<Emphasis Type="Italic">Hymenobacter daeguensis</Emphasis> sp. nov. isolated from river water

A Gram-stain-negative, non-motile, non-spore-forming, rod-shaped, aerobic bacterial strain, designated 16F3Y-2^T, was isolated from the Han River, South Korea, and was characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 16F3Y-2^T belonged to the family Cytophagaceae in the phylum Bacteroidetes and was most closely related to ‘Hymenobacter terrae’ DG7A (98.01%), H. soli PB17^T (97.26%), H. glaciei VUG-A130^T (96.78%), H. antarcticus VUG-A42aa^T (96.72%), H. ruber PB156^T (96.61%), and H. saemangeumensis GSR0100^T (95.77%). The G+C content of the genomic DNA of strain 16F3Y-2^T was 62.9 mol%. The isolate contained MK-7 as the predominant respiratory quinone, and summed feature 3 (C_16:1 ω7c/C_16:1 ω6c; 35.5%), C_15:0 iso (16.9%), C_16:1 ω5c (10.9%), and C_15:0 anteiso (9.9%) as major fatty acids. The major polar lipid was phosphatidylethanolamine. Phenotypic and chemotaxonomic data supported the affiliation of strain 16F3Y-2^T with the genus Hymenobacter. However, strain 16F3Y-2^T exhibited relatively low levels of DNA-DNA relatedness with ‘H. terrae’ KCTC 32554 (44.1%) and H. soli KCTC 12607^T (24.3%), clearly indicating that the isolate constitutes a new genospecies. Strain 16F3Y-2^T could be differentiated from its phylogenetic neighbors on the basis of several phenotypic, genotypic, and chemotaxonomic features. Therefore, strain 16F3Y-2^T represents a novel species in the genus Hymenobacter, for which the name Hymenobacter daeguensis sp. nov. is proposed. The type strain is 16F3Y-2^T (=KCTC 52537^T =JCM 31654^T).     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

CAT and MDH improve the germination and alleviate the oxidative stress of cryopreserved <Emphasis Type="Italic">Paeonia</Emphasis> and <Emphasis Type="Italic">Magnolia</Emphasis> pollen

Researches have reported that reactive oxygen species (ROS)-induced oxidative stress plays an important role in cell cryodamage during cryopreservation. In the current study, pollen from Magnolia denudata and Paeonia lactiflora ‘Zi Feng Chao Yang’ was cryopreserved and incubated with exogenous catalase (CAT) and malate dehydrogenase (MDH) immediately after thawing. The effect of CAT and MDH on the germination of cryopreserved pollen was measured. Based on that, the ROS level, lipid peroxidation and antioxidants activities in fresh pollen, cryopreserved pollen added with or without CAT or MDH were determined to investigate their relationship with oxidative stress. Pollen from Magnolia and Paeonia showed a significant loss of germination, but marked increase of ROS and malondialdehyde (MDA) production after cryostorage. Antioxidant profiles in them were also enhanced. CAT and MDH addition increased the post-LN pollen germination of Magnolia and Paeonia significantly. Their germination rate achieved the highest with 100 IU ml^?1 MDH and 400 IU ml^?1 CAT application, respectively. Compared to their untreated controls, ROS and MDA accumulation reduced significantly in cryopreserved Magnolia pollen treated with 100 IU ml^?1 MDH, while superoxide dismutase (SOD) activity improved markedly. In the case of Paeonia, significantly lower level of ROS and MDA, but higher activity of CAT and SOD were observed in cryopreserved pollen treated with 400 IU ml^?1 CAT. In conclusion, pollen deterioration after cryopreservation is associated with ROS-induced oxidative stress. Exogenous CAT and MDH can reduce the oxidative damage through the activity stimulation of antioxidant enzymes, and play a protective role in the pollen during cryopreservation.     

Responses of the antioxidative and biotransformation enzymes in the aquatic fungus <Emphasis Type="Italic">Mucor hiemalis</Emphasis> exposed to cyanotoxins

Objectives

To investigate antioxidative and biotransformation enzyme responses in Mucor hiemalis towards cyanotoxins considering its use in mycoremediation applications.

Results

Catalase (CAT), glutathione reductase (GR), and glutathione peroxidase (GPx) in M. hiemalis maintained their activities at all tested microcystin-LR (MC-LR) exposure concentrations. Cytosolic glutathione S-transferase (GST) activity decreased with exposure to 100 µg MC-LR l^?1 while microsomal GST remained constant. Cylindrospermopsin (CYN) at 100 µg l^?1 led to an increase in CAT activity and inhibition of GR, as well as to a concentration-dependent GPx inhibition. Microsomal GST was inhibited at all concentrations tested. β-N-methylamino-l-alanine (BMAA) inhibited GR activity in a concentration-dependent manner, however, CAT, GPx, and GST remained unaffected.

Conclusions

M. hiemalis showed enhanced oxidative stress tolerance and intact biotransformation enzyme activity towards MC-LR and BMAA in comparison to CYN, confirming its applicability in bioreactor technology in terms of viability and survival in their presence.

     

Metabolic compensation in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> catalase-deficient mutants

Multigenic families are widely represented in the genomes of higher plants, and are required for the reliability of cellular functions. Damage of individual genes can be compensated by diverse metabolic alterations, but the exact mechanisms of such compensations still remain not fully understood. Here we present novel data regarding the mechanisms of metabolic compensation in photorespiratory knock-out mutants cat2, cat3 and cat2cat3 of Arabidopsis thaliana, which are lacking activity of catalase isoforms CAT2 and CAT3. It was found that cultivation of the mutants under low light at optimal or increased temperature did not result in any morphological, physiological or biochemical signs of oxidative stress. Each of the mutant lines shows specific features of the compensatory mechanisms. Increased activity of CAT3 isoenzyme was found in the cat2 mutant, whereas cat3 and cat2cat3 demonstrate induction of CAT1, an isoform normally absent in young leaves, as well as activation of peroxidases, namely APX and POD. Comparison of these results and earlier published data revealed that the lack of CAT2 and CAT3 isoforms is compensated by preferential activation of non-enzymatic and enzymatic protection mechanisms, respectively.     

Deletion of <Emphasis Type="Italic">ldh</Emphasis>A and <Emphasis Type="Italic">ald</Emphasis>H genes in <Emphasis Type="Italic">Klebsiella</Emphasis><Emphasis Type="Italic">pneumoniae</Emphasis> to enhance 1,3-propanediol production

Objectives

To improve 1,3-propanediol (1,3-PD) production and reduce byproduct concentration during the fermentation of Klebsiella pneumonia.

Results

Klebsiella. pneumonia 2-1ΔldhA, K. pneumonia 2-1ΔaldH and K. pneumonia 2-1ΔldhAΔaldH mutant strains were obtained through deletion of the ldhA gene encoding lactate dehydrogenase required for lactate synthesis and the aldH gene encoding acetaldehyde dehydrogenase involved in the synthesis of ethanol. After fed-batch fermentation, the production of 1,3-PD from glycerol was enhanced and the concentrations of byproducts were reduced compared with the original strain K. pneumonia 2-1. The maximum yields of 1,3-PD were 85.7, 82.5 and 87.5 g/l in the respective mutant strains.

Conclusion

Deletion of either aldH or ldhA promoted 1,3-PD production in K. pneumonia.

     

A correlation between host-mediated expression of parasite genes as tandem inverted repeats and abrogation of development of female <Emphasis Type="Italic">Heterodera glycines</Emphasis> cyst formation during infection of <Emphasis Type="Italic">Glycine max</Emphasis>

Host-mediated (hm) expression of parasite genes as tandem inverted repeats was investigated as a means to abrogate the formation of mature Heterodera glycines (soybean cyst nematode) female cysts during its infection of Glycine max (soybean). A Gateway^®-compatible hm plant transformation system was developed specifically for these experiments in G. max. Three steps then were taken to identify H. glycines candidate genes. First, a pool of 150 highly conserved H. glycines homologs of genes having lethal mutant phenotypes or phenocopies from the free living nematode Caenorhabditis elegans were identified. Second, annotation of those 150 genes on the Affymetrix^® soybean GeneChip^® allowed for the identification of a subset of 131 genes whose expression could be monitored during the parasitic phase of the H. glycines life cycle. Third, a microarray analyses identified a core set of 32 genes with induced expression (>2.0-fold, log base 2) during the parasitic stages of infection. H. glycines homologs of small ribosomal protein 3a and 4 (Hg-rps-3a [accession number CB379877] and Hg-rps-4 [accession number CB278739]), synaptobrevin (Hg-snb-1 [accession number BF014436]) and a spliceosomal SR protein (Hg-spk-1 [accession number BI451523.1]) were tested for functionality in hm expression studies. Effects on H. glycines development were observed 8 days after infection. Experiments demonstrated that 81–93% fewer females developed on transgenic roots containing the genes engineered as tandem inverted repeats. The effect resembles RNA interference. The methodology has been used here as an alternative approach to engineer resistance to H. glycines.     

<Emphasis Type="Italic">Hymenobacter terrigena</Emphasis> sp. nov., isolated from soil

A Gram-stain-negative, non-motile, non-spore-forming, rodshaped, aerobic bacterial strain, designated S1-2-2-5^T, was isolated from the Jeollabuk-do province, Republic of Korea, and was characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain S1-2-2-5^T belonged to the family Cytophagaceae in phylum Bacteroidetes, and was most closely related to Hymenobacter terrae DG7A^T (98.2%), Hymenobacter rubidus DG7B^T (98.0%), Hymenobacter soli PB17^T (97.7%), Hymenobacter daeguensis 16F3Y-2^T (97.2%) and Hymenobacter saemangeumensis GSR0100^T (97.0%). The G + C content of the genomic DNA of strain S1-2-2-5^T was 59.4 mol%. The detection of menaquinone MK-7 as the predominant respiratory quinone, a fatty acid profile with summed feature 3 (C_16:1ω7c/C_16:1ω6c; 32.0%), C_15:0 iso (19.0%), and C_15:0 anteiso (15.0%) as the major components, and a polar lipid profile with phosphatidylethanolamine as the major component supported the affiliation of strain S1-2-2-5^T to the genus Hymenobacter. The DNA-DNA relatedness between strain S1-2-2-5^T and H. terrae KCTC 32554^T, H. rubidus KCTC 32553^T, H. soli KCTC 12607^T, H. daeguensis KCTC 52537^T, and H. saemangeumensis KACC 16452^T were 49.5, 48.2, 34.1, 28.1, and 31.8% respectively, clearly showing that the isolate is not related to them at the species level. Strain S1-2-2-5^T could be clearly differentiated from its closest neighbors on the basis of its phenotypic, genotypic and chemotaxonomic features. Therefore, strain S1-2-2-5^T represents a novel species of the genus Hymenobacter, for which the name Hymenobacter terrigena sp. nov. is proposed. The type strain is S1-2-2-5^T (= KCTC 52737^T = JCM 32195^T).     

<Emphasis Type="Italic">Candida krusei</Emphasis> isolated from fruit juices ultrafiltration membranes promotes colonization of <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 and <Emphasis Type="Italic">Salmonella enterica</Emphasis> on stainless steel surfaces

To clarify the interactions between a common food spoilage yeast and two pathogenic bacteria involved in outbreaks associated with fruit juices, the present paper studies the effect of the interplay of Candida krusei, collected from UF membranes, with Escherichia coli O157:H7 and Salmonella enterica in the overall process of adhesion and colonization of abiotic surfaces. Two different cases were tested: a) co-adhesion by pathogenic bacteria and yeasts, and b) incorporation of bacteria to pre-adhered C. krusei cells. Cultures were made on stainless steel at 25°C using apple juice as culture medium. After 24 h of co-adhesion with C. krusei, both E. coli O157:H7 and S. enterica increased their counts 1.05 and 1.11 log CFU cm², respectively. Similar increases were obtained when incorporating bacteria to pre-adhered cells of Candida. Nevertheless C. krusei counts decreased in both experimental conditions, in a) 0.40 log CFU cm² and 0.55 log CFU cm² when exposed to E. coli O157:H7 and S. enterica and in b) 0.18 and 0.68 log CFU cm², respectively. This suggests that C. krusei, E. coli O157:H7, and S. enterica have a complex relationship involving physical and chemical interactions on food contact surfaces. This study supports the possibility that pathogen interactions with members of spoilage microbiota, such as C. krusei, might play an important role for the survival and dissemination of E. coli O157:H7 and Salmonella enterica in food-processing environments. Based on the data obtained from the present study, much more attention should be given to prevent the contamination of these pathogens in acidic drinks.     

<Emphasis Type="Italic">Nonomuraea rhizosphaerae</Emphasis> sp. nov., an actinomycete isolated from the rhizosphere soil of a rubber tree (<Emphasis Type="Italic">Hevea brasiliensis</Emphasis> Muell. Arg)

A novel actinomycete, designated strain NEAU-mq18^T, was isolated from the rhizosphere soil of a rubber tree (Hevea brasiliensis Muell. Arg) collected from Xianglu Mountain in Heilongjiang Province, northeast China, and subjected to a polyphasic taxonomic study. The 16S rRNA gene sequence analysis showed that the isolate belongs to the genus Nonomuraea with high sequence similarity to Nonomuraea guangzhouensis NEAU-ZJ3^T (98.5%). Phylogenetic analysis based on 16S rRNA gene sequences indicated that the strain clusters phylogenetically with N. guangzhouensis NEAU-ZJ3^T and Nonomuraea glycinis NEAU-BB2C19^T. Moreover, key chemotaxonomic properties including the major menaquinones, fatty acid composition and phospholipid profile also confirmed the affiliation of the strain to the genus Nonomuraea. However, some physiological, morphological and biochemical properties, and low DNA–DNA relatedness values, enabled the strain to be differentiated from closely related species of the genus Nonomuraea. Thus, strain NEAU-mq18^T is concluded to represent a novel species of the genus Nonomuraea, for which the name Nonomuraea rhizosphaerae sp. nov. is proposed. The type strain is NEAU-mq18^T (=CGMCC 4.7431^T=DSM 105761^T).     

Performance evaluation of a rapid whole-blood immunoassay for the detection of IgG antibodies against <Emphasis Type="Italic">Helicobacter pylori</Emphasis> in daily clinical practice

Background

A growing number of rapid Helicobacter pylori antibody tests are commercially available now, however, some of these tests are often used without sufficient evaluation. The aim of this study was to evaluate the performance of a commercially available rapid whole-blood immunoassay (gabControl^® H. pylori; gabmed GmbH, Köln, Germany), for the qualitative detection of IgG antibodies against H. pylori with the ¹³C-urea breath test (¹³C-UBT) serving as a reference method.

Methods

A total of 108 consecutive outpatients, who were referred for ¹³C-UBT by general practitioners and specialists, were also tested for H. pylori infection by the gabControl^® H. pylori immunoassay. The clinical performance of this rapid whole-blood test was evaluated by determining the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) compared to the ¹³C-UBT. The agreement between the two tests was calculated using Cohen’s Kappa (κ) with 95 % confidence intervals (CI).

Results

The agreement between the gabControl^® H. pylori assay and the ¹³C-UBT was 0.62 [95 % confidence intervals (CIs) 0.47–0.76; P < 0.001]. With the ¹³C-UBT serving as the non-invasive gold standard method of H. pylori diagnosis, the gabControl^® H. pylori assay demonstrated a sensitivity and specificity of 91.4 and 76.7 %, respectively, with a PPV of 65.3 % and a NPV of 94.9 %. Seventeen (15.7 %) individuals with a positive H. pylori anamnesis showed a negative ¹³C-UBT and were typed positive by the gabControl^® H. pylori assay. Of these, 13 (76.5 %) and 3 individuals (17.6 %) had completed one and two eradication therapies, respectively.

Conclusions

The gabControl^® H. pylori immunoassay is a rapid and easy to use first line screening tool for H. pylori IgG antibody detection in daily clinical practice. However, this assay should not be used for confirmation of the successful H. pylori eradication after antibiotic treatment.