Combined use of <Emphasis Type="Italic">GAP</Emphasis> and <Emphasis Type="Italic">AOX1</Emphasis> promoters and optimization of culture conditions to enhance expression of <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> lipase

Rhizo mucor miehei lipase (RML) is an industrially important enzyme, but its application is limited due to its high cost. In this study, a series of measures such as codon optimization, propeptide addition, combined use of GAP and AOX1 promoters, and optimization of culture conditions were employed to increase the expression of RML. Three transformants of the constitutive-inducible combined Pichia pastoris strains were generated by transforming the pGAPZαA-rml vector into the pPIC9K-rml/GS115 strain, which resulted in high-expression yields of RML. Using the shake flask method, highest enzyme activity corresponding to 140 U/mL was observed in the strain 3-17, which was about sixfold higher than that of pPIC9K-rml/GS115 or pGAPZαA-rml/GS115. After optimization of culture conditions by response surface methodology, the lipolytic activity of strain 3-17 reached 175 U/mL in shake flasks. An increase in the copy number simultaneously with the synergistic effect provided by two promoters led to enhanced degree of protein expression.     

<Emphasis Type="Italic">In situ</Emphasis> fermentation dynamics during production of <Emphasis Type="Italic">gundruk</Emphasis> and <Emphasis Type="Italic">khalpi</Emphasis>, ethnic fermented vegetable products of the Himalayas

Gundruk is a fermented leafy vegetable and khalpi is a fermented cucumber product, prepared and consumed in the Himalayas. In situ fermentation dynamics during production of gundruk and khalpi was studied. Significant increase in population of lactic acid bacteria (LAB) was found during first few days of gundruk and khlapi fermentation, respectively. Gundruk fermentation was initiated by Lactobacillus brevis, Pediococcus pentosaceus and finally dominated by Lb. plantarum. Similarly in khalpi fermentation, heterofermentative LAB such as Leuconostoc fallax, Lb. brevis and P. pentosaceus initiated the fermentation and finally completed by Lb. plantarum. Attempts were made to produce gundruk and khalpi using mixed starter culture of LAB previously isolated from respective products. Both the products prepared under lab condition had scored higher sensory-rankings comparable to market products.     

Assessment of the antifungal activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Pediococcus</Emphasis> spp. for use as bioprotective cultures in dairy products

Fungi are commonly involved in dairy product spoilage and the use of bioprotective cultures can be a complementary approach to reduce food waste and economic losses. In this study, the antifungal activity of 89 Lactobacillus and 23 Pediococcus spp. isolates against three spoilage species, e.g., Yarrowia lipolytica, Rhodotorula mucilaginosa and Penicillium brevicompactum, was first evaluated in milk agar. None of the tested pediococci showed antifungal activity while 3, 23 and 43 lactobacilli isolates showed strong antifungal activity or total inhibition against Y. lipolytica, R. mucilaginosa and P. brevicompactum, respectively. Then, the three most promising strains, Lactobacillus paracasei SYR90, Lactobacillus plantarum O_VI9 and Lactobacillus rhamnosus BIO_III28 at initial concentrations of 10⁵ and 10⁷ CFU/ml were tested as bioprotective cultures against the same fungal targets in a yogurt model during a 5-week storage period at 10 °C. While limited effects were observed at 10⁵ CFU/ml inoculum level, L. paracasei SYR90 and L. rhamnosus BIO_III28 at 10⁷ CFU/ml respectively retarded the growth of R. mucilaginosa and P. brevicompactum as compared to a control without selected cultures. In contrast, growth of Y. lipolytica was only slightly affected. In conclusion, these selected strains may be good candidates for bioprotection of fermented dairy products.     

Clinical strains of <Emphasis Type="Italic">Lactobacillus</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> and protect <Emphasis Type="Italic">Galleria mellonella</Emphasis> against experimental candidiasis

Candida albicans is the most common human fungal pathogen and can grow as yeast or filaments, depending on the environmental conditions. The filamentous form is of particular interest because it can play a direct role in adherence and pathogenicity. Therefore, the purpose of this study was to evaluate the effects of three clinical strains of Lactobacillus on C. albicans filamentation as well as their probiotic potential in pathogen-host interactions via an experimental candidiasis model study in Galleria mellonella. We used the reference strain Candida albicans ATCC 18804 and three clinical strains of Lactobacillus: L. rhamnosus strain 5.2, L. paracasei strain 20.3, and L. fermentum strain 20.4. First, the capacity of C. albicans to form hyphae was tested in vitro through association with the Lactobacillus strains. After that, we verified the ability of these strains to attenuate experimental candidiasis in a Galleria mellonella model through a survival curve assay. Regarding the filamentation assay, a significant reduction in hyphae formation of up to 57% was observed when C. albicans was incubated in the presence of the Lactobacillus strains, compared to a control group composed of only C. albicans. In addition, when the larvae were pretreated with Lactobacillus spp. prior to C. albicans infection, the survival rate of G. mellonela increased in all experimental groups. We concluded that Lactobacillus influences the growth and expression C. albicans virulence factors, which may interfere with the pathogenicity of these microorganisms.     

<Emphasis Type="Italic">HMG1A</Emphasis> and <Emphasis Type="Italic">PPARG</Emphasis> are differently expressed in the liver of fat and lean broilers

The expression of nine functional candidates for QT abdominal fat weight and relative abdominal fat content was investigated by real-time polymerase chain reaction (PCR) in the liver, adipose tissue, colon, muscle, pituitary gland and brain of broilers. The high mobility group AT-hook 1 (HMG1A) gene was up-regulated in liver with a ratio of means of 2.90 (P?≤?0.01) in the «fatty» group (relative abdominal fat content 3.5?±?0.18%, abdominal fat weight 35.4?±?6.09 g) relative to the «lean» group (relative abdominal fat content 1.9?±?0.56%, abdominal fat weight 19.2?±?5.06 g). Expression of this gene was highly correlated with the relative abdominal fat content (0.70, P?≤?0.01) and abdominal fat weight (0.70, P?≤?0.01). The peroxisome proliferator-activated receptor gamma (PPARG) gene was also up-regulated in the liver with a ratio of means of 3.34 (P?≤?0.01) in the «fatty» group relative to the «lean» group. Correlation of its expression was significant with both the relative abdominal fat content (0.55, P?≤?0.05) and the abdominal fat weight (0.57, P?≤?0.01). These data suggest that the HMG1A and PPARG genes were candidate genes for abdominal fat deposition in chickens. Searching of rSNPs in regulatory regions of the HMG1A and PPARG genes could provide a tool for gene-assisted selection.     

Isolation and identification of <Emphasis Type="Italic">Salmonella</Emphasis> spp. from red foxes (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) and badgers (<Emphasis Type="Italic">Meles meles</Emphasis>) in northern Italy

Background

Salmonella spp. have been isolated from a wide range of wild animals. Opportunistic wild carnivores such as red foxes (Vulpes vulpes) and badgers (Meles meles) may act as environmental indicators or as potential sources of salmonellosis in humans. The present study characterizes Salmonella spp. isolated from the intestinal contents of hunted or dead red foxes (n?=?509) and badgers (n?=?17) in northern Italy.

Findings

Thirty-one strains of Salmonella belonging to 3 Salmonella enterica subspecies were isolated. Fourteen different serovars of S. enterica subsp. enterica were identified, among which were serovars often associated with human illness.

Conclusions

Wild opportunistic predators can influence the probability of infection of both domestic animals and humans through active shedding of the pathogen to the environment. The epidemiological role of wild carnivores in the spread of salmonellosis needs to be further studied.

     

In vitro virulence characteristics of rare serovars of <Emphasis Type="Italic">Salmonella enterica</Emphasis> isolated from sand lizards (<Emphasis Type="Italic">Lacerta agilis</Emphasis> L.)

The aim of this study was to estimate virulence potential of Salmonella enterica strains colonizing the gut of free-living sand lizards (Lacerta agilis L.). The strains belonged to three Salmonella serovars: Abony, Schleissheim, and Telhashomer. Adhesion and invasion abilities of the strains were determined in quantitative assays using the gentamicin protection method. Induction of apoptosis was assessed using HeLa cell monolayers. PCR assays were used for detection of 26 virulence genes localised within mobile elements: pathogenicity islands, virulence plasmids, and prophage sequences. In vitro studies revealed that all strains had adhesion and invasion abilities to human epithelial cells. The isolates were cytotoxic and induced apoptosis of the cells. The serovars differed in the number of virulence-associated genes: up to 18 genes were present in Salmonella Schleissheim, 17 in Salmonella Abony, whereas as few as six genes were found in Salmonella Telhashomer. Generally, Salmonella Abony and Salmonella Schleissheim did not differ much in gene content connected with the presence SPI-1 to -5. All of the strains lacked genes localised within bacteriophages and plasmids. The presence of virulence-associated genes and in vitro pathogenicity assays suggest that Salmonella sp. strains originating from autochthonous, free-living lizards can potentially infect and cause disease in humans.     

<Emphasis Type="Italic">Zingiber officinale</Emphasis> extends <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> life span in xenobiotic-induced oxidative stress conditions

Background

The possibility of dietary ginger to enhance oxidative stress resistance and to extend life span was studied on Drosophila melanogaster.

Methods

Oxidative stress was induced by a reducing agent dithiothreitol. Experimental groups of male D. melanogaster were cultured on media containing: 1) no additive; 2) dithiothreitol, added into the nutritional mixture to the final concentration of 10 mM; 3) 25 mg of ginger powder g^–1 of the nutritional mixture; and 4) 10 mM of dithiothreitol and 25 mg of ginger powder g^–1 of the nutritional mixture. The number of alive fruit flies was inspected daily, and mean life span was determined for each experimental group.

Results

The addition of dithiothreitol to D. melanogaster nutritional mixture was established to result in an increase in concentration of two markers of oxidative stress conditions (thiobarbituric acid reactive substances as products of lipid peroxidation and carbonylated proteins as products of protein oxidation) in fly tissues. It was followed by significant reduction of mean life span and maximum life span of the last 10% of flies. Plant preparation, being added simultaneously with dithiothreitol, significantly diminished the negative effects of this xenobiotic. In conditions of additional stress load induced by hydrogen peroxide or high temperature, survival of insects treated with dithiothreitol on the background of ginger powder was the highest.

Conclusions

Thus, the presented data give the evidence that ginger preparations can reduce oxidative stress outcomes and significantly increase the life expectancy of fruit flies in stress conditions.

     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Effect of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Tennozu-SU2 on <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium Infection in Human Enterocyte-Like HT-29-Luc Cells and BALB/c Mice

The probiotic properties and inhibitory effect on Salmonella Typhimurium adhesion on human enterocyte-like HT-29-Luc cells of three Lactobacillus plantarum strains isolated from fermented fish, beach sand and a coastal plant were determined. Compared with the type strain L. plantarum NBRC 15891^T, which was isolated from pickled cabbage, L. plantarum Tennozu-SU2 isolated from the acorn of a coastal tree showed high autoaggregation in de Man, Rogosa and Sharpe (MRS) broth and an antagonistic effect against S. Typhimurium in brain heart infusion (BHI) broth. Furthermore, heat-killed L. plantarum Tennozu-SU2 cells inhibited S. Typhimurium adhesion on HT-29-Luc cells. Both live and heat-killed L. plantarum Tennozu-SU2 cells showed an inhibitory effect on gut colonisation in BALB/c mice, as assessed by viable Salmonella count in faecal samples and by invasion into liver and spleen tissues. The properties shown in this study suggest that L. plantarum Tennozu-SU2 is useful as a starter and probiotic bacteria in functional food material.     

Ectomycorrhizae of <Emphasis Type="Italic">Tuber huidongense</Emphasis> and <Emphasis Type="Italic">T. liyuanum</Emphasis> with <Emphasis Type="Italic">Castanea mollissima</Emphasis> and <Emphasis Type="Italic">Pinus armandii</Emphasis>

Tuber huidongense and T. liyuanum are common commercial white truffles in China that belong to the Rufum and Puberulum groups of the genus Tuber, respectively. Their mycorrhizae were successfully synthesized with two native trees—Castanea mollissima and Pinus armandii—under greenhouse conditions. The identities of the mycorrhizae were confirmed through internal transcribed spacer (ITS) sequence analyses, and their morphological characteristics were described. All of the obtained mycorrhizae have an interlocking pseudoparenchymatous mantle, which is a typical feature of truffle mycorrhizae. The mycorrhizae of T. huidongense on the two trees have hyaline branched emanating hyphae, similar to the documented mycorrhizae of the Rufum group. The unramified, spiky, and hyaline cystidia on the mycorrhizae of T. liyuanum with both C. mollissima and P. armandii further confirmed that this characteristic is constant for the mycorrhizae of the Puberulum group. The successful mycorrhizal syntheses on the two nut-producing trees will be of economic importance in the cultivation of the two truffles.     

<Emphasis Type="Italic">Diplazium</Emphasis> hybrids involving <Emphasis Type="Italic">D. plantaginifolium</Emphasis> and <Emphasis Type="Italic">D</Emphasis>. <Emphasis Type="Italic">ternatum</Emphasis> from Mexico and Central America

Here we evaluate the origins and relationships of Mexican and Central American Diplazium hybrids derived from crosses involving either D. plantaginifolium or D. ternatum. Based on study of live plants and herbarium specimens, we distinguish D. ×verapax from the similar D. riedelianum and present evidence that the former is a sterile hybrid derived from a cross between D. plantaginifolium and D. werckleanum. We also describe new hybrids, D. ×torresianum and D. ×subternatum from Mexico and northern Central America. Both involve D. ternatum as one parent. Diplazium. cristatum is the other putative parent of D. ×torresianum, and D. plantaginifolium is the second parent of D. ×subternatum. We also designate lectotypes for D. cordovense and D. dissimile.     

<Emphasis Type="Italic">Sporidiobolus johnsonii</Emphasis> and <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> revisited

The relationship between Sporidiobolus johnsonii and S. salmonicolor was investigated using rDNA sequence data. Two statistically well-supported clades were obtained. One clade included the type strain of S. johnsonii and the other included the type strain of S. salmonicolor. However, some mating strains of S. salmonicolor were found in the S. johnsonii group. These strains belonged to mating type A2 and were sexually compatible with mating type A1 strains from the S. salmonicolor group. DNA–DNA reassociation values were high within each clade and moderate between the two clades. In the re-investigation of teliospore germination, we observed that the basidia of S. salmonicolor were two-celled. In S. johnsonii, basidia were not formed and teliospore germination resulted in direct formation of yeast cells. We hypothesize that the S. johnsonii clade is becoming genetically isolated from the S. salmonicolor group and that a speciation process is presently going on. We suspect that the observed sexual compatibility between strains of the S. johnsonii and S. salmonicolor groups and the possible genetic flow between the two species has little biological relevance because distinct phenotypes have been fixed in the two taxa and intermediate (hybrid) sequences for LSU and ITS rDNAs have not been detected.     

Dietary Flexibility and Feeding Strategies of <Emphasis Type="Italic">Eulemur</Emphasis>: A Comparison with <Emphasis Type="Italic">Propithecus</Emphasis>

Despite the great variety of habitats in Madagascar, Eulemur has successfully populated most forested habitats on the island. Although the high dietary flexibility of Eulemur is often credited as one of the drivers of its evolutionary success, other behavioral evidence suggests a limited capacity for dietary switching. To shed light on the feeding strategies of Eulemur, we compared the dietary flexibility between populations of this genus with that of another widespread lemur taxon, Propithecus. We hypothesized that Eulemur would show greater dietary flexibility than Propithecus, which has a digestive system specialized for folivory, and that Eulemur living in dry forests would switch its diet from fruit to other food seasonally. To examine these hypotheses, we performed a phylogenetic least-squares analysis on 10 populations of Eulemur and 7 of Propithecus to assess the contribution of environmental variables and body mass on their dietary flexibility while controlling for phylogenetic relatedness. Eulemur relied heavily on fruit and did not show large variations in primary food over the year. Propithecus consumed leaves and fruits equally and exhibited considerable flexibility across seasons. Therefore, in contrast to our predictions, the anatomical specialization for fiber digestion heightens dietary flexibility in Propithecus. At the intrageneric level, we found similar ecogeographic variation; populations of both genera with heavier body mass consumed more fruit. As we predicted, Eulemur in drier habitats switched the diet from fruit to alternative food more frequently. To compensate for low dietary flexibility, Eulemur mostly adopts a power-feeding strategy by which it increases energy expenditure to acquire patchily distributed fruit resources.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

Assessing the impacts of temperature and storage on <Emphasis Type="Italic">Escherichia coli</Emphasis>, <Emphasis Type="Italic">Salmonella</Emphasis>, and <Emphasis Type="Italic">L. monocytogenes</Emphasis> decay in dairy manure

Elevated levels of animal waste-borne pathogen in ambient water is a serious human health issue. Mitigating influx of pathogens from animal waste such as dairy manure to soil and water requires improving our existing knowledge of pathogen reductions in dairy manure treatment methods. This study was conducted to enhance the  understanding of human pathogen decay in liquid dairy manure in anaerobic (AN) and limited aerobic (LA) storage conditions. The decay of three pathogens (Escherichia coli, Salmonella spp., and Listeria monocytogenes) was assessed in bench-scale batch reactors fed with liquid slurry. A series of temperatures (30, 35, 42, and 50 °C) conditions were tested to determine the impacts of temperature on Escherichia coli, Salmonella, and L. monocytogenes decay in AN and LA conditions. Results showed prolonged survival of E. coli compared to Salmonella and L. monocytogenes in both LA and AN environments. Variations in survival among pathogens with temperature and environmental conditions (i.e., LA and AN) indicated the necessity of developing improved dairy manure waste treatment methods for controlling animal waste-borne pathogens. The results of this study will help in improving the current understanding of human pathogen decay in dairy manure for making informed decisions of animal manure treatment by stakeholders.     

Horizontal transfer of the C-termini of <Emphasis Type="Italic">tccC</Emphasis> genes in <Emphasis Type="Italic">Photorhabdus</Emphasis> and <Emphasis Type="Italic">Xenorhabdus</Emphasis>

The high molecular weight insecticidal toxin complexes (Tcs), including four toxin-complex loci (tca, tcb, tcc and tcd), were first identified in Photorhabdus luminescens W14. Each member of tca, tcb or tcc is required for oral toxicity of Tcs. However, the sequence sources of the C-termini of tccC3, tccC4, tccC6 and tccC7 are unknown. Here, we performed a whole genome survey to identify the orthologs of Tc genes, and found 165 such genes in 14 bacterial genomes, including 40 genes homologous to tccC1-7 in P. luminescens TT01. The sequence sources of the C-termini of tccC2-6 were determined by sequence analysis. Further phylogenetic investigations suggested that the C-termini of 6 tccC genes experienced horizontal gene transfer events.