Erzsébet Baka

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.

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)).

Enhancing recombinant protein solubility with ubiquitin-like small archeal modifying protein fusion partners.

A variety of protein expression tags with different biochemical properties has been used to enhance the yield and solubility of recombinant proteins. Ubiquitin, SUMO (small ubiquitin-like modifier) and prokaryotic ubiquitin like MoaD (molybdopterin synthase, small subunit) fusion tags are getting more popular because of their small size. In this paper we report on the use of ubiquitin-like small archaeal modifier proteins (SAMPs) as fusion tags since they proved to increase expression yield, stability and solubility in our experiments. Equally important, they did not co-purify with proteins of the expression host and there was information that their specific JAB1/MPN/Mov34 metalloenzyme (JAMM) protease can recognize the C-terminal VSGG sequence when SAMPs fused, either branched or linearly to target proteins, and cleave it specifically. SAMPs and JAMM proteases from Haloferax volcanii, Thermoplasma acidophilum, Methanococcoides burtonii and Nitrosopumilus maritimus were selected, cloned, expressed heterologously in Escherichia coli and tested as fusion tags and cleaving proteases, respectively. Investigated SAMPs enhanced protein expression and solubility on a wide scale. T. acidophilum SAMPs Ta0895 and Ta01019 were the best performing tags and their effect was comparable to the widely used maltose binding protein (MBP) and N utilization substance protein A (NusA) tags. Moreover, H. volcanii SAMP Hvo_2619 contribution was mediocre, whereas M. burtonii Mbur_1415 could not be expressed. Out of four investigated JAMM proteases, only Hvo_2505 could cleave fusion tags. Interestingly, it was found active not only on its own partner substrate Hvo_2619, but it also cleaved off Ta0895.

The first transformation method for the thermo-acidophilic archaeon Thermoplasma acidophilum

A transformation method yielding up to 10(4) transformants per μg circular DNA was developed for Thermoplasma acidophilum. The method is based on a natural DNA uptake process in which T. acidophilum cells keep their integrity and turn competent at pH 3.5 and 58°C. Shuttle vector maintenance could not be detected, since the used Nov(R) gyraseB gene integrated into its chromosomal counterpart by homologous recombination.

Draft Genome Sequence of the Lignocellulose Decomposer Thermobifida fusca Strain TM51.

ABSTRACT Here, we present the complete genome sequence of Thermobifida fusca strain TM51, which was isolated from the hot upper layer of a compost pile in Hungary. T. fusca TM51 is a thermotolerant, aerobic actinomycete with outstanding lignocellulose-decomposing activity.

Micrococcoides hystricis gen. Nov., sp. nov., a novel member of the family micrococcaceae, phylum Actinobacteria

A Gram-stain-positive bacterium, designated TSL3T, was isolated from faeces of a porcupine, Hystrix indica, from the Budapest Zoo and Botanical Garden, Hungary. On the basis of 16S rRNA gene sequence analysis, the strain is phylogenetically related to the family Micrococcaceae. The highest 16S rRNA gene sequence similarity was found with Micrococcus terreus V3M1T (96.50 %) followed by Arthrobacter humicola KV-653T (96.43 %). Cells of strain TSL3T were aerobic, non-motile and coccoid-shaped. The main fatty acids were anteiso-C15 : 0 (54.4 %), iso-C16 : 0 (18.2 %) and iso C15 : 0 (9.7 %). The major menaquinone was MK-7, and the polar lipid profile included phosphatidylglycerol, diphosphatidylglycerol, dimannosylglyceride, trimannosyldiacylglycerol, phosphatidylinositol, three unknown phospholipids and two unknown glycolipids. Strain TSL3T showed the peptidoglycan structure A4alpha l-Lys - Gly - l-Glu. The DNA G+C content of strain TSL3T was 58.4 mol%. Phenotypic and genotypic characterisation clearly showed that strain TSL3T could be differerentiated from the members of other genera in the family Micrococcaceae. According to these results, strain TSL3T represents a novel genus and species, for which the name Micrococcoides hystricis gen. nov., sp. nov. is proposed. The type strain is TSL3T (=DSM 29785T=NCAIM B. 02604T).