Flavobacterium hungaricum sp. nov. a novel soil inhabitant, cellulolytic bacterium isolated from plough field.

A Gram-negative bacterial strain, named Kb82, was isolated from agricultural soil and a polyphasic approach was used for characterisation and to determine its taxonomic position. Based on 16S rRNA gene sequence analysis, the highest similarity was found with Flavobacterium artemisiae SYP-B1015 (98.2%). The highest ANI (83.3%) and dDDH (26.5%) values were found with Flavobacterium ginsenosidimutans THG 01 and Flavobacterium fluviale HYN0086T, respectively. The isolate is aerobic with rod-shaped cells, positive for catalase and negative for oxidase tests. The DNA G+C content is 34.7 mol%. The only isoprenoid quinone is menaquinone 6 (MK-6). The major fatty acids are iso-C15:0, summed feature 3 (C16:1 ω7c/C16:1 ω6c) and iso-C17:0 3OH. The major polar lipid is phosphatidylethanolamine. On the bases of phenotypic characteristics and analysis of 16S rRNA gene sequences, it is concluded that strain Kb82T represents a novel species in the Flavobacterium genus, for which the name Flavobacterium hungaricum sp. nov. is proposed. The type strain of the species is strain Kb82T (= LMG 31576T = NCAIM B.02635T).

Comparison Evaluation of the Biological Effects of Sterigmatocystin and Aflatoxin B1 Utilizing SOS-Chromotest and a Novel Zebrafish (Danio rerio) Embryo Microinjection Method.

Aflatoxin B1 (AFB1) is a potent mycotoxin and natural carcinogen. The primary producers of AFB1 are Aspergillus flavus and A. parasiticus. Sterigmatocystin (STC), another mycotoxin, shares its biosynthetic pathway with aflatoxins. While there are abundant data on the biological effects of AFB1, STC is not well characterised. According to published data, AFB1 is more harmful to biological systems than STC. It has been suggested that STC is about one-tenth as potent a mutagen as AFB1 as measured by the Ames test. In this research, the biological effects of S9 rat liver homogenate-activated and non-activated STC and AFB1 were compared using two different biomonitoring systems, SOS-Chromotest and a recently developed microinjection zebrafish embryo method. When comparing the treatments, activated STC caused the highest mortality and number of DNA strand breaks across all injected volumes. Based on the E. coli SOS-Chromotest, the two toxins exerted the same genotoxicities. Moreover, according to the newly developed zebrafish microinjection method, STC appeared more toxic than AFB1. The scarce information correlating AFB1 and STC toxicity suggests that AFB1 is a more potent genotoxin than STC. Our findings contradict this assumption and illustrate the need for more complex biomonitoring systems for mycotoxin risk assessment.

Sphingobacterium hungaricum sp. nov. a novel species on the borderline of the genus Sphingobacterium.

A Gram-reaction-negative bacterial strain, designated Kb22T, was isolated from agricultural soil and characterized using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, the strain shows highest similarity (94.39 %) to Sphingobacterium nematocida M-SX103T. The highest average nucleotide identity value (71.83 %) was found with Sphingobacterium composti T5-12T, and the highest amino acid identity value (66.65 %) was found with Sphingobacterium olei HAL-9T. Cells are aerobic, non-motile rods. The isolate was found to be positive for catalase and oxidase tests. The assembled genome of strain Kb22T has a total length of 4,06 Mb, the DNA G+C content is 38.1 mol%. The only isoprenoid quinone is menaquinone 7 (MK-7). The major fatty acids are iso-C15:0 (28.4%), summed feature 3 (C16:1 ω7c and/or iso-C15:0 2-OH) (25.7 %) and iso-C17:0 3-OH (19.7 %). Based on phenotypic characteristics and phylogenetic results, it is concluded that strain Kb22T is a member of the genus Sphingobacterium and represents a novel species for which the name Sphingobacterium hungaricum sp. nov. is proposed. The type strain of the species is strain Kb22T (=LMG 31574T=NCAIM B.02638T).

Sphingobacterium pedocola sp. nov. a novel halotolerant bacterium isolated from agricultural soil.

A Gram-reaction-negative halotolerant bacterial strain, designated Ka21T, was isolated from agricultural soil and characterised using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, highest similarity was found with Sphingobacterium alkalisoli Y3L14T (96.72%). Cells were observed to be aerobic, non-motile rods. The isolate was found to be able to grow between 0 and 10% of NaCl concentration. The assembled genome of strain Ka21T has a total length of 5.2 Mb with a G + C content of 41.0 mol%. According to the genome analysis, Ka21T encodes several glycoside hydrolases that may play a role in the degradation of accumulated plant biomass in the soil. Based on phenotypic characteristics and phylogenetic analysis, it is concluded that strain Ka21T represents a novel species in the Sphingobacterium genus for which the name Sphingobacterium pedocola sp. nov. is proposed. The type strain of the species is strain Ka21T (= LMG 31575T = NCAIM B.02636T).

Cellvibrio polysaccharolyticus sp. nov., a cellulolytic bacterium isolated from agricultural soil.

A novel Gram-reaction-negative bacterial strain, designated Ka43T, was isolated from agricultural soil and characterised using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, the strain shows highest similarity (97.1 %) to Cellvibrio diazotrophicus E50T. Cells of strain Ka43T are aerobic, motile, short rods. The major fatty acids are summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH), C18 : 1 ω7c and C16 : 0. The only isoprenoid quinone is Q-8. The polar lipid profile includes phosphatidylethanolamine, phosphatidylglycerol, four phospholipids, two lipids and an aminolipid. The assembled genome of strain Ka43T has a total length of 4.2 Mb and the DNA G+C content is 51.6 mol%. Based on phenotypic data, including chemotaxonomic characteristics and analysis of the 16S rRNA gene sequences, it was concluded that strain Ka43T represents a novel species in the genus Cellvibrio, for which the name Cellvibrio polysaccharolyticus sp. nov. is proposed. The type strain of the species is strain Ka43T (=LMG 31577T=NCAIM B.02637T).

Effects of Single and Repeated Oral Doses of Ochratoxin A on the Lipid Peroxidation and Antioxidant Defense Systems in Mouse Kidneys.

Ochratoxin-A (OTA) is a carcinogenic and nephrotoxic mycotoxin, which may cause health problems in humans and animals, and it is a contaminant in foods and feeds. The purpose of the present study is to evaluate the effect of oral OTA exposure on the antioxidant defense and lipid peroxidation in the kidney. In vivo administration of OTA in CD1, male mice (1 or 10 mg/kg body weight in a single oral dose for 24 h and repeated daily oral dose for 72 h or repeated daily oral dose of 0.5 mg/kg bodyweight for 21 days) resulted in a significant elevation of OTA levels in blood plasma. Some histopathological alterations, transcriptional changes in the glutathione system, and oxidative stress response-related genes were also found. In the renal cortex, the activity of the glutathione-system-related enzymes and certain metabolites of the lipid peroxidation (conjugated dienes, trienes, and thiobarbituric reactive substances) also changed.

Aflatoxin B1 and Zearalenone-Detoxifying Profile of Rhodococcus Type Strains.

Aflatoxin B1 (AFB1) and zearalenone (ZON) are dangerous mycotoxins due to their carcinogenicity or oestrogenicity. To alleviate negative effects on humans and animals, successful detoxification tools are needed. The application of microorganisms to biodegrade mycotoxins can be an effective way in food and feed industry enhancing food safety. Several Rhodococcus strains are effective in the degradation of aromatic mycotoxins and their application in mycotoxin biodetoxification processes is a promising field of biotechnology. In this study, we investigated the AFB1 and ZON detoxification ability of 42 type strains of Rhodococcus species. Samples were analysed by high-performance liquid chromatograph equipped with fluorescence detector for mycotoxin concentration and SOS-chromotest was used for monitoring remaining genotoxicity. Out of the 42 Rhodococcus strains, 18 could eliminate more than 90% of the applied AFB1 and the genotoxicity was ceased by 15 strains in 72 h (R. imtechensis JCM 13270T, R. erythropolis JCM 3201T, R. tukisamuensis JCM 11308T, R. rhodnii JCM 3203T, R. aerolatus JCM 19485T, R. enclensis DSM 45688T, R. lactis DSM 45625T, R. trifolii DSM 45580T, R. qingshengii DSM 45222T, R. artemisiae DSM 45380T, R. baikonurensis DSM 44587T, R. globerulus JCM 7472T, R. kroppenstedtii JCM 13011T, R. pyridinivorans JCM 10940T, R. corynebacterioides JCM 3376T). In case of ZON, only R. percolatus JCM 10087T was able to degrade more than 90% of the compound and to reduce the oestrogenicity with 70%.

Xylanibacillus composti gen. nov., sp. nov., isolated from compost.

A novel Gram-stain-positive bacterial strain, designated as K13T, was isolated from compost and characterized using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, the strain showed highest similarity (93.8 %) to Paenibacillus nanensis MX2-3T. Cells of strain K13T were aerobic, motile rods. The major fatty acids were anteiso C15 : 0 (34.4 %), iso C16 : 0 (17.3 %) and C16 : 0 (10.0 %). The major menaquinone was MK-7, the polar lipid profile included diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine and an aminophospholipid. The DNA G+C content was 52.3 %. Based on phenotypic, including chemotaxonomic characteristics and analysis of the 16S rRNA gene sequences, it was concluded that strain K13T represents a novel genus, for which the name Xylanibacillus gen. nov., sp. nov. is proposed. The type species of the genus is Xylanibacillus composti, the type strain of which is strain K13T (=DSM 29793T=NCAIM B.02605T).

Cloning, Expression and Biochemical Characterization of Endomannanases from Thermobifida Species Isolated from Different Niches.

Thermobifidas are thermotolerant, compost inhabiting actinomycetes which have complex polysaccharide hydrolyzing enzyme systems. The best characterized enzymes of these hydrolases are cellulases from T. fusca, while other important enzymes especially hemicellulases are not deeply explored. To fill this gap we cloned and investigated endomannanases from those reference strains of the Thermobifida genus, which have published data on other hydrolases (T. fusca TM51, T. alba CECT3323, T. cellulosilytica TB100T and T. halotolerans YIM90462T). Our phylogenetic analyses of 16S rDNA and endomannanase sequences revealed that T. alba CECT3323 is miss-classified; it belongs to the T. fusca species. The cloned and investigated endomannanases belong to the family of glycosyl hydrolases 5 (GH5), their size is around 50 kDa and they are modular enzymes. Their catalytic domains are extended by a C-terminal carbohydrate binding module (CBM) of type 2 with a 23-25 residues long interdomain linker region consisting of Pro, Thr and Glu/Asp rich repetitive tetrapeptide motifs. Their polypeptide chains exhibit high homology, interdomain sequence, which don't show homology to each other, but all of them are built up from 3-6 times repeated tetrapeptide motifs) (PTDP-Tc, TEEP-Tf, DPGT-Th). All of the heterologously expressed Man5A enzymes exhibited activity only on mannan. The pH optima of Man5A enzymes from T. halotolerans, T. cellulosilytica and T. fusca are slightly different (7.0, 7.5 and 8.0, respectively) while their temperature optima span within the range of 70-75°C. The three endomannanases exhibited very similar kinetic performances on LBG-mannan substrate: 0.9-1.7mM of KM and 80-120 1/sec of turnover number. We detected great variability in heat stability at 70°C, which was influenced by the presence of Ca2+. The investigated endomannanases might be important subjects for studying the structure/function relation behind the heat stability and for industrial applications to hemicellulose degradation.

A new ochratoxin A biodegradation strategy using Cupriavidus basilensis Or16 strain.

Ochratoxin-A (OTA) is a mycotoxin with possibly carcinogenic and nephrotoxic effects in humans and animals. OTA is often found as a contaminant in agricultural commodities. The aim of the present work was to evaluate OTA-degrading and detoxifying potential of Cupriavidus basilensis OR16 strain. In vivo administration of OTA in CD1 male mice (1 or 10 mg/kg body weight for 72 hours or 0.5 mg/kg body weight for 21 days) resulted in significant elevation of OTA levels in the blood, histopathological alterations- and transcriptional changes in OTA-dependent genes (annexinA2, clusterin, sulphotransferase and gadd45 and gadd153) in the renal cortex. These OTA-induced changes were not seen in animals that have been treated with culture supernatants in which OTA was incubated with Cupriavidus basilensis OR16 strain for 5 days. HPLC and ELISA methods identified ochratoxin α as the major metabolite of OTA in Cupriavidus basilensis OR16 cultures, which is not toxic in vivo. This study has demonstrated that Cupriavidus basilensis OR16 efficiently degrade OTA without producing toxic adventitious metabolites.

Application of a yeast estrogen reporter system for screening zearalenone degrading microbes.

The aim of this study was to screen microbes for their zearalenone degrading potential and to select microbes whose activities do not create toxic or endocrine disrupting metabolites. Bioluminescent bioreporters (Saccharomyces cerevisiae BLYES and BLYR) were successfully used to monitor toxin degradation; the results of zearalenone biodegradation experiments were confirmed by parallel chemical analysis (HPLC-FLD) and immunoanalytical (ELISA) tests. Using the BLYES/BLYR bioreporters, the most appropriate microbes (ones that produced minimal toxic products and products with lower estrogenic potential) could be selected. The most promising strains belong to Streptomyces and Rhodococcus genera. Our findings demonstrate the benefit of using biological tests beside the analytical method, since bioreporters were able to monitor the samples for toxicity and estrogenic potential even after substantial degradation. We conclude that the BLYES/BLYR bioreporter system is a cost effective, fast and reliable tool for screening zearalenone-degrading microbes.

A new zearalenone biodegradation strategy using non-pathogenic Rhodococcus pyridinivorans K408 strain.

Zearalenone (hereafter referred to as ZEA) is a nonsteroidal estrogenic mycotoxin produced by several Fusarium spp. on cereal grains. ZEA is one of the most hazardous natural endocrine disrupting chemicals (EDC) which induces hyper estrogenic responses in mammals. This can result in reproductive disorders in farm animals as well as in humans. Consequently, detoxification strategies for contaminated crops are crucial for food safety. In this study we have developed a bacterial based detoxification system using a non-pathogen Rhodococcus pyridinivorans K408 strain. Following 5 days treatment of ZEA with R. pyridinivorans K408 strain HPLC analyses showed an 87.21% ZEA-degradation efficiency of the bacterial enzyme systems. In another approach, the strain biotransformation ability has also been confirmed by a bioluminescent version of the yeast estrogen screening system (BLYES), which detected an 81.75% of biodegradability of ZEA, in a good agreement with the chemical analyses. Furthermore, the capacity of R. pyridinivorans to eliminate the estrogenic effects of ZEA was tested by using an immature uterotrophic assay. Prepubertal female rats were treated with vehicle (olive oil), 17ß-estradiol, ZEA (0.1-1-5-10 mg/kg body weight) and LB broth containing 500 mg/l ZEA that has already been incubated with or without Rhodococcus pyridinivorans K408 strain. Uterine weights were measured and the mRNA level changes relating to apelin, aquaporin 5, complement component 2, and calbindin-3 genes were measured by qRT-PCR. These genes represent the major pathways that are affected by estromimetic compounds. Zearalenone feeding significantly increased the uterus weight in a dose dependent manner and at the same time upregulated complement component 2 and calbindin-3 expression as well as decreased apelin and aquaporin 5 mRNA levels comparable to that seen in 17ß-estradiol exposed rats. In contrast, LB broth in which ZEA was incubated with Rhodococcus pyridinivorans K408 prior to the feeding did not display any estrogenic effect neither on uterine weight nor on the expression of estrogen-regulated genes. Consequently, the identification of Rhodococcus pyridinivorans K408 strain in ZEA biodegradation proved to be a very efficient biological tool that is able to eliminate the complete estrogenic effects of ZEA. It is also remarkable that this biotransformation pathway of ZEA did not result in any residual estrogenic effects.

Quantification of subfamily I.2.C catechol 2,3-dioxygenase mRNA transcripts in groundwater samples of an oxygen-limited BTEX-contaminated site.

Low dissolved oxygen concentration of subsurface environments is a limiting factor for microbial aromatic hydrocarbon degradation, and to date, there are only a limited number of available reports on functional genes and microbes that take part in the degradation of aromatic hydrocarbons under hypoxic conditions. Recent discoveries shed light on the prevalence of subfamily I.2.C catechol 2,3-dioxygenases in petroleum hydrocarbon contaminated hypoxic groundwaters, and their considerable environmental importance was suggested. Here, we report on a Hungarian aromatic hydrocarbon (methyl-substituted benzene derivatives, mostly xylenes) contaminated site where we investigated this presumption. Groundwater samples were taken from the center and the edge of the contaminant plume and beyond the plume. mRNA transcripts of subfamily I.2.C catechol 2,3-dioxygenases were detected in considerable amounts in the contaminated samples by qPCR analysis, while activity of subfamily I.2.A, which includes the largest group of extradiol dioxygenases described by culture-dependent studies and thought to be widely distributed in BTEX-contaminated environments, was not observed. Bacterial community structure analyses showed the predominance of genus Rhodoferax related species in the contaminated samples.

Olivibacter oleidegradans sp. nov., a hydrocarbon-degrading bacterium isolated from a biofilter clean-up facility on a hydrocarbon-contaminated site.

A novel hydrocarbon-degrading, Gram-negative, obligately aerobic, non-motile, non-sporulating, rod-shaped bacterium, designated strain TBF2/20.2(T), was isolated from a biofilter clean-up facility set up on a hydrocarbon-contaminated site in Hungary. It was characterized by using a polyphasic approach to determine its taxonomic position. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate is affiliated with the genus Olivibacter in the family Sphingobacteriaceae. It was found to be related most closely to Olivibacter ginsengisoli Gsoil 060(T) (93.3% 16S rRNA gene sequence similarity). Strain TBF2/20.2(T) grew at pH 6-9 (optimally at pH 6.5-7.0) and at 15-42 °C (optimally at 30-37 °C). The major fatty acids were iso-C(15:0) (39.4%), summed feature 3 (iso-C(15:0) 2-OH and/or C(16:1)ω7c; 26.0%), iso-C(17:0) 3-OH (14.5%) and C(16:0) (4.5%). The major menaquinone was MK-7 and the predominant polar lipid was phosphatidylethanolamine. The DNA G+C content of strain TBF2/20.2(T) was 41.2 mol%. Physiological and chemotaxonomic data further confirmed the distinctiveness of strain TBF2/20.2(T) from recognized members of the genus Olivibacter. Thus, strain TBF2/20.2(T) is considered to represent a novel species of the genus Olivibacter, for which the name Olivibacter oleidegradans sp. nov. is proposed. The type strain is TBF2/20.2(T) (=NCAIM B 02393(T) =CCM 7765(T)).

Chryseobacterium hungaricum sp. nov., isolated from hydrocarbon-contaminated soil.

The taxonomic position of a strain isolated from kerosene-contaminated soil in Hungary and formerly misidentified as Brevundimonas vesicularis was examined using a polyphasic approach. The isolate, designated CHB-20p(T), could be clearly assigned to the genus Chryseobacterium (family Flavobacteriaceae) on the basis of 16S rRNA gene sequence similarity. Strain CHB-20p(T), a moderate oil degrader, was a Gram-negative, aerobic, mesophilic microbe with a temperature optimum of 28-30 degrees C. Predominant fatty acids were iso-C(15 : 0), summed feature 3 (comprising C(16 : 1)omega7c and/or iso-C(15 : 0) 2-OH) and iso-C(17 : 0) 3-OH. Menaquinone-6 (MK-6) was the predominant respiratory quinone; MK-5 was present as a minor component. The almost complete 16S rRNA gene sequence of strain CHB-20p(T) shared 94-97 % similarity with sequences of the type strains of species of the genus Chryseobacterium. DNA-DNA relatedness between strain CHB-20p(T) and its closest relative, Chryseobacterium caeni, was lower than 46 %. Moreover, several diagnostic phenotypic properties distinguished strain CHB-20p(T) from C. caeni. On the basis of biochemical, chemotaxonomic and genotypic data, isolate CHB-20p(T) represents a novel species within the genus Chryseobacterium, Chryseobacterium hungaricum sp. nov.; the type strain is CHB-20p(T) (=NCAIM B2269(T)=DSM 19684(T)).