Discovering the deep evolutionary roots of serum amyloid A protein family.

Deep evolutionary origin of the conserved animal serum amyloid A (SAA) apolipoprotein family leading to yet unknown highly similar SAA-like sequences occurring in certain bacterial genomes is demonstrated in this contribution. Horizontal gene transfer event of corresponding genes between gut bacteria and non-vertebrate animals was discovered in the reconstructed phylogenetic tree obtained with maximum likelihood and neighbor-joining methods, respectively. This detailed phylogeny based on totally 128 complete sequences comprised diverse serum amyloid A isoforms from various animal vertebrate and non-vertebrate phyla and also corresponding genes coding for highly similar proteins from animal gut bacteria. Typical largely conserved sequence motifs and a peculiar structural fold consisting mainly of four α-helices in a bundle within all reconstructed clades of the SAA protein family are discussed with respect to their supposed biological functions in various organisms that contain corresponding genes.

Deep Insights into the Specific Evolution of Fungal Hybrid B Heme Peroxidases.

In this study, we focus on a detailed bioinformatics analysis of hyBpox genes, mainly within the genomes of Sclerotiniaceae (Ascomycota, Leotiomycetes), which is a specifically evolved fungal family of necrotrophic host generalists and saprophytic or biotrophic host specialists. Members of the genus Sclerotium produce only sclerotia and no fruiting bodies or spores. Thus, their physiological role for peroxidases remains open. A representative species, S. cepivorum, is a dangerous plant pathogen causing white rot in Allium species, particularly in onions, leeks, and garlic. On a worldwide basis, the white rot caused by this soil-borne fungus is apparently the most serious threat to Allium-crop production. We have also found very similar peroxidase sequences in the related fungus S. sclerotiorum, although with minor yet important modifications in the architecture of its active centre. The presence of ScephyBpox1-specific mRNA was confirmed by transcriptomic analysis. The presence of Hybrid B peroxidase at the protein level as the sole extracellular peroxidase of this fungus was confirmed in the secretome of S. cepivorum through detailed proteomic analyses. This prompted us to systematically search for all available genes coding for Hybrid B heme peroxidases in the whole fungal family of Sclerotiniaceae. We present here a reconstruction of their molecular phylogeny and analyse the unique aspects of their conserved-sequence features and structural folds in corresponding ancestral sequences.

A new type strain of Pseudomonas putida NM-CH-I15-I isolated from a nickel-contaminated soil in southwest Slovakia harbouring the czcA-NM15I resistance determinant.

A soil bacterium MR-CH-I15-I was on the base of phylogenetic analysis of almost the whole (1,492 bp) 16S rRNA sequence and sequences of selected 9 marker genes identified as Pseudomonas putida strain NM-CH-I15-I. The bacterium exhibited typical morphological features and biochemical properties for this species, the highest resistance to nickel and copper and multidrug resistance to different antibiotic groups. In addition, the whole czcA-NM15I heavy-metal resistance gene sequence (3,126 bp, 1,042 amino acids, MW 112, 138 Da) was obtained and on the base of phylogenetic analysis was assigned to CzcA protein from Pseudomonas reidholzensis with 93% similarity. This gene was significantly induced mainly by the addition of zinc, cadmium and cobalt and in a lesser extent of nickel. Furthermore, an increased expression of the CzcA-NM15I protein was confirmed by immunoblot analysis after heterologous expression of the czcA-NM15I synthetic variant gene in E. coli BL21 (DE3). Finally, the location of amino acids (R83, R673, D402, D408, D619, E415, E568) in the homology model of the CzcA-NM15I protein suggested that these amino acids may play an important role in the transport of cations such as cobalt, zinc or cadmium. This soil bacterium can represent a new type strain of P. putida NM-CH-I15-I.

The Heavy-Metal Resistance Determinant of Newly Isolated Bacterium from a Nickel-Contaminated Soil in Southwest Slovakia.

A bacterial isolate MR-CH-I2 [KC809939] isolated from soil contaminated mainly by high nickel concentrations in southwest Slovakia was previously found carrying nccA-like heavy-metal resistance determinant, marked as MR-CH-I2-HMR [KF218096]. According to phylogenetic analysis of short (696 bp) 16S rDNA (16S rRNA) sequences this bacterium was tentatively assigned to Uncultured beta proteobacterium clone GC0AA7ZA05PP1 [JQ913301]. nccA-like gene product was on the same base of its partial (581 bp) sequences tentatively assigned to CzcA family heavy metal efflux pump [YP_001899332] from Ralstonia picketii 12J with 99% similarity. In this study the bacterium MR-CH-I2 and its heavy-metal resistance determinant were more precisely identified. This bacterial isolate was on the base of phylogenetic analysis of almost the whole (1,500 bp) 16S rDNA (16S rRNA) sequence, MR-CH-I2 [MF102046], and sequence for gyrB gene and its product respectively, MR-CH-I2-gyrB [MF134666], assigned to R. picketii 12J [CP001068] with 99 and 100% similarities, respectively. In addition, the whole nccA-like heavy-metal resistance gene sequence (3,192 bp), marked as MR-CH-I2-nccA [KR476581], was obtained and on the base of phylogenetic analysis its assignment was confirmed to MULTISPECIES: cation efflux system protein CzcA [WP_004635342] from Burkholderiaceae with 98% similarity. Furthermore, although the bacterium carried one high molecular plasmid of about 50 kb in size, nccA-like gene was not located on this plasmid. Finally, the results from RT-PCR analysis showed that MR-CH-I2-nccA gene was significantly induced only by the addition of nickel.

A simple strategy for investigating the diversity and hydrocarbon degradation abilities of cultivable bacteria from contaminated soil.

The use of indigenous bacterial strains is a valuable bioremediation strategy for cleaning the environment from hydrocarbon pollutants. The isolation and selection of hydrocarbon-degrading bacteria is therefore crucial for obtaining the most promising strains for site decontamination. Two different media, a minimal medium supplemented with a mixture of polycyclic aromatic hydrocarbons and a MS medium supplemented with triphenyltetrazolium chloride, were used for the isolation of bacterial strains from two hydrocarbon contaminated soils and from their enrichment phases. The hydrocarbon degradation abilities of these bacterial isolates were easily and rapidly assessed using the 2,6-dichlorophenol indophenol assay. The diversity of the bacterial communities isolated from these two soil samples and from their enrichment phases was evaluated by the combination of a bacterial clustering method, fluorescence ITS-PCR, and bacterial identification by 16S rRNA sequencing. Different PCR-based assays were performed in order to detect the genes responsible for hydrocarbon degradation. The best hydrocarbon-degrading bacteria, including Arthrobacter sp., Enterobacter sp., Sphingomonas sp., Pseudomonas koreensis, Pseudomonas putida and Pseudomonas plecoglossicida, were isolated directly from the soil samples on minimal medium. The nahAc gene was detected only in 13 Gram-negative isolates and the sequences of nahAc-like genes were obtained from Enterobacter, Stenotrophomonas, Pseudomonas brenneri, Pseudomonas entomophila and P. koreensis strains. The combination of isolation on minimal medium with the 2,6-dichlorophenol indophenol assay was effective in selecting different hydrocarbon-degrading strains from 353 isolates.

Isolation, identification, and characterization of Vibrio cholerae from the Danube River in Slovakia.

The occurrence of Vibrio cholerae, an important aquatic pathogen, was assessed in the surface water of the Danube River near Bratislava. The isolates were distinguished by biochemical tests and grouped by ARDRA to three clusters corresponding to three species (V. cholerae, Vibrio metschnikovii, and Aeromonas spp.). The identification of V. cholerae was confirmed by multiplex PCR using primer pairs targeted to ompW gene (membrane protein), ctxA gene (toxicity gene), and toxR gene (regulatory gene). None from the isolated V. cholerae from surface water contained ctxA gene; seven of them possessed toxR gene. Serotyping of V. cholerae isolates with polyvalent O antiserum and O/139 antiserum was negative. All isolates of V. cholerae were susceptible to chloramphenicol, rifampicin, tetracycline, variable to ampicillin, and resistant to kanamycin and streptomycin.

Investigation of microbial community isolated from indoor artworks and air environment: identification, biodegradative abilities, and DNA typing.

This study deals with establishing the characteristics of a microbial community isolated from indoor artworks and the surrounding air environment. It is one of the few studies on microbial degradation of indoor artworks. It shows the potential biodegradative risk that can occur if artworks are not exhibited and conserved in an appropriate environment. The microbial community isolated from the indoor artworks and air environment was examined by cultural and molecular methods. Different plate assays were used to screen the biodegradative activity of the isolated microflora: Remazol Brilliant Blue R, phenol red, and Azure B for the ligninolytic properties; Ostazin brilliant red H-3B for cellulose degradation; CaCO3 glucose agar for solubilization activity; and B4 agar for biomineralization. To type the bacterial and fungal isolates, 2 PCR methods, repetitive extragenic palindromes (REP) and random amplified microsatellite polymorphisms (RAMP) were used. The art objects were principally colonized by fungi. The most commonly isolated strains were represented by hyphomycetes of the genera Penicillium, Aspergillus, Cladosporium, and Chaetomium. Members of these genera showed intensive biodegradation activity, both on wood and on stone. Bacteria were predominant in the air, exhibiting complex communities, both in the air and on the artworks. The most frequently isolated genera were Bacillus and Staphylococcus with extensive biodegradation abilities. REP-PCR revealed high variability within strains belonging to the same genus. RAMP is a new PCR-based method, used in this research for the first time to cluster the microfilamentous fungi and to characterize and select especially Penicillium and Aspergillus strains, which were isolated in a large number.

Assessment of environmental enterococci: bacterial antagonism, pathogenic capacity and antibiotic resistance.

The properties of 166 environmental strains belonging to the seven enterococcal species were studied. Enterococci originated mainly from surface- and waste-waters. They were screened for the presence of enterocins, virulence factors, and antibiotic resistance. The presence of different enterocin genes (entA, entB, entP, ent31, entL50AB) was frequently observed in our enterococcal isolates, 109 strains contained at least one enterocin gene. The distribution of enterocin genes varied according to the species, the genes were present mainly in E. hirae and E. faecium. By enterocin spot assay, 10 isolates inhibited the growth of Listeria strains. To evaluate the pathogenic ability of isolates, the distribution of selected virulence genes (cylA, gelE and esp) was investigated, eleven strains were positive in some of these genes, five of them belonged to E. faecalis. Regarding the antibiotic resistance of isolates, only two strains were multiresistant and two strains (E. hirae and E. casseliflavus) were resistant to vancomycin.

Evaluation of different PCR-based approaches for the identification and typing of environmental enterococci.

The aim of the work was the evaluation of different PCR-based methods to found an appropriate identification and typing strategy for environmental enterococci. Environmental enterococci were isolated mainly from surface- and waste-waters. Species identification was provided by combination of phenotypic (Micronaut System, Merlin) and molecular detection methods (fluorescent ITS-PCR, ddl-PCR, REP-PCR, AFLP). Very similar results were observed among molecular methods, however several discrepancies were recognized during comparison of molecular and biochemical identification. Seven enterococcal species (E. faecium, E. hirae, E. casseliflavus, E. mundtii, E. faecalis, E. durans and E. gallinarum) were identified within 166 environmental isolates. The results obtained in this work attest the importance of PCR-based methods for identification and typing of environmental enterococci. The fluorescent ITS-PCR (fITS-PCR) showed the best results in order to identify the enterococci strains, the method used the automated capillary electrophoresis to separate the PCR products in a very rapid and precise way. The AFLP method was suitable to identify and characterize the isolates, while the REP-PCR can be used for species identification.

Function of a novel cadmium-induced yodA protein in Escherichia coli.

Cells of Escherichia coli increase greatly the synthesis of a small primarily cytoplasmic protein as soon as the cell growth rate falls below the maximal growth rate supported by cadmium exposure, after which the mature product is exported to the periplasm. This protein was previously identified as the product of the E. coli yodA open reading frame. We now report the isolation of an E. coli mutant defective in YodA synthesis because of insertional inactivation of the corresponding gene. In experiments to test the ability of both the wild-type and yodA mutant E. coli cells to bind cadmium, we have used gamma-labeled [(109)Cd]. Whereas the wild-type E. coli strain was able to bind metal, the yodA mutant strain failed to do so. In addition, analysis of such a mutant demonstrated that it grows at a rate distinguishable from that of the isogenic parent in the presence of cadmium ions. However, challenging cells with hydrogen peroxide and additional metals such as zinc, copper, cobalt, and nickel did not significantly affect the growth rate of the mutant. This growth phenotype was found to be the result of the loss of its ability to bind cadmium. These results suggest that the role of YodA protein might be to decrease the concentration level of cadmium ions in E. coli cells during cadmium stress by its ability to bind heavy metal.

Lag period of 14CO2 evolution from dioctyl sulpho[2,3-14C]succinate in relation to adaptation of bacterium, Comamonas terrigena, to dialkyl esters of sulphosuccinate.

Comamonas terrigena, strain N3H, which was isolated from soil polluted with crude oil products, degraded dioctyl sulphosuccinate, a synthetic commercial surfactant. The primary degradation of this compound, the cleavage of ester bonds between octyl groups and sulphosuccinate, lasted significantly shorter time than the subsequent breakdown of the sulphosuccinate moiety of dioctyl sulpho[2,3-(14)C]succinate. (14)CO(2) evolution had a significant shorter lag period with cells in Tris/phosphate medium, without inorganic sulphate and adapted to surfactant, than unadapted cells. The acceleration of the primary degradation by adapted cells also suggest that some enzymes involved in surfactant degradation are inducible. The bacterium may be useful for bioremediation.

The cadmium-stress stimulon of Escherichia coli K-12.

The influence of cadmium on stress protein production in Escherichia coli K-12 (strain MG1655) was analysed using two-dimensional polyacrylamide gel electrophoresis and the gene-protein database of E. coli K-12. Cadmium (273 microM) caused complete but transient inhibition of growth accompanied by the synthesis of cadmium-induced proteins (CDPs). It was found that some CDPs induced during the growth-arrested phase belong to the heat-shock, oxidation stress, SOS and stringent response regulons, while others are general stress inducible proteins (e.g. H-NS, UspA). In addition, trigger factor, adenylate kinase, W-protein, the cold shock protein G041.2, and seven unknown proteins whose synthesis is not known to be controlled by a global regulator, were identified as immediate responders to cadmium exposure. The rate of synthesis of most of the immediate responders to cadmium exposure decreased when the growth of the cells resumed. However, seven CDPs, including those encoded by argI, tyrA and xthA, maintained a high production rate during growth in the presence of cadmium. Two of the unidentified proteins were N-terminally sequenced by Edman degradation. The N-terminal amino acid sequence of one of these proteins (designated F023.3) matches the E. coli open reading frame o216. This ORF is similar to the N-terminal third of the copper-binding protein amine oxidases (encoded by maoA) of both E. coli and Klebsiella pneumoniae (K. aerogenes). The other N-terminally sequenced protein (designated C044.6) matches perfectly the product of the metK gene, S-adenosylmethionine synthetase I. In comparison to untreated cells, cadmium-stressed cells were found to recover more rapidly during subsequent stress conditions, such as ethanol, osmotic, heat shock, and nalidixic acid treatment. The role of the CDPs is discussed in view of their physiological assignments in the cell.