Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml⁻¹, a half-saturation of signal at 0.26 ng ml⁻¹, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Development and testing of a DNA macroarray to assess nitrogenase (nifH) gene diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml(-1), a half-saturation of signal at 0.26 ng ml(-1), and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Physiological and genetic correlates of boldness: characterising the mechanisms of behavioural variation in rainbow trout, Oncorhynchus mykiss

Bold, risk-taking animals have previously been putatively linked with a proactive stress coping style whereas it is suggested shyer, risk-averse animals exhibit a reactive coping style. The aim of this study was to investigate whether differences in the expression of bold-type behaviour were evident within and between two lines of rainbow trout, Oncorhynchus mykiss, selectively bred for a low (LR) or high (HR) endocrine response to stress, and to link boldness and stress responsiveness with the expression of related candidate genes. Boldness was determined in individual fish over two trials by measuring the latency to approach a novel object. Differences in plasma cortisol concentrations and the expression of eight novel candidate genes previously identified as being linked with divergent behaviours or stress were determined. Bold and shy individuals, approaching the object within 180 s or not approaching within 300 s respectively, were evident within each line, and this was linked with activity levels in the HR line. Post-stress plasma cortisol concentrations were significantly greater in the HR line compared with the LR line, and six of the eight tested genes were upregulated in the brains of LR fish compared with HR fish. However, no direct relationship between boldness and either stress responsiveness or gene expression was found, although clear differences in stress physiology and, for the first time, gene expression could be identified between the lines. This lack of correlation between physiological and molecular responses and behavioural variation within both lines highlights the complexity of the behavioural-physiological complex.     

Scales Tell a Story on the Stress History of Fish

Fish faced with stressful stimuli launch an endocrine stress response through activation of the hypothalamic-pituitary-interrenal (HPI-) axis to release cortisol into the blood. Scientifically validated biomarkers to capture systemic cortisol exposure over longer periods of time are of utmost importance to assess chronic stress in governmental, wildlife, aquaculture and scientific settings. Here we demonstrate that cortisol in scales of common carp (Cyprinus carpio L.) is the long-sought biomarker for chronic stress. Undisturbed (CTR) and daily stressed (STRESS) carp were compared. Dexamethasone (DEX) or cortisol (CORT) fed fish served as negative and positive controls, respectively. Scale cortisol was quantified with a validated ultra-performance liquid chromatography tandem mass spectrometry method. An increase in scale cortisol content was found in STRESS and CORT but not in CTR and DEX fish. Scale cortisol content reflects its accumulation in a stressor and time dependent manner and validates the scale cortisol content as biomarker for chronic stress. Plasma analyses confirmed that (i) CTR, DEX and CORT treatments were effective, (ii) plasma cortisol of STRESS fish showed no signs of chronic HPI-axis activation, and (iii) plasma cortisol is a poor predictor for chronic stress. The expression of HPI key genes crf, pomc, and star were up-regulated in STRESS fish in the absence of a plasma cortisol response, as was the target gene of cortisol encoding subunit α1 of the Na⁺/K⁺-ATPase in gills. When lost, scales of fish regenerate fast. Regenerated scales corroborate our findings, offering (i) unsurpassed time resolution for cortisol incorporation and as such for stressful events, and (ii) the possibility to investigate stress in a well defined and controlled environment and time frame creating novel opportunities for bone physiological research. We conclude that the cortisol content in ontogenetic and regenerated scales is an innovative biomarker for chronic stress offering ample applications in science and industry.     

Engineering of a robust Escherichia coli chassis and exploitation for large-scale production processes

In large-scale bioprocesses microbes are exposed to heterogeneous substrate availability reducing the overall process performance. A series of deletion strains was constructed from E. coli MG1655 aiming for a robust phenotype in heterogeneous fermentations with transient starvation. Deletion targets were hand-picked based on a list of genes derived from previous large-scale simulation runs. Each gene deletion was conducted on the premise of strict neutrality towards growth parameters in glucose minimal medium. The final strain of the series, named E. coli RM214, was cultivated continuously in an STR-PFR (stirred tank reactor – plug flow reactor) scale-down reactor. The scale-down reactor system simulated repeated passages through a glucose starvation zone. When exposed to nutrient gradients, E. coli RM214 had a significantly lower maintenance coefficient than E. coli MG1655 (Δm_s = 0.038 g_Glucose/g_CDW/h, p < 0.05). In an exemplary protein production scenario E. coli RM214 remained significantly more productive than E. coli MG1655 reaching 44% higher eGFP yield after 28 h of STR-PFR cultivation. This study developed E. coli RM214 as a robust chassis strain and demonstrated the feasibility of engineering microbial hosts for large-scale applications.     

Gene Expression of CRAL_TRIO Family Proteins modulated by Vitamin E Deficiency in Zebrafish (Danio Rerio)

An evaluation of the impact of vitamin E deficiency on expression of the alpha-tocopherol transfer protein (α-TTP) and related CRAL_TRIO genes was undertaken using livers from adult zebrafish based on the hypothesis that increased lipid peroxidation would modulate gene expression. Zebrafish were fed either a vitamin E sufficient (E+) or deficient (E–) diet for 9 months, then fish were euthanized, and livers were harvested. Livers from the E+ relative to E– fish contained 40-times more α-tocopherol (P <0.0001) and one fourth the malondialdehyde (P = 0.0153). RNA was extracted from E+ and E– livers, then subject to evaluation of gene expression of ttpa and other genes of the CRAL_TRIO family, genes of antioxidant markers, and genes related to lipid metabolism. Ttpa expression was not altered by vitamin E status. However, one member of the CRAL_TRIO family, tyrosine-protein phosphatase non-receptor type 9 gene (ptpn9a), showed a 2.4-fold increase (P = 0.029) in E– relative to E+ livers. Further, we identified that the gene for choline kinase alpha (chka) showed a 3.0-fold increase (P = 0.010) in E– livers. These outcomes are consistent with our previous findings that show vitamin E deficiency increased lipid peroxidation causing increases in phospholipid turnover.     

A Simplified Method for Gene Knockout and Direct Screening of Recombinant Clones for Application in Paenibacillus polymyxa

Background

Paenibacillus polymyxa is a bacterium widely used in agriculture, industry, and environmental remediation because it has multiple functions including nitrogen fixation and produces various biologically active compounds. Among these compounds are the antibiotics polymyxins, and the bacterium is currently being reassessed for medical application. However, a lack of genetic tools for manipulation of P. polymyxa has limited our understanding of the biosynthesis of these compounds.

Methods and Principal Findings

To facilitate an understanding of the genetic determinants of the bacterium, we have developed a system for marker exchange mutagenesis directly on competent cells of P. polymyxa under conditions where homologous recombination is enhanced by denaturation of the suicide plasmid DNA. To test this system, we targeted P. polymyxa α-and β-amylase genes for disruption. Chloramphenicol or erythromycin resistance genes were inserted into the suicide plasmid pGEM7Z-f+ (Promega). To mediate homologous recombination and replacement of the targeted genes with the antibiotic resistance genes nucleotide sequences of the α-and β-amylase genes were cloned into the plasmid flanking the antibiotic resistance genes.

Conclusions

We have created a simple system for targeted gene deletion in P. polymyxa E681. We propose that P. polymyxa isogenic mutants could be developed using this system of marker exchange mutagenesis. α-and β-amylase genes provide a useful tool for direct recombinant screening in P. polymyxa.     

Gene doctoring: a method for recombineering in laboratory and pathogenic Escherichia coli strains

Background  

Homologous recombination mediated by the λ-Red genes is a common method for making chromosomal modifications in Escherichia coli. Several protocols have been developed that differ in the mechanisms by which DNA, carrying regions homologous to the chromosome, are delivered into the cell. A common technique is to electroporate linear DNA fragments into cells. Alternatively, DNA fragments are generated in vivo by digestion of a donor plasmid with a nuclease that does not cleave the host genome. In both cases the λ-Red gene products recombine homologous regions carried on the linear DNA fragments with the chromosome. We have successfully used both techniques to generate chromosomal mutations in E. coli K-12 strains. However, we have had limited success with these λ-Red based recombination techniques in pathogenic E. coli strains, which has led us to develop an enhanced protocol for recombineering in such strains.     

Effects of copper on the acute cortisol response and associated physiology in rainbow trout

The aim of this study was to determine the effects of chronic waterborne copper (Cu) exposure on the acute stress-induced cortisol response and associated physiological consequences in rainbow trout (Oncorhynchus mykiss). Trout were exposed to 30 μg Cu/L in moderately hard water (120 mg/L as CaCO(3)) for 40 days, following which time the acute cortisol response was examined with a series of stressors. At 40 days, a 65% increase in Cu was observed in the gill, but no accumulation was observed in the liver, brain or head kidney. Stressors such as air exposure or confinement did not elicit an increase in circulating cortisol levels for Cu-exposed fish, in contrast to controls. However, this inhibitory effect on the acute cortisol response appeared to have few implications on the ability of Cu-exposed fish to maintain ion and carbohydrate homeostasis. For example, plasma Na(+), Ca(2+) and glucose levels as well as hepatic glycogen levels were the same post-stress in control and Cu-exposed fish. Trout were also challenged with exposure to 50% seawater for 48 h, where Cu-exposed trout maintained plasma Na(+), glucose and hepatic glycogen levels. However, Cu-exposed fish experienced decreased plasma K(+) levels throughout the Cu exposure and stress tests. In conclusion, chronic Cu exposure resulted in the abolition of an acute cortisol response post-stress. There was no Cu accumulation in the hypothalamus-pituitary-interrenal axis (HPI axis) suggesting this was not a direct toxic effect of Cu on the cortisol regulatory pathway. However, the lack of an acute cortisol response in Cu-exposed fish did not impair the ability of the fish to maintain ion and carbohydrate homeostasis. This effect on cortisol may be a strategy to reduce costs during the chronic stress of Cu exposure, and not endocrine disruption as a result of toxic injury.     

ArrayD: A general purpose software for Microarray design

Background  

Microarray is a high-throughput technology to study expression of thousands of genes in parallel. A critical aspect of microarray production is the design aimed at space optimization while maximizing the number of gene probes and their replicates to be spotted.     

Acute exposure to offshore produced water has an effect on stress- and secondary stress responses in three-spined stickleback Gasterosteus aculeatus

Pollution is one of today's greatest problems, and the release of contaminants into the environment can cause adverse changes in vitally important biological pathways. In this study, we exposed three-spined stickleback Gasterosteus aculeatus to produced water (PW), i.e. wastewater from offshore petroleum production. PW contains substances such as alkylphenols (APs) and aromatic hydrocarbons (PAHs) known to induce toxicant stress and endocrine disruption in a variety of organisms. Following exposure to PW, a standardized confinement treatment was applied as a second stressor (PW-stress), testing how fish already under stress from the pollutant would respond to an additional stressor. The endpoint for analysis was a combination of blood levels of cortisol and glucose, in addition to transcribed levels of a set of genes related to toxicant stress, endocrine disruption and general stress. The findings of this study indicate that low doses of PW do not induce vitellogenin in immature female stickleback, but do cause an upregulation of cytochrome (CYP1A) and UDP-glucuronsyltransferase (UDP-GT), two biomarkers related to toxicant stress. However, when the second stressor was applied, both genes were downregulated, indicating that the confinement exposure had a suppressive effect on the expression of toxicant biomarkers (CYP1A and UDP-GT). Further, two of the stress related genes, heat shock protein 90 (HSP90) and stress-induced phosphoprotein (STIP), were upregulated in both PW- and PW-stress-treatment, but not in the water control confinement treatment, indicating that PW posed as a larger stress-factor than confinement for these genes. The confinement stressor caused an increased level of glucose in both control and PW-treated fish, indicating hyperglycemia, a commonly reported stress response in fish.     

The identification of apolipoprotein genes in rare minnow (Gobiocypris rarus) and their expression following perfluorooctanoic acid exposure

Apolipoproteins play important roles in lipid transport and uptake in vertebrates, and they are associated with pathogenesis of many cardiovascular diseases. However, the diverse apolipoproteins in individual fish species have not been extensively characterized. Partial cDNA sequences encoding ApoA-IV, ApoE, ApoM, ApoL, and ApoO, and full-length cDNA sequences encoding ApoA-I were cloned from rare minnow (Gobiocypris rarus). Sequence analysis showed that these genes, as well as fragments of other known apolipoprotein genes (ApoC-I, ApoC-II, ApoB) of rare minnow had a high similarity (91–96%) to their orthologues in the spotted barbel Hemibarbus mylodon (Teleostei:Cypriniformes). The expression of these nine genes and their possible upstream genes, PPARα, PPARγ, and HNF4α, were investigated in rare minnow after subacute exposure to perfluorooctanoic acid (PFOA) for 14 days. Results showed that the expression of mRNA for ApoA-I, ApoC-II, and ApoM was significantly downregulated in all PFOA-treated animals. Only fish receiving the highest dose of PFOA showed downregulation of the expression of ApoA-IV and ApoC-I, while fish treated with 10 mg PFOA/L showed upregulation of expression of ApoE. Expression of ApoB, ApoO, and ApoL was unchanged between control and treated groups. In addition, the expression of PPARα was increased in all dosed fish, while the mRNAs for PPARγ and HNF4α were significantly altered with 30 and 3 mg PFOA/L doses, respectively. Therefore, subacute exposure to PFOA resulted in alteration of expression of apolipoproteins and related genes. These changes in gene expression may further influence lipid metabolism or other physiological functions in fish.     

Analysis of ROH patterns in the Noriker horse breed reveals signatures of selection for coat color and body size

Overlapping runs of homozygosity (ROH islands) shared by the majority of a population are hypothesized to be the result of selection around a target locus. In this study we investigated the impact of selection for coat color within the Noriker horse on autozygosity and ROH patterns. We analyzed overlapping homozygous regions (ROH islands) for gene content in fragments shared by more than 50% of horses. Long‐term assortative mating of chestnut horses and the small effective population size of leopard spotted and tobiano horses resulted in higher mean genome‐wide ROH coverage (S_ROH) within the range of 237.4–284.2 Mb, whereas for bay, black and roan horses, where rotation mating is commonly applied, lower autozygosity (S_ROH from 176.4–180.0 Mb) was determined. We identified seven common ROH islands considering all Noriker horses from our dataset. Specific islands were documented for chestnut, leopard spotted, roan and bay horses. The ROH islands contained, among others, genes associated with body size (ZFAT, LASP1 and LCORL/NCAPG), coat color (MC1R in chestnut and the factor PATN1 in leopard spotted horses) and morphogenesis (HOXB cluster in all color strains except leopard spotted horses). This study demonstrates that within a closed population sharing the same founders and ancestors, selection on a single phenotypic trait, in this case coat color, can result in genetic fragmentation affecting levels of autozygosity and distribution of ROH islands and enclosed gene content.     

Development of a DNA macroarray for simultaneous detection of multiple foodborne pathogenic bacteria in fresh chicken meat

A DNA macroarray was developed to provide the ability to detect multiple foodborne pathogens in fresh chicken meat. Probes targeted to the 16S rRNA and genus- and species-specific genes, including fimY, ipaH, prfA, and uspA, were selected for the specific detection of Salmonella spp., Shigella spp., Listeria monocytogenes, and Escherichia coli, respectively. The combination of target gene amplification by PCR and a DNA macroarray in our system was able to distinguish all target bacteria from pure cultures with a detection sensitivity of 10⁵ c.f.u. ml^?1. The DNA macroarray was also applied to 10 fresh chicken meat samples. The assay validation demonstrated that by combining the enrichment steps for the target bacteria and the DNA macroarray, all 4 target bacteria could be detected simultaneously from the fresh chicken samples. The sensitivity of L. monocytogenes and Shigella boydii detection in the fresh chicken samples was at least 10 and 3 c.f.u. of the initial contamination in 25 g samples, respectively. The advantages of our developed protocol are high accuracy and time reduction when compared to conventional culture. The macroarray developed in our investigation was cost effective compared to modern oligonucleotide microarray techniques because there was no expensive equipment required for the detection of multiple foodborne pathogens.     

Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch,Perca fluviatilis

The effects of acute stress on immune status and its regulation by cortisol/corticosteroid receptors have received little attention in percids. To address that question, we investigated the physiological and immune responses of Eurasian perch, Perca fluviatilis to acute stress. We exposed immature perch to an 1-min exondation and measured at 1 h, 6 h, 24 h and 72 h post-stress: (1) stress-related parameters including plasma cortisol and glucose levels, (2) immune parameters in the plasma and in the spleen (complement, respiratory burst and lysozyme activity, total immunoglobulins; gene expression of lysozyme, complement unit 3, apolipoprotein A1 and 14 kDa, hepcidin and chemotaxin) (3) the corticosteroid receptors gene expression in the spleen after having cloned them. In addition, the in vitro effects of cortisol on the spleen immune parameters were also investigated.Plasma cortisol and glucose levels increased markedly 1 h post-stress and returned at basal levels after 24 h. P. fluviatilis mineralocorticoid receptor, but not glucocorticoid receptors, was significantly up-regulated both in vivo after the stress and in vitro by cortisol at a physiological concentration (100 ng/ml). The plasma immune parameters were not significantly affected by the stress. In contrast, spleno-somatic index, spleen lysozyme activity, lysozyme and hepcidin gene expression were depleted and total immunoglobulins increased along the whole time-course (1–72 h). But, these immune parameters were not regulated in vitro by cortisol at physiological or supra-physiological doses.Our results indicate that handling stress may affect spleen antibacterial defences without clear effects on circulating immune compounds and that the elevation of plasma cortisol after handling stress may not be related to the regulation of this splenic response.