István Szabó

Investigation of catechol 2,3-dioxygenase and 16S rRNA gene diversity in hypoxic, petroleum hydrocarbon contaminated groundwater.

Detection of catechol 2,3-dioxygenase genes in aromatic hydrocarbon contaminated environments gives the opportunity to measure the diversity of bacteria involved in the degradation of the contaminants under aerobic conditions. In this study, we investigated the diversity and distribution of Comamonadaceae family (Betaproteobacteria) related catechol 2,3-dioxygenase genes, which belong to the I.2.C subfamily of extradiol dioxygenase genes. These catabolic genes encode enzymes supposed to function under hypoxic conditions as well, and may play a notable role in BTEX degradation in oxygen limited environments. Therefore, their diversity was analyzed in oxygen limited, petroleum hydrocarbon contaminated groundwater by terminal restriction fragment length polymorphism and cloning. Subfamily I.2.C related catechol 2,3-dioxygenase genes were detected in every investigated groundwater sample and a dynamic change was observed in the case of the structure of C23O gene possessing bacterial communities. To link the metabolic capability to the microbial structure, 16S rRNA gene-based clone libraries were generated and it was concluded that Betaproteobacteria were abundant in the bacterial communities of the contaminated samples. These results support the opinion that Betaproteobacteria may play a significant role in BTEX degradation under hypoxic conditions.

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.

The Occurrence of Multidrug-Resistant Pseudomonas aeruginosa on Hydrocarbon-Contaminated Sites

The main aim of this paper was the comprehensive estimation of the occurrence rate and the antibiotic-resistance conditions of opportunistic pathogen Pseudomonas aeruginosa in hydrocarbon-contaminated environments. From 2002 to 2007, 26 hydrocarbon-contaminated sites of Hungary were screened for the detection of environmental isolates. Altogether, 156 samples were collected and examined for the determination of appearance, representative cell counts, and antibiotic-resistance features of P. aeruginosa. The detected levels of minimal inhibitory concentrations of ten different drugs against 36 environmental strains were compared to the results of a widely used reference strain ATCC 27853 and four other clinical isolates of P. aeruginosa. Based on our long-term experiment, it can be established that species P. aeruginosa was detectable in case of 61.5% of the investigated hydrocarbon-contaminated sites and 35.2% of the examined samples that shows its widespread occurrence in polluted soil–groundwater systems. In the course of the antibiotic-resistance assay, our results determined that 11 of the examined 36 environmental strains had multiple drug-resistance against several clinically effective antimicrobial classes: cephalosporins, wide spectrum penicillins, carbapenems, fluoroquinolones, and aminoglycosides. The fact that these multiresistant strains were isolated from 8 different hydrocarbon-contaminated sites, mainly from outskirts, confirms that multiple drug-resistance of P. aeruginosa is widespread not only in clinical, but also in natural surroundings as well.

Germline VHL gene mutations in Hungarian families with von Hippel-Lindau disease and patients with apparently sporadic unilateral pheochromocytomas

OBJECTIVE Von Hippel-Lindau (VHL) disease is a hereditary tumor syndrome caused by mutations or deletions of the VHL tumor-suppressor gene. Germline VHL gene alterations may be also present in patients with apparently sporadic pheochromocytoma (ASP), although a wide variation in mutation frequencies has been reported in different patient cohorts. DESIGN Herein, we report the analysis of the VHL gene in Hungarian families with VHL disease and in those with ASP. METHODS Seven families (35 members) with VHL disease and 37 unrelated patients with unilateral ASP were analyzed. Patients were clinically evaluated and the VHL gene was analyzed using direct sequencing, multiplex ligation-dependent probe amplification, and real-time PCR with SYBR Green chemistry. RESULTS Disease-causing genetic abnormalities were identified in each of the seven VHL families and in 3 out of the 37 patients with ASP (one nonsense and six missense mutations, two large gene deletions and one novel 2 bp deletion). Large gene deletions and other genetic alterations resulting in truncated VHL protein were found only in families with VHL type 1, whereas missense mutations were associated mainly, although not exclusively, with VHL type 2B and type 2C. CONCLUSIONS The spectrum of VHL gene abnormalities in the Hungarian population is similar to that observed in Western, Japanese, or Chinese VHL kindreds. The presence of VHL gene mutations in 3 out of the 37 patients with ASP suggests that genetic testing is useful not only in patients with VHL disease but also in those with ASP.

De Novo Genome Project of Cupriavidus basilensis OR16

Here we report on the complete genome sequence of Cupriavidus basilensis OR16 NCAIM BO2487. The genome of strain OR16 contains 7,534 putative coding sequences, including a large set of xenobiotics-degrading genes and a unique glucose dehydrogenase gene that is absent from other Cupriavidus genomes.

One-year monitoring of meta-cleavage dioxygenase gene expression and microbial community dynamics reveals the relevance of subfamily I.2.C extradiol dioxygenases in hypoxic, BTEX-contaminated groundwater.

Aromatic hydrocarbons including benzene, toluene, ethyl-benzene, and xylene (BTEX) are frequent contaminants of groundwater, the major drinking water resource. Bioremediation is the only sustainable process to clean up these environments. Microbial degradation of BTEX compounds occurs rapidly under aerobic conditions but, in subsurface environments, the availability of oxygen is commonly restricted. Even so, the microaerobic degradation of aromatic compounds is still poorly understood. Hence, the dynamics of a bacterial community and the expression of meta-cleavage dioxygenase genes, with particular emphasis on subfamily I.2.C extradiol dioxygenase genes, were assessed over a 13-month period in a hypoxic, aromatic hydrocarbon-contaminated shallow groundwater by using sequence-aided terminal-restriction fragment length polymorphism (T-RFLP) and single-nucleotide primer extension (SNuPE), respectively. The bacterial 16S rRNA fingerprinting revealed the predominance of members of Rhodoferax, Azoarcus, Pseudomonas, and unknown bacteria related to Rhodocyclaceae. It was observed that mRNA transcripts of subfamily I.2.C extradiol dioxygenase genes were detected constantly over the monitoring period, and the detected sequences clustered into six distinct clusters. In order to reveal changes in the expression of these clusters over the monitoring period a SNuPE assay was developed. This quasi fingerprinting of functional gene expression provided the opportunity to link the investigated function to specific microbial populations. The results obtained can improve our understanding of aromatic hydrocarbon degradation under oxygen limitation and may benefit bioremediation research by demonstrating the usefulness of SNuPE for the monitoring of microbial populations involved in degradation process.

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)).

Polyphasic analysis of an Azoarcus-Leptothrix dominated bacterial biofilm developed on stainless steel surface in a gasoline contaminated hypoxic groundwater.

Pump and treat systems are widely used for hydrocarbon-contaminated groundwater remediation. Although biofouling (formation of clogging biofilms on pump surfaces) is a common problem in these systems, scarce information is available regarding the phylogenetic and functional complexity of such biofilms. Extensive information about the taxa and species as well as metabolic potential of a bacterial biofilm developed on the stainless steel surface of a pump submerged in a gasoline-contaminated hypoxic groundwater is presented. Results shed light on a complex network of interconnected hydrocarbon-degrading chemoorganotrophic and chemolitotrophic bacteria. It was found that besides the well-known hydrocarbon-degrading aerobic/facultative anaerobic biofilm-forming organisms (e.g., Azoarcus, Leptothrix, Acidovorax, Thauera, Pseudomonas, etc.), representatives of Fe2+-and Mn2+-oxidizing (Thiobacillus, Sideroxydans, Gallionella, Rhodopseudomonas, etc.) as well as of Fe3+- and Mn4+-respiring (Rhodoferax, Geobacter, Magnetospirillum, Sulfurimonas, etc.) bacteria were present in the biofilm. The predominance of β-Proteobacteria within the biofilm bacterial community in phylogenetic and functional point of view was revealed. Investigation of meta-cleavage dioxygenase and benzylsuccinate synthase (bssA) genes indicated that within the biofilm, Azoarcus, Leptothrix, Zoogloea, and Thauera species are most probably involved in intrinsic biodegradation of aromatic hydrocarbons. Polyphasic analysis of the biofilm shed light on the fact that subsurface microbial accretions might be reservoirs of novel putatively hydrocarbon-degrading bacterial species. Moreover, clogging biofilms besides their detrimental effects might supplement the efficiency of pump and treat systems.

Hyperthyroidism caused by a germline activating mutation of the thyrotropin receptor gene: Difficulties in diagnosis and therapy

BACKGROUND Germline activating mutations of the thyrotropin receptor (TSHR) gene have been considered as the only known cause of sporadic nonautoimmune hyperthyroidism in the pediatric population. Here we describe the long-term follow-up and evaluation of a patient with sporadic nonautoimmune primary hyperthyroidism who was found to have a de novo germline activating mutation of the TSHR gene. SUMMARY The patient was an infant who presented at the age of 10 months in an unconscious state with exsiccation, wet skin, fever, and tachycardia. Nonautoimmune primary hyperthyroidism was diagnosed, and brain magnetic resonance imaging and computed tomography showed also Arnold-Chiari malformation type I. Continuous propylthiouracil treatment resulted in a prolonged clinical cure lasting for 10 years. At the age of 11 years and 5 months the patient underwent subtotal thyroidectomy because of symptoms of trachea compression caused by a progressive multinodular goiter. However, 2 months after surgery, hormonal evaluation indicated recurrent hyperthyroidism and the patient was treated with propylthiouracil during the next 4 years. At the age of 15 years the patient again developed symptoms of trachea compression. Radioiodine treatment resulted in a regression of the recurrent goiter and a permanent cure of hyperthyroidism without relapse during the last 3 years of his follow-up. Sequencing of exon 10 of the TSHR gene showed a de novo heterozygous germline I630L mutation, which has been previously described as activating mutation at somatic level in toxic thyroid nodules. CONCLUSIONS The I630L mutation of the TSHR gene occurs not only at somatic level in toxic thyroid nodules, but also its presence in germline is associated with nonautoimmune primary hyperthyroidism. Our case report demonstrates that in this disorder a continuous growth of the thyroid occurs without any evidence of elevated TSH due to antithyroid drug overdosing. This may justify previous recommendations for early treatment of affected patients with removal of as much thyroid tissue as possible.

Verticum-type systems applied to ecological monitoring

In the paper ecological interaction chains of the type resource - producer - primary user - secondary consumer are considered. The dynamic behaviour of these four-level chains is modelled by a system of differential equations, the linearization of which is a verticum-type system introduced for the study of industrial verticums. Applying the technique of such systems, for the monitoring of the considered ecological system, an observer system is constructed, which makes it possible to recover the whole state process from the partial observation of the ecological interaction chain.