An Empirical Strategy for Characterizing Bacterial Proteomes across Species in the Absence of Genomic Sequences

Global protein identification through current proteomics methods typically depends on the availability of sequenced genomes. In spite of increasingly high throughput sequencing technologies, this information is not available for every microorganism and rarely available for entire microbial communities. Nevertheless, the protein-level homology that exists between related bacteria makes it possible to extract biological information from the proteome of an organism or microbial community by using the genomic sequences of a near neighbor organism. Here, we demonstrate a trans-organism search strategy for determining the extent to which near-neighbor genome sequences can be applied to identify proteins in unsequenced environmental isolates. In proof of concept testing, we found that within a CLUSTAL W distance of 0.089, near-neighbor genomes successfully identified a high percentage of proteins within an organism. Application of this strategy to characterize environmental bacterial isolates lacking sequenced genomes, but having 16S rDNA sequence similarity to Shewanella resulted in the identification of 300–500 proteins in each strain. The majority of identified pathways mapped to core processes, as well as to processes unique to the Shewanellae, in particular to the presence of c-type cytochromes. Examples of core functional categories include energy metabolism, protein and nucleotide synthesis and cofactor biosynthesis, allowing classification of bacteria by observation of conserved processes. Additionally, within these core functionalities, we observed proteins involved in the alternative lactate utilization pathway, recently described in Shewanella

Draft genome sequences of four Enterococcus faecium strains isolated from Argentine cheese

We report the draft genome sequences of four Enterococcus faecium strains isolated from Argentine regional cheeses. These strains were selected based on their technological properties, i.e., their ability to produce aroma compounds (diacetyl, acetoin, and 2,3-butanediol) from citrate. The goal of our study is to provide further genetic evidence for the rational selection of entero-cocci strains based on their pheno- and genotype in order to be used in cheese production.Fil: Martino, Gabriela Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Martino, Gabriela Paula. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina.Fil: Quintana, Ingrid M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Quintana, Ingrid M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina.Fil: Espariz, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Espariz, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina.Fil: Blancato, Victor. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Blancato, Victor. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina.Fil: Gallina Nizo, Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina.Fil: Gallina Nizo, Gabriel. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina.Fil: Esteban, Luis. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina.Fil: Esteban, Luis. Universidad Nacional de Rosario. Facultad de Ciencias Médicas; Argentina.Fil: Magni, Christian. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Magni, Christian. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina

Acinetobacter baumannii NCIMB8209: a rare environmental strain displaying extensive insertion sequence-mediated genome remodeling resulting in the loss of exposed cell structures and defensive mechanisms

Acinetobacter baumannii represents nowadays an important nosocomial pathogen of poorly defined reservoirs outside the clinical setting. Here, we con ducted whole-genome sequencing analysis of the Acinetobacter sp. NCIMB8209 col lection strain, isolated in 1943 from the aerobic degradation (retting) of desert gu ayule shrubs. Strain NCIMB8209 contained a 3.75-Mb chromosome and a plasmid of 134 kb. Phylogenetic analysis based on core genes indicated NCIMB8209 affiliation to A. baumannii, a result supported by the identification of a chromosomal blaOXA 51-like gene. Seven genomic islands lacking antimicrobial resistance determinants, 5 regions encompassing phage-related genes, and notably, 93 insertion sequences (IS) were found in this genome. NCIMB8209 harbors most genes linked to persistence and virulence described in contemporary A. baumannii clinical strains, but many of the genes encoding components of surface structures are interrupted by IS. More over, defense genetic islands against biological aggressors such as type 6 secretion systems or CRISPR-cas are absent from this genome. These findings correlate with a low capacity of NCIMB8209 to form biofilm and pellicle, low motility on semisolid medium, and low virulence toward Galleria mellonella and Caenorhabditis elegans. Searching for catabolic genes and concomitant metabolic assays revealed the ability of NCIMB8209 to grow on a wide range of substances produced by plants, including aromatic acids and defense compounds against external aggressors. All the above features strongly suggest that NCIMB8209 has evolved specific adaptive features to a particular environmental niche. Moreover, they also revealed that the remarkable genetic plasticity identified in contemporary A. baumannii clinical strains represents an intrinsic characteristic of the species.Fil: Repizo, Guillermo Daniel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Repizo, Guillermo Daniel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Espariz, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Espariz, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Seravalle, Joana L. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Seravalle, Joana L. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Díaz Miloslavich, Juan Ignacio. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Díaz Miloslavich, Juan Ignacio. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Steimbrüch, Bruno A. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Steimbrüch, Bruno A. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Shuman, Howard A. University of Chicago. Department of Microbiology; United States.Fil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina

The environmental Acinetobacter baumannii isolate DSM30011 reveals clues into the preantibiotic era genome diversity, virulence potential, and niche range of a predominant nosocomial pathogen

Acinetobacter baumannii represents nowadays an important nosocomial opportunistic pathogen whose reservoirs outside the clinical setting are obscure. Here, we traced the origins of the collection strain A. baumannii DSM30011 to an isolate first reported in 1944, obtained from the enriched microbiota responsible of the aerobic decomposition of the resinous desert shrub guayule. Whole-genome sequencing and phylogenetic analysis based on core genes confirmed DSM30011 affiliation to A. baumannii. Comparative studies with 32 complete A. baumannii genomes revealed the presence of 12 unique accessory chromosomal regions in DSM30011 including five encompassing phage-related genes, five containing toxin genes of the type-6 secretion system, and one with an atypical CRISPRs/cas cluster. No antimicrobial resistance islands were identified in DSM30011 agreeingwithageneralantimicrobialsusceptibilityphenotypeincludingfolatesynthesisinhibitors.Themarginalampicillinresistance of DSM30011 most likely derived from chromosomal ADC-type ampC and blaOXA-51-type genes. Searching for catabolic pathways genes revealed several clusters involved in the degradation of plant defenses including woody tissues and a previously unreported atu locus responsible of aliphatic terpenes degradation, thus suggesting that resinous plants may provide an effective niche for this organism. DSM30011 also harbored most genes and regulatory mechanisms linked to persistence and virulence in pathogenicAcinetobacterspecies. This strain thus revealed important clues into the genomic diversity, virulence potential, and niche ranges of the preantibiotic era A. baumannii population, and may provide an useful tool for our understanding of the processes that led to the recent evolution of this species toward an opportunistic pathogen of humans.This article has been accepted for publication in Genome Biology and Evolution Published by Oxford University Press.Fil: Repizo, Guillermo Daniel. University of Lyon - Centre National de la Recherche Scientifique (CNRS). Laboratory of Molecular Microbiology and Structural Biochemistry; France.Fil: Repizo, Guillermo Daniel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR - CONICET); Argentina.Fil: Repizo, Guillermo Daniel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR - CONICET); Argentina.Fil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Borges, Vítor. National Institute of Health. Department of Infectious Diseases. Bioinformatics Unit; Portugal.Fil: Cameranesi, María Marcela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR - CONICET); Argentina.Fil: Cameranesi, María Marcela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Taib, Najwa. Université Lyon - Centre National de la Recherche Scientifique (CNRS). Laboratoire de Biométrie et Biologie Évolutive; France.Fil: Espariz, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR - CONICET); Argentina.Fil: Espariz, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Brochier-Armanet, Céline. Université Lyon - Centre National de la Recherche Scientifique (CNRS). Laboratoire de Biométrie et Biologie Évolutive; France.Fil: Gomes, João Paulo. National Institute of Health. Department of Infectious Diseases. Bioinformatics Unit; Portugal.Fil: Salcedo, Suzana P. University of Lyon - Centre National de la Recherche Scientifique (CNRS). Laboratory of Molecular Microbiology and Structural Biochemistry; France

Copper Handling in the Salmonella Cell Envelope and Its Impact on Virulence

Copper (Cu) plays a key role at the host–pathogen interface as both an essential element and a toxic element. Intracellular strains of pathogenic Salmonella have acquired the periplasmic Cu chaperone, CueP, and the thiol oxidoreductases complex Scs, while losing the ancestral Cu-detoxification Cus system. Coregulation of these species-specific factors link Cu with redox stress and allows Salmonella to counteract Cu toxicity during infection.Fil: Checa, Susana Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Giri, German Francisco. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Espariz, Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Argüello, José M.. Worcester Polytechnic Institute; Estados UnidosFil: Soncini, Fernando Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Genetic engineering of Lactococcus lactis co-producing antigen and the mucosal adjuvant 3′ 5′- cyclic di adenosine monophosphate (c-di-AMP) as a design strategy to develop a mucosal vaccine prototype

Lactococcus lactis is a promising candidate for the development of mucosal vaccines. More than 20 years of experimental research supports this immunization approach. In addition, 30 5 0 - cyclic di-adenosine monophosphate (c-di-AMP) is a bacterial second messenger that plays a key role in the regulation of diverse physiological functions (potassium and cellular wall homeostasis, among others). Moreover, recent studies showed that c-di-AMP has a strong mucosal adjuvant activity that promotes both humoral and cellular immune responses. In this study, we report the development of a novel mucosal vaccine prototype based on a genetically engineered L. lactis strain. First, we demonstrate that homologous expression of cdaA gen in L. lactis is able to increase c-di-AMP levels. Thus, we hypothesized that in vivo synthesis of the adjuvant can be combined with production of an antigen of interest in a separate form or jointly in the same strain. Therefore, a specifically designed fragment of the trans-sialidase (TScf) enzyme from the Trypanosoma cruzi parasite, the etiological agent of Chagas disease, was selected to evaluate as proof of concept the immune response triggered by our vaccine prototypes. Consequently, we found that oral administration of a L. lactis strain expressing antigenic TScf combined with another L. lactis strain producing the adjuvant c-di-AMP could elicit a TS-specific immune response. Also, an additional L. lactis strain containing a single plasmid with both cdaA and tscf genes under the Pcit and Pnis promoters, respectively, was also able to elicit a specific immune response. Thus, the current report is the first one to describe an engineered L. lactis strain that simultaneously synthesizes the adjuvant c-di-AMP as well as a heterologous antigen in order to develop a simple and economical system for the formulation of vaccine prototypes using a food grade lactic acid bacterium.Para citar este articulo: Quintana I, Espariz M, Villar SR, González FB, Pacini MF, Cabrera G, Bontempi I, Prochetto E, Stülke J, Perez AR, Marcipar I, Blancato V and Magni C (2018) Genetic Engineering of Lactococcus lactis Co-producing Antigen and the Mucosal Adjuvant 30 5 0 - cyclic di Adenosine Monophosphate (c-di-AMP) as a Design Strategy to Develop a Mucosal Vaccine Prototype. Front. Microbiol. 9:2100. doi: 10.3389/fmicb.2018.02100Fil: Quintana, Ingrid M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Quintana, Ingrid M. Georg-August-Universität Göttingen. Göttinger Zentrum für Molekulare Biowissenschaften (GZMB). Department of General Microbiology; Germany.Fil: Espariz, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Espariz, Martín. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina.Fil: Villar, Silvina Raquel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER – CONICET); Argentina.Fil: Villar, Silvina Raquel. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Centro de Investigación y Producción de Reactivos Biológicos; Argentina.Fil: González, Florencia Belén. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER – CONICET); Argentina.Fil: Pacini, Maria F. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER – CONICET); Argentina.Fil: Cabrera, Gabriel. Universidad Nacional del Litoral. Laboratorio de Tecnología Inmunológica; Argentina.Fil: Cabrera, Gabriel. Universidad Nacional del Litoral. Facultad de Ciencias Médicas; Argentina.Fil: Bontempi, Iván. Universidad Nacional del Litoral. Laboratorio de Tecnología Inmunológica; Argentina.Fil: Bontempi, Iván. Universidad Nacional del Litoral. Facultad de Ciencias Médicas; Argentina.Fil: Prochetto, Estefanía. Universidad Nacional del Litoral. Laboratorio de Tecnología Inmunológica; Argentina.Fil: Stülke, Jörg. Georg-August-Universität Göttingen. Göttinger Zentrum für Molekulare Biowissenschaften (GZMB). Department of General Microbiology; Germany.Fil: Perez, Ana R. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Inmunología Clínica y Experimental de Rosario (IDICER – CONICET); Argentina.Fil: Perez, Ana R. Universidad Nacional de Rosario. Facultad de Ciencias Médicas. Centro de Investigación y Producción de Reactivos Biológicos; Argentina.Fil: Marcipar, Iván. Universidad Nacional del Litoral. Laboratorio de Tecnología Inmunológica; Argentina.Fil: Marcipar, Iván. Universidad Nacional del Litoral. Facultad de Ciencias Médicas; Argentina.Fil: Blancato, Victor. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Blancato, Victor. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina.Fil: Magni, Christian. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET). Laboratorio de Fisiología y Genética de Bacterias Lácticas; Argentina.Fil: Magni, Christian. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Laboratorio de Biotecnología e Inocuidad de los Alimentos; Argentina

Biochemical and Genetic Characterization of the Enterococcus faecalis Oxaloacetate Decarboxylase Complex

<p>Enterococcus faecalis encodes a biotin-dependent oxaloacetate decarboxylase (OAD), which is constituted by four subunits: E. faecalis carboxyltransferase subunit OadA (termed Ef-A), membrane pump Ef-B, biotin acceptor protein Ef-D, and the novel subunit Ef-H. Our results show that in E. faecalis, subunits Ef-A, Ef-D, and Ef-H form a cytoplasmic soluble complex (termed Ef-AHD) which is also associated with the membrane. In order to characterize the role of the novel Ef-H subunit, coexpression of oad genes was performed in Escherichia coli, showing that this subunit is vital for Ef-A and Ef-D interaction. Diminished growth of the oadA and oadD single deletion mutants in citrate-supplemented medium indicated that the activity of the complex is essential for citrate utilization. Remarkably, the oadB-deficient strain was still capable of growing to wild-type levels but with a delay during the citrate-consuming phase, suggesting that the soluble Ef-AHD complex is functional in E. faecalis. These results suggest that the Ef-AHD complex is active in its soluble form, and that it is capable of interacting in a dynamic way with the membrane-bound Ef-B subunit to achieve its maximal alkalinization capacity during citrate fermentation.</p>