Metabolic and physiological adaptations of microalgal growth-promoting bacterium Azospirillum brasilense growing under biogas atmosphere: a microarray-based transcriptome analysis.

Using microalgal growth-promoting bacteria (MGPB) to improve the cultured microalga metabolism during biotechnological processes is one of the most promising strategies to enhance their benefits. Nonetheless, the culture condition effect used during the biotechnological process on MGPB growth and metabolism is key to ensure the expected positive bacterium growth and metabolism of microalgae. In this sense, the present research study investigated the effect of the synthetic biogas atmosphere (75% CH4-25% CO2) on metabolic and physiological adaptations of the MGPB Azospirillum brasilense by a microarray-based transcriptome approach. A total of 394 A. brasilense differentially expressed genes (DEGs) were found: 201 DEGs (34 upregulated and 167 downregulated) at 24 h and 193 DEGs (140 upregulated and 53 downregulated) under the same conditions at 72 h. The results showed a series of A. brasilense genes regulating processes that could be essential for its adaptation to the early stressful condition generated by biogas. Evidence of energy production is shown by nitrate/nitrite reduction and activation of the hypothetical first steps of hydrogenotrophic methanogenesis; signal molecule modulation is observed: indole-3-acetic acid (IAA), riboflavin, and vitamin B6, activation of Type VI secretion system responding to IAA exposure, as well as polyhydroxybutyrate (PHB) biosynthesis and accumulation. Moreover, an overexpression of ipdC, ribB, and phaC genes, encoding the key enzymes for the production of the signal molecule IAA, vitamin riboflavin, and PHB production of 2, 1.5 and 11 folds, respectively, was observed at the first 24 h of incubation under biogas atmosphere Overall, the ability of A. brasilense to metabolically adapt to a biogas atmosphere is demonstrated, which allows its implementation for generating biogas with high calorific values and the use of renewable energies through microalga biotechnologies.

Antibacterial effect of acetic acid during an outbreak of carbapenem-resistant Acinetobacter baumannii in an ICU (II).

INTRODUCTION: Acetic acid (AA) has been commonly used in medicine as an antiseptic agent for the past 6000 years. This study evaluated the antibacterial effect of AA during an outbreak in an intensive care unit (ICU) facility in Baja California Sur, México. METHODOLOGY: Thirty-five environmental samples were collected, subsequently, disinfection with AA (4%) was performed, and two days later the same areas were sampled inside the ICU facility. Carbapenem-resistant A. baumannii (CRAB) was detected with loop-mediated isothermal amplification assay (Garciglia-Mercado et al. companion paper), targeting blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP and blaVIM genes. CRAB isolates before and after disinfection were compared by PFGE. RESULTS: Eighteen (54.5%) and five (14.3%) of thirty-five environmental samples were identified as Acinetobacter baumannii before and after disinfection, respectively, showing a significant decrease of 85.7% (p < 0.05) both by Loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). Furthermore, the presence of blaOXA-23-like and blaOXA-58-like genes significantly decreased (p < 0.05) both by LAMP and PCR methods. PFGE genotype showed high similarity among CRAB isolates before and after disinfection, suggesting wide clonal dissemination in the ICU facility. CONCLUSIONS: This study demonstrated the novel application of AA with the LAMP assays developed for detecting CRAB. AA promises to be a cheap and efficacious disinfectant alternative to both developed and especially developing countries, preventing the spread of this organism in the environment and to other susceptible patients in health care settings.

Development of a LAMP method for detection of carbapenem-resistant Acinetobacter baumannii during a hospital outbreak.

INTRODUCTION: Carbapenem-resistant A. baumannii (CRAB) represents a public health threat increasing worldwide. We assess the suitability of a loop-mediated isothermal amplification (LAMP) method for on-site screening of CRAB in a hospital facility. METHODOLOGY: A set of six primers were designed for recognizing eight distinct sequences on six targets: blaOXA-23-like, blaOXA-24-like, blaOXA-51-like, blaOXA-58-like, blaIMP, and blaVIM. A LAMP method was developed, optimized and evaluated for the identification of CRAB in thirty-three environmental samples from an outbreak in an Intensive Care Unit (ICU) facility. RESULTS: The sensitivity of the LAMP assay for the detection of A. baumannii was ten-fold higher than the PCR assay (1.0 ng.µL-1). The LAMP assays showed a higher detection rate for CRAB samples and robust diagnosis performance in comparison to a conventional PCR, with clinical sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100% for blaOXA-23-like, blaOXA-51-like and blaVIM. CONCLUSIONS: The developed LAMP assays are powerful tools that can be useful in on-site screening of CRAB causing local outbreaks in clinics and hospitals facilities where costs and equipment restraints are imperative.

Involvement of OpsLTP1 from Opuntia streptacantha in abiotic stress adaptation and lipid metabolism.

Plant lipid transfer proteins (LTPs) exhibit the ability to transfer lipids between membranes in vitro, and have been implicated in diverse physiological processes associated to plant growth, reproduction, development, biotic and abiotic stress responses. However, their mode of action is not yet fully understood. To explore the functions of the OpsLTP1 gene encoding a LTP from cactus pear Opuntia streptacantha Lem., we generated transgenic Arabidopsis thaliana (L.) Heynh. plants to overexpress OpsLTP1 and contrasted our results with the loss-of-function mutant ltp3 from A. thaliana under abiotic stress conditions. The ltp3 mutant seeds showed impaired germination under salt and osmotic treatments, in contrast to OpsLTP1 overexpressing lines that displayed significant increases in germination rate. Moreover, stress recovery assays showed that ltp3 mutant seedlings were more sensitive to salt and osmotic treatments than wild-type plants suggesting that AtLTP3 is required for stress-induced responses, while the OpsLTP1 overexpressing line showed no significant differences. In addition, OpsLTP1 overexpressing and ltp3 mutant seeds stored lower amount of total lipids compared with wild-type seeds, showing changes primarily on 16C and 18C fatty acids. However, ltp3 mutant also lead changes in lipid profile and no over concrete lipids which may suggest a compensatory activation of other LTPs. Interestingly, linoleic acid (18:2ω6) was consistently increased in neutral, galactoglycerolipids and phosphoglycerolipids of OpsLTP1 overexpressing line indicating a role of OpsLTP1 in the modulation of lipid composition in A. thaliana.

Isolation of the sex-determining gene Sex-lethal (Sxl) in Penaeus (Litopenaeus) vannamei (Boone, 1931) and characterization of its embryogenic, gametogenic, and tissue-specific expression.

The Pacific white shrimp Penaeus vannamei is the most cultured shrimp species around the world. Because females grow larger than males, the culture of 'only females' is of great interest, but knowledge on sex determination and differentiation is required for producing only females. In an effort to obtain information associated with reproduction in P. vannamei, transcriptomic data from female gonads was generated, and partial sequences of a transcript were identified as Sex-lethal (Sxl). Its characterization indicated that, differently from other penaeids in which this gene has been isolated, there are six isoforms of the Sxl transcript in P. vannamei (PvanSxl 1-6). These isoforms result from alternative splicing at three splice sites (SS1, SS2, SS3). The first splice-site is unique to P. vannamei, as it has not been reported for other Arthropod species; the second splice-site (SS2) is common among crustaceans, and the third splice-site (SS3) is also unique to P. vannamei and when spliced-out, it is always together with SS2. All isoforms are expressed during embryogenesis as well as gametogenesis of both genders. The two shorter isoforms, PvanSxl-5 and PvanSxl-6, which result from the splicing of SS2 and SS3, were found mostly expressed in adult testis, but PvanSxl-6 was also expressed in oocytes during gametogenesis. During oogenesis, the second largest isoform, PvanSxl-2, which splices-out only SS1, and PvanSxl-4 that splices-out SS1 and SS2 were highly expressed. These two isoforms were also highly expressed during embryonic development. In situ hybridization allowed pinpointing more specifically the cells where the PvanSxl transcripts were expressed. During embryogenesis, hybridization was observed from the one-cell stage embryo to late gastrula. In the female gonad in previtellogenesis, hybridization occurred in the nucleus of oocytes, whereas in secondary vitellogenesis the transcript also hybridized cytoplasmic granules and cortical crypts. Finally, in situ hybridization corroborated the expression of PvanSxl also in the male gonad during spermatogenesis, mostly occurring in the cytoplasm from spermatogonia and spermatocytes.

Tryptophan, thiamine and indole-3-acetic acid exchange between Chlorella sorokiniana and the plant growth-promoting bacterium Azospirillum brasilense.

During synthetic mutualistic interactions between the microalga Chlorella sorokiniana and the plant growth-promoting bacterium (PGPB) Azospirillum brasilense, mutual exchange of resources involved in producing and releasing the phytohormone indole-3-acetic acid (IAA) by the bacterium, using tryptophan and thiamine released by the microalga, were measured. Although increased activities of tryptophan synthase in C. sorokiniana and indole pyruvate decarboxylase (IPDC) in A. brasilense were observed, we could not detect tryptophan or IAA in the culture medium when both organisms were co-immobilized. This indicates that no extra tryptophan or IAA is produced, apart from the quantities required to sustain the interaction. Over-expression of the ipdC gene occurs at different incubation times: after 48 h, when A. brasilense was immobilized alone and grown in exudates of C. sorokiniana and at 96 h, when A. brasilense was co-immobilized with the microalga. When A. brasilense was cultured in exudates of C. sorokiniana, increased expression of the ipdC gene, corresponding increase in activity of IPDC encoded by the ipdC gene, and increase in IAA production were measured during the first 48 h of incubation. IAA production and release by A. brasilense was found only when tryptophan and thiamine were present in a synthetic growth medium (SGM). The absence of thiamine in SGM yielded no detectable IAA. In summary, this study demonstrates that C. sorokiniana can exude sufficient tryptophan and thiamine to allow IAA production by a PGPB during their interaction. Thiamine is essential for IAA production by A. brasilense and these three metabolites are part of a communication between the two microorganisms.

A novel CHH gene from the Pacific white shrimp Litopenaeus vannamei was characterized and found highly expressed in gut and less in eyestalk and other extra-eyestalk tissues.

The crustacean hyperglycemic hormone (CHH) family is an important group of neuropeptides involved in controlling growth, reproduction, and stress response in decapod species. In this study, a new gene containing 4 exons-3 introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions was isolated from Litopenaeus vannamei. Two full length transcripts of this CHH were isolated from eyestalk and pericardial tissue of males and females using rapid amplification of cDNA ends (RACE). Transcripts sequences were 1578bp in length in males pericardial tissues and in males and females eyestalk with 100% identity, but the transcript isolated from females pericardial tissues was shorter (974bp). The differences in transcripts length is a result of two polyadenylation sites present in the 3'UTR resulting in two transcription termination signals. Transcript sequences encoded one unique protein that can be classified as type I CHH subfamily because of the 4 exons and 3 introns structure, although the CPRP region is not-well conserved and there is no amidation in the C-terminal of the deduced amino acid sequence. Furthermore, there is a glycine inserted in the mature peptide not at position 12 as in type II CHHs but after amino acid 31 and the phylogenetic analysis did not group the peptide within type I, but closer to type II CHHs. We demonstrated by endpoint-PCR, qPCR, and in situ hybridization (ISH), that this gene is expressed in neuroendocrine organs known to express CHHs in penaeid shrimp, including X-organ and optic nerve in eyestalk, supraesophageal ganglion (SoG), but it is also expressed in other organs as gill, gut, pericardial cavity, as well as in terminal ampoule or spermatophore and vas deferens of males.

In silico epitope analysis of unique and membrane associated proteins from Mycobacterium avium subsp. paratuberculosis for immunogenicity and vaccine evaluation.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis disease affecting ruminants worldwide. The aim of this study was to identify potential candidate antigens and epitopes by bio and immuno-informatic tools which could be later evaluated as vaccines and/or diagnosis. 110 protein sequences were selected from MAP K-10 genome database: 48 classified as putative enzymes involved in surface polysaccharide and lipopolysaccharide synthesis, as membrane associated and secreted proteins, 32 as conserved membrane proteins, and 30 as absent from other mycobacterial genomes. These 110 proteins were preliminary screened for Major Histocompatibility Complex (MHC) class II affinity and promiscuity using ProPred program. In addition, subcellular localization and host protein homology was analyzed. From these analyses, 23 MAP proteins were selected for a more accurate inmunoinformatic analysis (i.e. T cell and B cell epitopes analysis) and for homology with mycobacterial proteins. Finally, eleven MAP proteins were identified as potential candidates for further immunogenic evaluation: six proteins (MAP0228c, MAP1239c, MAP2232, MAP3080, MAP3131 and MAP3890) were identified as presenting potential T cell epitopes, while 5 selected proteins (MAP0232c, MAP1240c, MAP1738, MAP2239 and MAP3641c) harbored a large numbers of epitopes predicted to induce both cell- and antibody-mediated immune responses. Moreover, immunogenicity of selected epitopes from MAP1239c were evaluated in IFN-γ release assay. In summary, eleven M. avium subsp. paratuberculosis proteins were identified by in silico analysis and need to be further evaluated for their immunodiagnostic and vaccine potential in field and mice model.

The Opuntia streptacantha OpsHSP18 gene confers salt and osmotic stress tolerance in Arabidopsis thaliana.

Abiotic stress limits seed germination, plant growth, flowering and fruit quality, causing economic decrease. Small Heat Shock Proteins (sHSPs) are chaperons with roles in stress tolerance. Herein, we report the functional characterization of a cytosolic class CI sHSP (OpsHSP18) from Opuntia streptacantha during seed germination in Arabidopsis thaliana transgenic lines subjected to different stress and hormone treatments. The over-expression of the OpsHSP18 gene in A. thaliana increased the seed germination rate under salt (NaCl) and osmotic (glucose and mannitol) stress, and in ABA treatments, compared with WT. On the other hand, the over-expression of the OpsHSP18 gene enhanced tolerance to salt (150 mM NaCl) and osmotic (274 mM mannitol) stress in Arabidopsis seedlings treated during 14 and 21 days, respectively. These plants showed increased survival rates (52.00 and 73.33%, respectively) with respect to the WT (18.75 and 53.75%, respectively). Thus, our results show that OpsHSP18 gene might have an important role in abiotic stress tolerance, in particular in seed germination and survival rate of Arabidopsis plants under unfavorable conditions.

Cytosolic manganese superoxide dismutase genes from the white shrimp Litopenaeus vannamei are differentially expressed in response to lipopolysaccharides, white spot virus and during ontogeny.

Manganese superoxide dismutase (MnSOD) is an antioxidant enzyme usually located in mitochondria. There are only a few examples of cytosolic MnSOD (cMnSOD). In the shrimp Litopenaeus vannamei, we have previously characterized three cMnSOD cDNAs and their differential tissue-specific expression. To obtain insights about their genomic organization, we characterized the three corresponding cMnSOD genes, named them cMnsod1, cMnsod2, and cMnsod3 and studied their specific expression during ontogeny, response to lipopolysaccharides (LPS) and white spot virus infection (WSSV) in hemocytes from shrimp. The first two genes contain five introns flanked by canonical 5'-GT-AG-3' intron splice-site junctions, while the third one is intron-less. We analyzed 995 nucleotides upstream cMnsod2, but no classical promoter sequences were found. The deduced products of the three cMnSOD genes differ in two amino acids and there are four silent changes. cMnsod3 expression is modulated by WSSV and cMnsod2 by LPS. cMnsod2 is expressed from eggs to post larval stage during ontogeny. This is the first report of crustacean cMnSOD multigenes that are differently induced during the defense response and ontogeny.

Shrimp invertebrate lysozyme i-lyz: gene structure, molecular model and response of c and i lysozymes to lipopolysaccharide (LPS).

The invertebrate lysozyme (i-lyz or destabilase) is present in shrimp. This protein may have a function as a peptidoglycan-breaking enzyme and as a peptidase. Shrimp is commonly infected with Vibrio sp., a Gram-negative bacteria, and it is known that the c-lyz (similar to chicken lysozyme) is active against these bacteria. To further understand the regulation of lysozymes, we determined the gene sequence and modeled the protein structure of i-lyz. In addition, the expression of i-lyz and c-lyz in response to lipopolysaccharide (LPS) was studied. The shrimp i-lyz gene is interrupted by two introns with canonical splice junctions. The expression of the shrimp i-lyz was transiently down-regulated after LPS injection followed by induction after 6 h in hepatopancreas. In contrast, c-lyz was up-regulated in hepatopancreas 4 h post-injection and slightly down-regulated in gills. The L. vannamei i-lyz does not contain the catalytic residues for muramidase (glycohydrolase) neither isopeptidase activities; however, it is known that the antibacterial activity does not solely rely on the enzymatic activity of the protein. The study of invertebrate lysozyme will increase our understanding of the regulatory process of the defense mechanisms.

Genome-wide analysis of the beta-glucosidase gene family in maize (Zea mays L. var B73).

The hydrolysis of beta-D: -glucosidic bonds which is required for the liberation of many physiologically important compounds is catalyzed by the enzyme beta-glucosidase (BGLU, EC 3.2.1.21). BGLUs are implicated in several processes in plants, such as the timely response to biotic and abiotic stresses through activation of phytohormones and defense compounds. We identified 26 BGLU isozymes in the genome of the maize inbred B73 and propose a standardized nomenclature for all Zea mays BGLU paralogs (Zmbglu1-Zmbglu26). We characterized their intron-exon structure, protein features, phylogenetic relationships, and measured their expression and activity in various tissues under different environmental conditions. Sequence alignments revealed some characteristic motifs (conserved amino acids) and specific differences among different isozymes. Analysis of putative signal peptides suggested that some BGLUs are plastidic, whereas others are mitochondrial, cytosolic, vacuolar or secreted. Microarray and RT-PCR analysis showed that each member of the Zmbglu family had a characteristic expression pattern with regard to tissue specificity and response to different abiotic conditions. The source of variance for gene expression was highest for the type of organ analyzed (tissue variance) than for the growth conditions (environmental variance) or genotype (genetic variance). Analysis of promoter sequences revealed that each Zmbglu paralog possesses a distinct set of cis elements and transcription factor binding sites. Since there are no two Zmbglu paralogs that have identical molecular properties, we conclude that gene subfunctionalization in maize occurs much more rapidly than gene duplication.

Tissue-specific expression and molecular modeling of cytosolic manganese superoxide dismutases from the white shrimp Litopenaeus vannamei.

Manganese superoxide dismutases (MnSODs) are usually mitochondrial enzymes, although there are few examples of cytosolic MnSODs (cMnSOD). We have previously characterized a cMnSOD cDNA from Litopenaeus vannamei hemocytes, and to obtain new insights into the tissue specific expression and the protein structure, we characterized three more different cMnSOD transcripts (cMnsod1, cMnsod2 and cMnsod3) and modeled the three-dimensional protein structure using human MnSOD as a template. The nucleotide sequences differ in seven positions. Four differences are silent; while three produce changes in amino acid sequence. cMnsod1, cMnsod2 and cMnsod3 are differentially expressed in nervous system, hepatopancreas and hemocytes. The structural protein model predicts bona fide MnSODs with proper coordination for the enzymatic activity.

The cytosolic manganese superoxide dismutase from the shrimp Litopenaeus vannamei: molecular cloning and expression.

Manganese containing superoxide dismutase (SOD) is normally a nuclear-encoded mitochondrial enzyme in eukaryotic organisms; however, a cytoplasmic manganese SOD (cMnSOD) was found in crustaceans that use hemocyanin as oxygen carrier. The complete cDNA and deduced amino acid sequence of a cMnSOD from Litopenaeus vannamei were determined. The coding sequence predicts a 287 residues protein with a unique 61 amino acids extension at the N-terminus and lacking a mitochondrial-targeting sequence. Phylogenetic analysis clusters cMnSODs and mitochondrial MnSODs in two separate groups. cMnSOD transcripts were detected in hemocytes, heart, hepatopancreas, intestine, nervous system, muscle, pleopods and gills. Since hemocytes are key defense cells and their reactions produce superoxide radicals, the infection by white spot syndrome virus on the cMnSOD transcript levels were investigated and found to increase transiently 1h post-infection and then decrease as the viral infection progressed to levels significantly lower than uninfected controls by 12h post-infection.

Catalase from the white shrimp Penaeus (Litopenaeus) vannamei: molecular cloning and protein detection.

Catalase is an antioxidant enzyme that plays a very important role in the protection against oxidative damage by breaking down hydrogen peroxide. It is a very highly conserved enzyme that has been identified from numerous species including bacteria, fungi, plants and animals, but the information about catalase in crustaceans is very limited. A cDNA containing the complete coding sequence for catalase from the shrimp Penaeus (Litopenaeus) vannamei was sequenced and the mRNA was detected by RT-PCR in selected tissues. Catalase was detected in hepatopancreas crude extracts by Western blot analysis with anti-human catalase polyclonal antibodies. The nucleotide sequence is 1692 bp long, including a 72-bp 5'-UTR, a coding sequence of 1515 bp and a 104-bp 3'-UTR. The deduced amino acid sequence corresponds to 505 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases and contains the catalytic residues His71, Asn144, and Tyr354. The predicted protein has a calculated molecular mass of 57 kDa; which coincides with the size of the subunit (approximately 55 kDa) and the tetrameric protein (approximately 230 kDa) detected in hepatopancreas extracts under native conditions. Catalase mRNA level was higher in hepatopancreas, followed by gills and was not detected in muscle.