Stress response and virulence factors in bacterial pathogens relevant for Chilean aquaculture: current status and outlook of our knowledge.

The study of the stress responses in bacteria has given us a wealth of information regarding the mechanisms employed by these bacteria in aggressive or even non-optimal living conditions. This information has been applied by several researchers to identify molecular targets related to pathogeny, virulence, and survival, among others, and to design new prophylactic or therapeutic strategies against them. In this study, our knowledge of these mechanisms has been summarized with emphasis on some aquatic pathogenic bacteria of relevance to the health and productive aspects of Chilean salmon farming (Piscirickettsia salmonis, Tenacibaculum spp., Renibacterium salmoninarum, and Yersinia ruckeri). This study will aid further investigations aimed at shedding more light on possible lines of action for these pathogens in the coming years.

Health Status of Mytilus chilensis from Intensive Culture Areas in Chile Assessed by Molecular, Microbiological, and Histological Analyses.

Shellfish farming is a relevant economic activity in Chile, where the inner sea in Chiloé island concentrates 99% of the production of the mussel Mytilus chilensis. This area is characterized by the presence of numerous human activities, which could harm the quality of seawater. Additionally, the presence of potentially pathogenic microorganisms can influence the health status of mussels, which must be constantly monitored. To have a clear viewpoint of the health status of M. chilensis and to study its potential as a host species for exotic diseases, microbiological, molecular, and histological analyses were performed. This study was carried out in October 2018, where M. chilensis gut were studied for: presence of food-borne bacteria (Vibrio parahaemolyticus, Escherichia coli, Salmonella spp.), exotic bacteria ("Candidatus Xenohaliotis californiensis"), viruses (abalone and Ostreid herpes virus), and protozoa (Marteilia spp., Perkinsus spp. and Bonamia spp.). Additionally, 18S rDNA metabarcoding and histology analyses were included to have a complete evaluation of the health status of M. chilensis. Overall, despite the presence of risk factors, abnormal mortality rates were not reported during the monitoring period and the histological examination did not reveal significant lesions. Pathogens of mandatory notification to World Organization for Animal Health (OIE) and the Chilean National Fisheries and Aquaculture Service (SERNAPESCA) were not detected, which confirms that M. chilensis have a good health status, highlighting the importance of an integrated vision of different disciplines to ensure the sustainability of this important mussel industry in Chile.

Phenolic and anthocyanin content characterization related to genetic diversity analysis of Solanum tuberosum subsp. tuberosum Chilotanum Group in southern Chile.

The potato (Solanum tuberosum L) is one of the four most important crops worldwide in production and consumption. It originated from South America along the Andes, where six hotspots of diversity known as subcenters of origin are described from Venezuela to Chiloe Island in Chile, and where the greatest diversity of potatoes in the world is found. Today, the use of ancestral genetic resources has gained significant relevance, recovering and producing foods with a greater nutrient content and beneficial to human health. Therefore, native potatoes possess a set of characteristics with great potential for use in potato breeding guided primarily to produce better feed, especially potatoes of the Chilotanum Group that are easily crossed with conventional varieties. The primary objective of this study was to evaluate 290 accessions of S. tuberosum subsp tuberosum belonging to the Chilotanum Group using a set of molecular markers and correlate them to its phenotypic traits for future use in breeding programs. For this purpose, 290 accessions were analysed through 22 specific microsatellites described previously, correlating them with flesh and skin colour, total phenolic content, and anthocyanin content. A division into groups considering all the 290 accessions resulted in two clusters using STRUCTURE analysis and seven different genetic clusters using UPGMA. The latter exhibited common phenotypic characteristics as well as anthocyanin content, strongly supporting a correlation between phenotypic traits and the genetic fingerprint. These results will enable breeders to focus on the development of potatoes with high polyphenol and anthocyanin content.

Stress response and virulence factors in bacterial pathogens relevant for Chilean aquaculture: current status and outlook of our knowledge

The study of the stress responses in bacteria has given us a wealth of information regarding the mechanisms employed by these bacteria in aggressive or even non-optimal living conditions. This information has been applied by several researchers to identify molecular targets related to pathogeny, virulence, and survival, among others, and to design new prophylactic or therapeutic strategies against them. In this study, our knowledge of these mechanisms has been summarized with emphasis on some aquatic pathogenic bacteria of relevance to the health and productive aspects of Chilean salmon farming (Piscirickettsia salmonis, Tenacibaculum spp., Renibacterium salmoninarum, and Yersinia ruckeri). This study will aid further investigations aimed at shedding more light on possible lines of action for these pathogens in the coming years.

Interferon Gamma Induces the Increase of Cell-Surface Markers (CD80/86, CD83 and MHC-II) in Splenocytes From Atlantic Salmon.

Type II interferon gamma (IFNγ) is a pleiotropic cytokine capable of modulating the innate and adaptive immune responses which has been widely characterized in several teleost families. In fish, IFNγ stimulates the expression of cytokines and chemokines associated with the pro-inflammatory response and enhances the production of nitrogen and oxygen reactive species in phagocytic cells. This work studied the effect of IFNγ on the expression of cell-surface markers on splenocytes of Atlantic salmon (Salmo salar). In vitro results showed that subpopulations of mononuclear splenocytes cultured for 15 days were capable of increasing gene expression and protein availability of cell-surface markers such as CD80/86, CD83 and MHC II, after being stimulated with recombinant IFNγ. These results were observed for subpopulations with characteristics associated with monocytes (51%), and features that could be related to lymphocytes (46.3%). In addition, a decrease in the expression of zbtb46 was detected in IFNγ-stimulated splenocytes. Finally, the expression of IFNγ and cell-surface markers was assessed in Atlantic salmon under field conditions. In vivo results showed that the expression of ifnγ increased simultaneously with the up-regulation of cd80/86, cd83 and mhcii during a natural outbreak of Piscirickettsia salmonis. Overall, the results obtained in this study allow us to propose IFNγ as a candidate molecule to stimulate the phenotypic progression of a small population of immune cells, which will increase antigen presenting cells markers. Thereby, modulatory strategies using IFNγ may generate a robust and coordinated immune response in fish against pathogens that affect aquaculture.

Draft Genome Sequence of Bacillus safensis RP10, Isolated from Soil in the Atacama Desert, Chile.

Genome analysis of Bacillus safensis RP10, a strain from the soil of Atacama Desert in northern Chile, reflects a bacterium adapted to live in soil containing high levels of heavy metals, high salt conditions, and low carbon and energy sources.

Multilocus sequence typing detects new Piscirickettsia salmonis hybrid genogroup in Chilean fish farms: Evidence for genetic diversity and population structure.

Piscirickettsia salmonisis the causative bacterial pathogen of piscirickettsiosis, a salmonid disease that causes notable mortalities in the worldwide aquaculture industry. Published research describes the phenotypic traits, virulence factors, pathogenicity and antibiotic-resistance potential for various P. salmonisstrains. However, evolutionary and genetic information is scarce for P. salmonis. The present study used multilocus sequence typing (MLST) to gain insight into the population structure and evolution of P. salmonis. Forty-two Chilean P. salmonisisolates, as well as the type strain LF-89T , were recovered from diseased Salmo salar, Oncorhynchus kisutchand Oncorhynchus mykissfrom two Chilean Regions. MLST assessed the loci sequences of dnaK, efp, fumC, glyA, murG, rpoD and trpB. Bioinformatics analyses established the genetic diversity among P. salmonis isolates (H = 0.5810). A total of 23 sequence types (ST) were identified, 53.48% of which were represented by ST1, ST5 and ST2. Population structure analysis through polymorphism patterns showed few polymorphic sites (218 nucleotides from 4,010 bp), while dN/dS ratio analysis indicated purifying selection for dnaK, epf, fumC, murG, and rpoD but neutral selection for the trpB loci. The standardized index of association indicated strong linkage disequilibrium, suggesting clonal population structure. However, recombination events were detected in a group of seven isolates. Findings included genogroups homologous to the LF-89T and EM-90 strains, as well as a seven-isolate hybrid genogroup recovered from both assessed regions (three O. mykiss and four S. salar isolates). The presented MLST scheme has comparative potential, with promising applications in studying distinct P. salmonis isolates (e.g., from different hosts, farms, geographical areas) and in understanding the epidemiology of this pathogen.

Draft genomes and reference transcriptomes extend the coding potential of the fish pathogen Piscirickettsia salmonis

Background: Draft and complete genome sequences from bacteria are key tools to understand genetic determinants involved in pathogenesis in several disease models. Piscirickettsia salmonis is a Gram-negative bacterium responsible for the Salmon Rickettsial Syndrome (SRS), a bacterial disease that threatens the sustainability of the Chilean salmon industry. In previous reports, complete and draft genome sequences have been generated and annotated. However, the lack of transcriptome data underestimates the genetic potential, does not provide information about transcriptional units and contributes to disseminate annotation errors. Results: Here we present the draft genome and transcriptome sequences of four P. salmonis strains. We have identified the transcriptional architecture of previously characterized virulence factors and trait-specific genes associated to cation uptake, metal efflux, antibiotic resistance, secretion systems and other virulence factors. Conclusions: This data has provided a refined genome annotation and also new insights on the transcriptional structures and coding potential of this fish pathogen.

Draft genome sequence of Exiguobacterium aurantiacum strain PN47 isolate from saline ponds, known as "Salar del Huasco", located in the Altiplano in the North of Chile

ABSTRACT In this report, we present a draft genome of 2,886,173 bp of an Exiguobacterium aurantiacum strain PN47 isolate from the sediment of a saline pond named "Salar del Huasco" in the Altiplano in the North of Chile. Strain PN47 encodes adaptive characteristics enabling survival in extreme environmental conditions of high heavy metal and salt concentrations and high alkalinity.

Draft genome sequence of Exiguobacterium aurantiacum strain PN47 isolate from saline ponds, known as "Salar del Huasco", located in the Altiplano in the North of Chile.

In this report, we present a draft genome of 2,886,173bp of an Exiguobacterium aurantiacum strain PN47 isolate from the sediment of a saline pond named "Salar del Huasco" in the Altiplano in the North of Chile. Strain PN47 encodes adaptive characteristics enabling survival in extreme environmental conditions of high heavy metal and salt concentrations and high alkalinity.

Tellurite: history, oxidative stress, and molecular mechanisms of resistance.

The perceived importance of tellurium (Te) in biological systems has lagged behind selenium (Se), its lighter sister in the Group 16 chalcogens, because of tellurium's lower crustal abundance, lower oxyanion solubility and biospheric mobility and the fact that, unlike Se, Te has yet to be found to be an essential trace element. Te applications in electronics, optics, batteries and mining industries have expanded during the last few years, leading to an increase in environmental Te contamination, thus renewing biological interest in Te toxicity. This chalcogen is rarely found in the nontoxic, elemental state (Te(0)), but its soluble oxyanions, tellurite (TeO(3)(2-)) and tellurate (TeO(4)(2-)), are toxic for most forms of life even at very low concentrations. Although a number of Te resistance determinants (Tel) have been identified in plasmids or in the bacterial chromosome of different species of bacteria, the genetic and/or biochemical basis underlying bacterial TeO(3)(2-) toxicity is still poorly understood. This review traces the history of Te in its biological interactions, its enigmatic toxicity, importance in cellular oxidative stress, and interaction in cysteine metabolism.

Cloning, purification and characterization of Geobacillus stearothermophilus V uroporphyrinogen-III C-methyltransferase: evaluation of its role in resistance to potassium tellurite in Escherichia coli.

The Geobacillus stearothermophilus V cobA gene encoding uroporphyrinogen-III C-methyltransferase (also referred to as SUMT) was cloned into Escherichia coli and the recombinant enzyme was overexpressed and purified to homogeneity. The enzyme binds S-adenosyl-L-methionine and catalyzes the production of III methyl uroporphyrinogen in vitro. E. coli cells expressing the G. stearothermophilus V cobA gene exhibited increased resistance to potassium tellurite and potassium tellurate. Site-directed mutagenesis of cobA abolished tellurite resistance of the mesophilic, heterologous host and SUMT activity in vitro. No methylated, volatile derivatives of tellurium were found in the headspace of tellurite-exposed cobA-expressing E. coli, suggesting that the role of SUMT methyltransferase in tellurite(ate) detoxification is not related to tellurium volatilization.

Cysteine metabolism-related genes and bacterial resistance to potassium tellurite.

Tellurite exerts a deleterious effect on a number of small molecules containing sulfur moieties that have a recognized role in cellular oxidative stress. Because cysteine is involved in the biosynthesis of glutathione and other sulfur-containing compounds, we investigated the expression of Geobacillus stearothermophilus V cysteine-related genes cobA, cysK, and iscS and Escherichia coli cysteine regulon genes under conditions that included the addition of K2TeO3 to the culture medium. Results showed that cell tolerance to tellurite correlates with the expression level of the cysteine metabolic genes and that these genes are up-regulated when tellurite is present in the growth medium.

Bacterial toxicity of potassium tellurite: unveiling an ancient enigma.

Biochemical, genetic, enzymatic and molecular approaches were used to demonstrate, for the first time, that tellurite (TeO(3) (2-)) toxicity in E. coli involves superoxide formation. This radical is derived, at least in part, from enzymatic TeO(3) (2-) reduction. This conclusion is supported by the following observations made in K(2)TeO(3)-treated E. coli BW25113: i) induction of the ibpA gene encoding for the small heat shock protein IbpA, which has been associated with resistance to superoxide, ii) increase of cytoplasmic reactive oxygen species (ROS) as determined with ROS-specific probe 2'7'-dichlorodihydrofluorescein diacetate (H(2)DCFDA), iii) increase of carbonyl content in cellular proteins, iv) increase in the generation of thiobarbituric acid-reactive substances (TBARs), v) inactivation of oxidative stress-sensitive [Fe-S] enzymes such as aconitase, vi) increase of superoxide dismutase (SOD) activity, vii) increase of sodA, sodB and soxS mRNA transcription, and viii) generation of superoxide radical during in vitro enzymatic reduction of potassium tellurite.

Catalases are NAD(P)H-dependent tellurite reductases.

Reactive oxygen species damage intracellular targets and are implicated in cancer, genetic disease, mutagenesis, and aging. Catalases are among the key enzymatic defenses against one of the most physiologically abundant reactive oxygen species, hydrogen peroxide. The well-studied, heme-dependent catalases accelerate the rate of the dismutation of peroxide to molecular oxygen and water with near kinetic perfection. Many catalases also bind the cofactors NADPH and NADH tenaciously, but, surprisingly, NAD(P)H is not required for their dismutase activity. Although NAD(P)H protects bovine catalase against oxidative damage by its peroxide substrate, the catalytic role of the nicotinamide cofactor in the function of this enzyme has remained a biochemical mystery to date. Anions formed by heavy metal oxides are among the most highly reactive, natural oxidizing agents. Here, we show that a natural isolate of Staphylococcus epidermidis resistant to tellurite detoxifies this anion thanks to a novel activity of its catalase, and that a subset of both bacterial and mammalian catalases carry out the NAD(P)H-dependent reduction of soluble tellurite ion (TeO(3)(2-)) to the less toxic, insoluble metal, tellurium (Te(o)), in vitro. An Escherichia coli mutant defective in the KatG catalase/peroxidase is sensitive to tellurite, and expression of the S. epidermidis catalase gene in a heterologous E. coli host confers increased resistance to tellurite as well as to hydrogen peroxide in vivo, arguing that S. epidermidis catalase provides a physiological line of defense against both of these strong oxidizing agents. Kinetic studies reveal that bovine catalase reduces tellurite with a low Michaelis-Menten constant, a result suggesting that tellurite is among the natural substrates of this enzyme. The reduction of tellurite by bovine catalase occurs at the expense of producing the highly reactive superoxide radical.

Identification of biogenic dimethyl selenodisulfide in the headspace gases above genetically modified Escherichia coli.

Escherichia coli JM109 cells were modified to express the genes encoded in a 3.8-kb chromosomal DNA fragment from a metalloid-resistant thermophile, Geobacillus stearothermophilus V. Manual headspace extraction was used to collect the gases for gas chromatography with fluorine-induced sulfur chemiluminescence analysis while solid-phase microextraction was used for sample collection in gas chromatography/mass spectrometry (GC/MS) analysis. When grown in the presence of selenate or selenite, these bacteria produced both organo-sulfur and organo-selenium in the headspace gases above the cultures. Organo-sulfur compounds detected were methanethiol, dimethyl sulfide, dimethyl disulfide, and dimethyl trisulfide. Organo-selenium compounds detected were dimethyl selenide and dimethyl diselenide. Two mixed sulfur-selenium compounds, dimethyl selenenyl sulfide and a chromatographically late-eluting compound, were detected. Dimethyl selenodisulfide, CH(3)SeSSCH(3), and dimethyl bis(thio)selenide, CH(3)SSeSCH(3), were synthesized and analyzed by GC/MS and fluorine-induced chemiluminescence to determine which corresponded to the late-eluting compound that was bacterially produced. CH(3)SeSSCH(3) was positively identified as the compound detected in bacterial headspace above Se-amended cultures. Using GC retention times, the boiling point of CH(3)SeSSCH(3) was estimated to be approximately 192 degrees C. This is the first report of CH(3)SeSSCH(3) produced by bacterial cultures.

The product of the qacC gene of Staphylococcus epidermidis CH mediates resistance to beta-lactam antibiotics in gram-positive and gram-negative bacteria.

We have characterized a natural isolate of Staphylococcus epidermidis resistant to heavy metals that carries a small 2391-bp plasmid, pSepCH, encoding the qacC gene. The S. epidermidis qacC gene confers resistance to a number of beta-lactam antibiotics and to ethidium bromide in its natural host and in Escherichia coli K12 and Salmonella enterica sv. Typhimurium. This is the first communication of a small multidrug resistance (SMR) pump involved in resistance to beta-lactam antibiotics. Experiments using tolC, ompW and ompD mutant strains of S. Typhimurium demonstrated that the beta-lactam antibiotic resistance conferred by this pump does not depend on these outer membrane proteins.

The Geobacillus stearothermophilus V iscS gene, encoding cysteine desulfurase, confers resistance to potassium tellurite in Escherichia coli K-12.

Many eubacteria are resistant to the toxic oxidizing agent potassium tellurite, and tellurite resistance involves diverse biochemical mechanisms. Expression of the iscS gene from Geobacillus stearothermophilus V, which is naturally resistant to tellurite, confers tellurite resistance in Escherichia coli K-12, which is naturally sensitive to tellurite. The G. stearothermophilus iscS gene encodes a cysteine desulfurase. A site-directed mutation in iscS that prevents binding of its pyridoxal phosphate cofactor abolishes both enzyme activity and its ability to confer tellurite resistance in E. coli. Expression of the G. stearothermophilus iscS gene confers tellurite resistance in tellurite-hypersensitive E. coli iscS and sodA sodB mutants (deficient in superoxide dismutase) and complements the auxotrophic requirement of an E. coli iscS mutant for thiamine but not for nicotinic acid. These and other results support the hypothesis that the reduction of tellurite generates superoxide anions and that the primary targets of superoxide damage in E. coli are enzymes with iron-sulfur clusters.