.

Genetic and Functional Characterization of an MCR-3-Like Enzyme-Producing Escherichia coli Isolate Recovered from Swine in Brazil.

A collection of 126 pigs was screened for carriage of colistin-resistant Enterobacteriaceae in a farm in Minas Gerais, Brazil. Out of this collection, eight colistin-resistant Escherichia coli isolates were recovered, including one from Minas Gerais State producing a new MCR-3 variant (MCR-3.12). Analysis of the lipopolysaccharide revealed that MCR-3.12 had a function similar to that of MCR-1 and MCR-2 as a result of the addition of a phosphoethanolamine group to the lipid A moiety. Genetic analysis showed that the mcr-3.12 gene was carried by an IncA/C2 plasmid and was embedded in an original genetic environment. This study reports the occurrence of the MCR-3-like determinant in South America and is the first to demonstrate the functionality of this group of enzymes as a phosphoethanolamine transferase.

GNPTAB missense mutations cause loss of GlcNAc-1-phosphotransferase activity in mucolipidosis type II through distinct mechanisms.

Mucolipidoses (ML) II and III alpha/beta are lysosomal storage diseases caused by pathogenic mutations in GNPTAB encoding the α/ß-subunit precursor of GlcNAc-1-phosphotransferase. To determine genotype-phenotype correlation and functional analysis of mutant GlcNAc-1-phosphotransferase, 13 Brazilian patients clinically and biochemical diagnosed for MLII or III alpha/beta were studied. By sequencing of genomic GNPTAB of the MLII and MLIII alpha/beta patients we identified six novel mutations: p.D76G, p.S385L, p.Q278Kfs*3, p.H588Qfs*27, p.N642Lfs*10 and p.Y1111*. Expression analysis by western blotting and immunofluorescence microscopy revealed that the mutant α/ß-subunit precursor p.D76G is retained in the endoplasmic reticulum whereas the mutant p.S385L is correctly transported to the cis-Golgi apparatus and proteolytically processed. Both mutations lead to complete loss of GlcNAc-1-phosphotransferase activity, consistent with the severe clinical MLII phenotype of the patients. Our study expands the genotypic spectrum of MLII and provides novel insights into structural requirements to ensure GlcNAc-1-phosphotransferase activity.

Content of cardiolipin of the membrane and sensitivity to cationic surfactants in Pseudomonas putida.

AIMS: To establish the role of cardiolipin (CL) of the membrane in response to the presence of tetradecyltrimethylammonium in Pseudomonas putida A (ATCC 12633). METHODS AND RESULTS: Two ORFs of Ps. putida A (ATCC 12633), which in Ps. putida KT2440 encode the putative CL synthase genes cls and cls2, were cloned, sequenced and mutated. Only the double mutant lacking cls and cls2 showed a reduction of the CL content, 83% lower than the amount produced by the wild-type. Accompanying this change was a 40% decrease in the content of unsaturated fatty acid. Consequently, the membrane of the mutant was more rigid than the one of the parental strain, as observed using fluorescence polarization techniques. The mutant strain showed reduced viability in the presence of tetradecyltrimethylammonium. The incorporation of exogenous CL into its membrane relieved sensitivity to the cationic detergent. CONCLUSIONS: Pseudomonas Putida cells with low levels of CL die in the presence of tetradecyltrimethylammonium, because they cannot counter the fluidizing effect of the cationic surfactant. SIGNIFICANCE AND IMPACT OF THE STUDY: The modification in the membrane phospholipids composition allows knowing the adaptation strategy of Ps. putida when these bacteria are exposed to cationic surfactant.

Enigmatic in vivo GlcNAc-1-phosphotransferase (GNPTG) transcript correction to wild type in two mucolipidosis III gamma siblings homozygous for nonsense mutations.

Mucolipidosis (ML) III gamma is a rare autosomal-recessive disorder caused by pathogenic mutations in the GNPTG gene. GNPTG encodes the γ-subunit of GlcNAc-1-phosphotransferase that catalyzes mannose 6-phosphate targeting signal synthesis on soluble lysosomal enzymes. ML III gamma patients are characterized by missorting of lysosomal enzymes. In this report, we describe the probable occurrence of mRNA editing in two ML III gamma patients. Patients A and B (siblings) presented at the adult age with a typical clinical picture of ML III gamma, mainly compromising bone and joints, and high levels of lysosomal enzymes in plasma and low levels in fibroblasts. Both were found to be homozygous for c.-112C>G and c.328G>T (p.Glu110Ter) mutations in genomic DNA (gDNA) analysis of GNPTG. Analysis of complementary DNA (cDNA), however, showed normal genotypes for both patients. Low GNPTG mRNA expression was observed in both patients. The mRNA editing can explain the differences found in patients A and B regarding gDNA and cDNA analysis, and the mild clinical phenotype associated with homozygosity for a nonsense mutation. Our results suggest that mRNA editing can be more frequent than expected in monogenic disorders and that GNPTG analysis should be performed on gDNA.

Analyses of disease-related GNPTAB mutations define a novel GlcNAc-1-phosphotransferase interaction domain and an alternative site-1 protease cleavage site.

Mucolipidosis II (MLII) and III alpha/beta are autosomal-recessive diseases of childhood caused by mutations in GNPTAB encoding the α/ß-subunit precursor protein of the GlcNAc-1-phosphotransferase complex. This enzyme modifies lysosomal hydrolases with mannose 6-phosphate targeting signals. Upon arrival in the Golgi apparatus, the newly synthesized α/ß-subunit precursor is catalytically activated by site-1 protease (S1P). Here we performed comprehensive expression studies of GNPTAB mutations, including two novel mutations T644M and T1223del, identified in Brazilian MLII/MLIII alpha/beta patients. We show that the frameshift E757KfsX1 and the non-sense R587X mutations result in the retention of enzymatically inactive truncated precursor proteins in the endoplasmic reticulum (ER) due to loss of cytosolic ER exit motifs consistent with a severe clinical phenotype in homozygosity. The luminal missense mutations, C505Y, G575R and T644M, partially impaired ER exit and proteolytic activation in accordance with less severe MLIII alpha/beta disease symptoms. Analogous to the previously characterized S399F mutant, we found that the missense mutation I403T led to retention in the ER and loss of catalytic activity. Substitution of further conserved residues in stealth domain 2 (I346 and W357) revealed similar biochemical properties and allowed us to define a putative binding site for accessory proteins required for ER exit of α/ß-subunit precursors. Interestingly, the analysis of the Y937_M972del mutant revealed partial Golgi localization and formation of abnormal inactive ß-subunits generated by S1P which correlate with a clinical MLII phenotype. Expression analyses of mutations identified in patients underline genotype-phenotype correlations in MLII/MLIII alpha/beta and provide novel insights into structural requirements of proper GlcNAc-1-phosphotransferase activity.

Sphingomyelin metabolism is involved in the differentiation of MDCK cells induced by environmental hypertonicity.

Sphingolipids (SLs) are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, SLs provide the structural framework for plasma membrane organization. Particularly, SM is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in an SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, Madin-Darby canine kidney (MDCK) cells acquire a differentiated phenotype with changes in SL metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM synthase 1 is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin and ß- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum.

INTRODUCTION: Kala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar. METHODS: To determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification. RESULTS: The nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival. CONCLUSIONS: NAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis.

New insights into chloramphenicol biosynthesis in Streptomyces venezuelae ATCC 10712.

Comparative genome analysis revealed seven uncharacterized genes, sven0909 to sven0915, adjacent to the previously identified chloramphenicol biosynthetic gene cluster (sven0916-sven0928) of Streptomyces venezuelae strain ATCC 10712 that was absent in a closely related Streptomyces strain that does not produce chloramphenicol. Transcriptional analysis suggested that three of these genes might be involved in chloramphenicol production, a prediction confirmed by the construction of deletion mutants. These three genes encode a cluster-associated transcriptional activator (Sven0913), a phosphopantetheinyl transferase (Sven0914), and a Na(+)/H(+) antiporter (Sven0915). Bioinformatic analysis also revealed the presence of a previously undetected gene, sven0925, embedded within the chloramphenicol biosynthetic gene cluster that appears to encode an acyl carrier protein, bringing the number of new genes likely to be involved in chloramphenicol production to four. Microarray experiments and synteny comparisons also suggest that sven0929 is part of the biosynthetic gene cluster. This has allowed us to propose an updated and revised version of the chloramphenicol biosynthetic pathway.

Genes that encodes NAGT, MIF1 and MIF2 are not virulence factors for kala-azar caused by Leishmania infantum

Introduction Kala-azar is a disease resulting from infection by Leishmania donovani and Leishmania infantum. Most patients with the disease exhibit prolonged fever, wasting, anemia and hepatosplenomegaly without complications. However, some patients develop severe disease with hemorrhagic manifestations, bacterial infections, jaundice, and edema dyspnea, among other symptoms, followed by death. Among the parasite molecules that might influence the disease severity are the macrophage migration inhibitory factor-like proteins (MIF1 and MIF2) and N-acetylglucosamine-1-phosphotransferase (NAGT), which act in the first step of protein N-glycosylation. This study aimed to determine whether MIF1, MIF2 and NAGT are virulence factors for severe kala-azar. Methods To determine the parasite genotype in kala-azar patients from Northeastern Brazil, we sequenced the NAGT genes of L. infantum from 68 patients as well as the MIF1 and MIF2 genes from 76 different subjects with diverse clinical manifestations. After polymerase chain reaction (PCR), the fragments were sequenced, followed by polymorphism identification. Results The nucleotide sequencing of the 144 amplicons revealed the absence of genetic variability of the NAGT, MIF1 and MIF2 genes between the isolates. The conservation of these genes suggests that the clinical variability of kala-azar does not depend upon these genes. Additionally, this conservation suggests that these genes may be critical for parasite survival. Conclusions NAGT, MIF1 and MIF2 do not alter the severity of kala-azar. NAGT, MIF1 and MIF2 are highly conserved among different isolates of identical species and exhibit potential for use in phylogenetic inferences or molecular diagnosis. .

A study of the role of the FOXP2 and CNTNAP2 genes in persistent developmental stuttering.

A number of speech disorders including stuttering have been shown to have important genetic contributions, as indicated by high heritability estimates from twin and other studies. We studied the potential contribution to stuttering from variants in the FOXP2 gene, which have previously been associated with developmental verbal dyspraxia, and from variants in the CNTNAP2 gene, which have been associated with specific language impairment (SLI). DNA sequence analysis of these two genes in a group of 602 unrelated cases, all with familial persistent developmental stuttering, revealed no excess of potentially deleterious coding sequence variants in the cases compared to a matched group of 487 well characterized neurologically normal controls. This was compared to the distribution of variants in the GNPTAB, GNPTG, and NAGPA genes which have previously been associated with persistent stuttering. Using an expanded subject data set, we again found that NAGPA showed significantly different mutation frequencies in North Americans of European descent (p=0.0091) and a significant difference existed in the mutation frequency of GNPTAB in Brazilians (p=0.00050). No significant differences in mutation frequency in the FOXP2 and CNTNAP2 genes were observed between cases and controls. To examine the pattern of expression of these five genes in the human brain, real time quantitative reverse transcription PCR was performed on RNA purified from 27 different human brain regions. The expression patterns of FOXP2 and CNTNAP2 were generally different from those of GNPTAB, GNPTG and NAPGA in terms of relatively lower expression in the cerebellum. This study provides an improved estimate of the contribution of mutations in GNPTAB, GNPTG and NAGPA to persistent stuttering, and suggests that variants in FOXP2 and CNTNAP2 are not involved in the genesis of familial persistent stuttering. This, together with the different brain expression patterns of GNPTAB, GNPTG, and NAGPA compared to that of FOXP2 and CNTNAP2, suggests that the genetic neuropathological origins of stuttering differ from those of verbal dyspraxia and SLI.

Transcriptional regulation of fatty acid biosynthesis in mycobacteria.

The main purpose of our study is to understand how mycobacteria exert control over the biosynthesis of their membrane lipids and find out the key components of the regulatory network that control fatty acid biosynthesis at the transcriptional level. In this article we describe the identification and purification of FasR, a transcriptional regulator from Mycobacterium sp. that controls the expression of the fatty acid synthase (fas) and the 4-phosphopantetheinyl transferase (acpS) encoding genes, whose products are involved in the fatty acid and mycolic acid biosynthesis pathways. In vitro studies demonstrated that fas and acpS genes are part of the same transcriptional unit and that FasR specifically binds to three conserved operator sequences present in the fas-acpS promoter region (Pfas). The construction and further characterization of a fasR conditional mutant confirmed that FasR is a transcriptional activator of the fas-acpS operon and that this protein is essential for mycobacteria viability. Furthermore, the combined used of Pfas-lacZ fusions in different fasR backgrounds and electrophoretic mobility shift assays experiments, strongly suggested that long-chain acyl-CoAs are the effector molecules that modulate the affinity of FasR for its DNA binding sequences and therefore the expression of the essential fas-acpS operon.

Mucolipidosis II and III alpha/beta in Brazil: analysis of the GNPTAB gene.

UNLABELLED: Mucolipidosis II and III (MLII and MLIII) alpha/beta are rare autosomal recessive lysosomal storage diseases (LSDs) caused by pathogenic variations in the GNPTAB gene. GNPTAB gene codes for the α and ß subunits of phosphotransferase, the enzyme responsible for synthesis of the mannose-6-phosphate (M6P) marker that directs lysosomal enzymes to the lysosome. OBJECTIVES: The objective of this study is to identify sequence variations of the GNPTAB gene in Brazilian patients with MLII and MLIII alpha/beta. METHOD: Sequencing of the GNPTAB gene was performed in samples of gDNA extracted from the peripheral blood of patients with MLII/III diagnosed at a national reference center for LSDs. RESULTS: Twelve unrelated patients, from several regions of Brazil, were included in this study. Only one was born of consanguineous parents. All patients were found to carry at least one nonpathogenic variation. Nine causal sequence variations were found: c.242G>T (p.W81L); c.1123C>T (p.R375X); c.1196C>T (p.S399F); c.1208T>C (p.I403T); c.1514G>A (p.C505Y); c.1759C>T (p.R587X); c.2808A>G (p.Y937_M972del, novel mutation); c. 2269_2273delGAAAC (p.E757KfsX2, novel mutation); and c.3503_3504delTC (p.L1168QfsX5). Both pathogenic variations were identified in 8 of 12 patients; in four patients, only one pathogenic variation was identified. Mutation c.3503_3504delTC, located in exon 19, was the most frequent pathogenic variation found (n=11/24 alleles). The deleterious effect of the c.2808A>C mutation on splicing was confirmed by cDNA analysis. DISCUSSION/CONCLUSIONS: Our findings confirm that the GNPTAB gene presents broad allelic heterogeneity and suggests that, in Brazilian ML II and III patients, screening for mutations should begin at exon 19 of the GNPTAB gene. Further analyses will be conducted on patients in whom both pathogenic mutations have not been found in this study.

Functional and topological analysis of phosphatidylcholine synthase from Sinorhizobium meliloti.

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of eubacteria. It can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation pathway or the phosphatidylcholine synthase (Pcs) pathway. Pcs belongs to the CDP-alcohol phosphotransferase superfamily and synthesizes PC and CMP in one step from CDP-diacylglycerol and choline. In this study, we aligned sequences of characterized Pcs enzymes to identify conserved amino acid residues. Alanine scanning mutagenesis was performed on 55 of these conserved residues. The mutation of nine residues caused a drastic to complete loss (<20% of wild type activity) of Pcs activity. Six of these essential residues were subjected to further mutagenesis studies replacing them by amino acids with similar properties or size. A topological analysis of sinorhizobial Pcs showed the presence of eight transmembrane helices, with the C- and N-terminus located in the cytoplasm. The majority of the conserved residues is predicted to be either located within the cytoplasmic loops or on the cytoplasmic side of the membrane which can be expected for an enzyme using one membrane-associated and one soluble substrate.

The phosphatidylcholine synthase of Pseudomonas putida A ATCC 12633 is responsible for the synthesis of phosphatidylcholine, which acts as a temporary reservoir for Al3+.

In Pseudomonas putida A ATCC 12633 cells grown with tetradecyltrimethylammonium bromide and exposed to Al(3)Cl, phosphatidylcholine (PC) levels increased, which alleviated stress caused by the Al(3+). Here we cloned and sequenced a gene from this strain that encodes a phosphatidylcholine synthase (PCS) and characterized a pcs-deficient mutant. In the pcs-deficient mutant, PC could not be detected, whereas the mutant could be successfully complemented and expressed the enzyme, indicating that PC synthesis occurs exclusively via the PCS pathway in this organism. Although under non-stressing growth conditions the pcs-deficient mutant showed growth like that of the wild-type strain, the mutant was much more sensitive when challenged with Al(3+), which strongly supports the supposition that PC is involved in the response of P. putida to Al(3+) and acts as a temporary reservoir of available ions through the formation of Al(3+) : PC complexes.

Role of the 4-phosphopantetheinyl transferase of Trichoderma virens in secondary metabolism and induction of plant defense responses.

Trichoderma virens is a ubiquitous soil fungus successfully used in biological control due to its efficient colonization of plant roots. In fungi, 4-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary and secondary metabolism. Therefore, we cloned the PPTase gene ppt1 from T. virens and generated PPTase-deficient (?ppt1) and overexpressing strains to investigate the role of this enzyme in biocontrol and induction of plant defense responses. The ?ppt1 mutants were auxotrophic for lysine, produced nonpigmented conidia, and were unable to synthesize nonribosomal peptides. Although spore germination was severely compromised under both low and high iron availability, mycelial growth occurred faster than the wild type, and the mutants were able to efficiently colonize plant roots. The ?ppt1 mutants were unable of inhibiting growth of phytopathogenic fungi in vitro. Arabidopsis thaliana seedlings co-cultivated with wild-type T. virens showed increased expression of pPr1a:uidA and pLox2:uidA markers, which correlated with enhanced accumulation of salicylic acid (SA), jasmonic acid, camalexin, and resistance to Botrytis cinerea. Co-cultivation of A. thaliana seedlings with ?ppt1 mutants compromised the SA and camalexin responses, resulting in decreased protection against the pathogen. Our data reveal an important role of T. virens PPT1 in antibiosis and induction of SA and camalexin-dependent plant defense responses.

Phosphatidylcholine levels of peanut-nodulating Bradyrhizobium sp. SEMIA 6144 affect cell size and motility.

Phosphatidylcholine, the major phospholipid in eukaryotes, is found in rhizobia and in many other bacteria interacting with eukaryotic hosts. Phosphatidylcholine has been shown to be required for a successful interaction of Bradyrhizobium japonicum USDA 110 with soybean roots. Our aim was to study the role of bacterial phosphatidylcholine in the Bradyrhizobium-peanut (Arachis hypogaea) symbiosis. Phospholipid N-methyltransferase (Pmt) and minor phosphatidylcholine synthase (Pcs) activities were detected in crude extracts of the peanut-nodulating strain Bradyrhizobium sp. SEMIA 6144. Our results suggest that phosphatidylcholine formation in Bradyrhizobium sp. SEMIA 6144 is mainly due to the phospholipid methylation pathway. Southern blot analysis using pmt- and pcs-probes of B. japonicum USDA 110 revealed a pcs and multiple pmt homologues in Bradyrhizobium sp. SEMIA 6144. A pmtA knockout mutant was constructed in Bradyrhizobium sp. SEMIA 6144 that showed a 50% decrease in the phosphatidylcholine content in comparison with the wild-type strain. The mutant was severely affected in motility and cell size, but formed wild-type-like nodules on its host plant. However, in coinoculation experiments, the pmtA-deficient mutant was less competitive than the wild type, suggesting that wild-type levels of phosphatidylcholine are required for full competitivity of Bradyrhizobium in symbiosis with peanut plants.

A eukaryote-like cardiolipin synthase is present in Streptomyces coelicolor and in most actinobacteria.

Cardiolipin (CL) is an anionic membrane lipid present in bacteria, plants, and animals, but absent from archaea. It is generally thought that bacteria use an enzyme belonging to the phospholipase D superfamily as cardiolipin synthase (Cls) catalyzing a reversible phosphatidyl group transfer from one phosphatidylglycerol (PG) molecule to another PG to form CL and glycerol. In contrast, in eukaryotes a Cls of the CDP-alcohol phosphatidyltransferase superfamily uses cytidine diphosphate-diacylglycerol (CDP-DAG) as the donor of the phosphatidyl group, which is transferred to a molecule of PG to form CL. Searching the genome of the actinomycete Streptomyces coelicolor A3(2) we identified a gene coding for a putative Cls of the CDP-alcohol phosphatidyltransferase superfamily (Sco1389). Here we show that expression of Sco1389 in a CL-deficient Rhizobium etli mutant restores CL formation. In an in vitro assay Sco1389 condenses CDP-DAG with PG to form CL and therefore catalyzes the same reaction as eukaryotic cardiolipin synthases. This is the first time that a CDP-alcohol phosphatidyltransferase from bacteria is shown to be responsible for CL formation. The broad occurrence of putative orthologues of Sco1389 among the actinobacteria suggests that CL synthesis involving a eukaryotic type Cls is common in actinobacteria.