The Application of MALDI-TOF MS for a Variability Study of Paenibacillus larvae.

In recent decades, the significant deterioration of the health status of honey bees has been observed throughout the world. One of the most severe factors affecting the health of bee colonies worldwide is American foulbrood disease. This devastating disease, with no known cure, is caused by the Gram-positive spore-forming bacteria of Paenibacillus larvae species. At present, DNA-based methods are being used for P. larvae identification and typing. In our study, we compare two of the most advanced DNA-based technologies (rep-PCR and 16S rRNA analyses) with MALDI-TOF MS fingerprinting to evaluate P. larvae variability in Central Europe. While 16S rRNA analysis presents a very limited variation among the strains, MALDI-TOF MS is observed to be more efficient at differentiating P. larvae. Remarkably, no clear correlation is observed between whole-genome rep-PCR fingerprinting and MALDI-TOF MS-based typing. Our data indicate that MALDI-TOF protein profiling provides accurate and cost-effective methods for the rapid identification of P. larvae strains and provides novel perspectives on strain diversity compared to conventional DNA-based genotyping approaches. The current study provides a good foundation for future studies.

Toxicity of oxalic acid and impact on some antioxidant enzymes on in vitro-reared honeybee larvae.

Nowadays, Varroa destructor is considered as a serious pest of honeybees (Apis mellifera) and its resistance to acaricides has been reported in Europe since the early 1990s. That is why new methods of treatment for Varroa mites are still in focus of many scientists. In our study, we determined the lethal concentration LC50 (72 h) of 2.425% oxalic acid solution following single spray exposure of honeybee larvae under laboratory conditions (Guideline OECD 237 2013). Potential sublethal effects of oxalic acid were monitored through the determination of the activity of antioxidant enzymes. Activation of primary antioxidant enzymes was observed at 1.75% of oxalic acid; 3.5% of oxalic acid brought on a statistically significant increase of glutathione S-transferase activity. This change was accompanied by an increase in thiobarbituric acid reactive substances, products of lipid peroxidation. Our results indicate that oxalic acid may be harmful to bee brood when present during application.

Analysis of the bacterial community from high alkaline (pH > 13) drainage water at a brown mud disposal site near Ziar nad Hronom (Banská Bystrica region, Slovakia) using 454 pyrosequencing.

Brown mud, as a waste product of the industrial process of aluminum production, represents a great environmental burden due to its toxicity to living organisms. However, some microorganisms are able to survive in this habitat, and they can be used in bioremediation processes. Traditional cultivation methods have a limited capacity to characterize bacterial composition in environmental samples. Recently, next-generation sequencing methods have provided new perspectives on microbial community studies. The aim of this study was to analyze the bacterial community in the drainage water of brown mud disposal site near Ziar nad Hronom (Banská Bystrica region, Slovakia) using 454 pyrosequencing. We obtained 9964 sequences assigned to 163 operational taxonomic units belonging to 10 bacterial phyla. The phylum Proteobacteria showed the highest abundance (80.39%) within the bacterial community, followed by Firmicutes (13.05%) and Bacteroidetes (5.64%). Other bacterial phyla showed an abundance lower than 1%. The classification yielded 85 genera. Sulfurospirillum spp. (45.19%) dominated the bacterial population, followed by Pseudomonas spp. (13.76%) and Exiguobacterium spp. (13.02%). These results indicate that high heavy metals content, high pH, and lack of essential nutrients are the drivers of a dramatic reduction of diversity in the bacterial population in this environment.

Formetanate toxicity and changes in antioxidant enzyme system of Apis mellifera larvae.

Substantial percentage of world food production depends on pollinating service of honeybees that directly depends on their health status. Among other factors, the success of bee colonies depends on health of developed larvae. The crucial phase of larval development is the first 6 days after hatching when a worker larva grows exponentially and larvae are potentially exposed to xenobiotics via diet. In the present study, we determined the lethal concentration LC50 (72 h) following single dietary exposure of honeybee larvae to formetanate under laboratory conditions, being also the first report available in scientific literature. Activities of antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and glutathione-S-transferase (GST) were also measured in the homogenates of in vitro reared honeybee larvae after single formetanate exposure. Decreased specific activity of SOD and increased activities of CAT and GST suggest the induction of oxidative stress. Higher levels of thiobarbituric reactive species in all samples supported this fact. Comparing determined larval toxicity (LC50 of 206.01 mg a.i./kg diet) with adult toxicity data, we can suppose that the larvae may be less sensitive to formetanate than the adult bees.

Actinomyces succiniciruminis sp. nov. and Actinomyces glycerinitolerans sp. nov., two novel organic acid-producing bacteria isolated from rumen.

Two bacterial strains, Am4 and G10 were isolated from rumen fluid of different ruminants: cow (Holstein-Friesian) and sheep (Slovenskè merino), respectively. They were isolated from different hosts and regions, but showed 99.2% similarity of the 16S rRNA genes. Both strains are versatile and ferment various sugars to mainly succinate and lactate and small amounts of acetate and formate. The 16S rRNA sequences of Am4 and G10 revealed that they belonged to the genus Actinomyces, and are related to Actinomyces ruminicola JCM 13352T with 97.0% and 97.4% similarity, respectively. DDH showed strain Am4 and G10 had only 55.8 and 43.3% similarity with the Actinomyces ruminicola JCM 13352T, and had 69.9% similarity among each other. Comparing strain Am4 and G10, gANI value and dDDH were 92.9% and 68.6%, respectively. Additionally, AAI between the strains was 95.8%. MLSA of housekeeping genes showed difference of metG and pheS. The G+C% contents of strain Am4 and G10 were 69.8% and 68.5%, respectively. MK-10(H4) was the principal quinone for strain Am4 (82%) and G10 (91%) with small amounts of MK-10(H8) and MK-10(H6) for both strains. Only MK-9(H4) was detected in strain Am4. MALDI-TOF analysis of protein profiles also revealed that Am4 and G10 are different from each other and from Actinomyces ruminicola JCM 13352T. Based on phylogenetic and physiological characteristics, together with genome comparison and MLSA we propose two novel species in the genus Actinomyces: Actinomyces succiniciruminis sp. nov. (type strain Am4T=TISTR 2317T=DSM 10376T) and Actinomyces glycerinitolerans sp. nov. (type strain G10T=TISTR 2318T=DSM 10377T).

Heterotrophic microflora of highly alkaline (pH > 13) brown mud disposal site drainage water near Ziar nad Hronom (Banska Bystrica region, Slovakia).

Brown mud is a waste by-product of alumina production by Bayer process. Due to extensive sodium hydroxide use in the process, brown mud disposal site near Ziar nad Hronom (Banska Bystrica region, Slovakia) and drainage water are ones of the greatest environmental burdens in Slovakia. Drainage water from this landfills has pH value higher than 13, and it contains many heavy metals and elevated salt content. In our experiments, relatively numerous bacterial population was detected in the drainage water with frequency of about 80 cfu/ml using cultivation approach. The alkalitolerant heterotrophic isolates were identified by combination of MALDI-TOF and 16S rDNA analysis. Drainage water population was dominated by Actinobacteria (Microbacterium spp. and Micrococcus spp.) followed by low G + C-content gram-positive bacteria (Bacillus spp.). Two isolates belonged to gram-negative bacteria only, identified as Brevundimonas spp. Phylogenetic and biochemical analyses indicate that nearly half of the bacteria isolated are probably representatives of a new species. Brown mud disposal site is proposed as a source of new bacterial taxa possibly used in bioremediation processes.

Telomere repeats and macronuclear DNA organization in the soil ciliate Kahliella matisi (Ciliophora, Hypotricha).

To better understand the structure of macronuclear chromosomes in ciliates, the organization of macronuclear DNA was investigated in the hypotrich Kahliella matisi. Total DNA of K. matisi separated by agarose gel electrophoresis showed continuous smear ranging in size from ∼500bp to ∼15kb. This fragmentation was found to be due to the presence of gene-sized macronuclear chromosomes. The sequence analysis of four randomly cloned macronuclear chromosomes showed that K. matisi telomeres consist of 5'-dC4A4-3' repeats and carry one or two open reading frames. The transcription unit was found to be flanked with non-coding AT rich 5' leader and 3' trailer. No consensus transcription-regulatory sequences were identified in 5' leader and only one of analyzed gene-sized chromosomes showed the presence of conserved poly(A) addition signal sequence in 3' trailer. All ORFs showed highest relatedness to Oxytricha trifallax macronuclear chromosomes with conserved exon/intron structure. Sequence comparisons indicate that macronuclear chromosome organization is at least partially conserved in ciliates.

The symbiotic intestinal ciliates and the evolution of their hosts.

The evolution of sophisticated differentiations of the gastro-intestinal tract enabled herbivorous mammals to digest dietary cellulose and hemicellulose with the aid of a complex anaerobic microbiota. Distinctive symbiotic ciliates, which are unique to this habitat, are the largest representatives of this microbial community. Analyses of a total of 484 different 18S rRNA genes show that extremely complex, but related ciliate communities can occur in the rumen of cattle, sheep, goats and red deer (301 sequences). The communities in the hindgut of equids (Equus caballus, Equus quagga), and elephants (Elephas maximus, Loxodonta africanus; 162 sequences), which are clearly distinct from the ruminant ciliate biota, exhibit a much higher diversity than anticipated on the basis of their morphology. All these ciliates from the gastro-intestinal tract constitute a monophyletic group, which consists of two major taxa, i.e. Vestibuliferida and Entodiniomorphida. The ciliates from the evolutionarily older hindgut fermenters exhibit a clustering that is specific for higher taxa of their hosts, as extant species of horse and zebra on the one hand, and Africa and Indian elephant on the other hand, share related ciliates. The evolutionary younger ruminants altogether share the various entodiniomorphs and the vestibuliferids from ruminants.

Two rep genes in small cryptic plasmid pKST21 of Escherichia coli.

The complete nucleotide sequence of a small cryptic plasmid pKST21 from Escherichia coli was determined. This plasmid is 1,460 bp long with an overall GC content of 51 %. Based on sequence analysis, the presence of two segments with different average GC density was observed. The segment with higher GC content revealed 98-90 % similarity to several small plasmids of E. coli and to pCR1 from Gram-positive Corynebacterium renale. Plasmid pKST21 possesses two conversely oriented open reading frames encoding proteins with a high degree of amino acid identity to Rep proteins involved in replication. ORF1 encodes replication protein similar to RepA protein of Bartonella tribocorum or Bacillus cereus plasmids or to the putative plasmid Rep protein from ecologically close Selenomonas ruminantium. ORF2 similarly encodes a replication protein, which shares 97 % homology with Rep protein from C. renale. Genetic diversity observed in plasmid pKST21 indicates a mosaic structure of the plasmid with different segments acquired from different sources. Deletion analysis showed that both fragments carrying the repA and repB genes are necessary for the replication of pKST21 in E. coli. The presence of plasmid with the same gene composition was revealed in 14 % of tested E. coli isolates from the rumen of sheep. All these strains produced identical ERIC-PCR profiles indicating isogenic origin of the strain and lack of horizontal gene transfer of pKST21 plasmid.

Water treatment using activated carbon supporting silver and magnetite.

Recent efforts in water purification have led to the development of novel materials whose unique properties can offer effective biocidal capabilities with greater ease of use and at lower cost. In this study, we introduce a novel procedure for the preparation of activated carbon (charcoal) composite in which magnetite and silver are incorporated (MCAG); we also describe the use of this material for the disinfection of surface water. The formation process of magnetic MCAG composite was studied using ultraviolet-visible spectroscopy. The results demonstrated the high sorption efficiency of AgNO3 to magnetic activated carbon. The antimicrobial capabilities of the prepared MCAG were examined and the results clearly demonstrate their inhibitory effect on total river water bacteria and on Pseudomonas koreensis and Bacillus mycoides cultures isolated from river water. The bacterial counts in river water samples were reduced by five orders of magnitude following 30 min of treatment using 1 g l⁻¹ of MCAG at room temperature. The removal of all bacteria from the surface water samples implies that the MCAG material would be a suitable disinfectant for such waters. In combination with its magnetic character, MCAG would be an excellent candidate for the simple ambulatory disinfection of surface water.

Variability of putative rep gene cassettes in Selenomonas ruminantium plasmids.

Characteristic feature of the most of Selenomonas ruminantium cryptic plasmids is the presence of short, conserved sequences encompassing the gene for replication protein creating a potential rep gene cassette. PCR-based experiment was designed to analyse the genetic organization of putative plasmid rep modules and to assess S. ruminantium plasmid biodiversity. Analysed PCR amplicons contained single open reading frames encoding for putative replication proteins. While most of the derived protein sequences were often found to be conserved among putative plasmid molecules, at noncoding regions, genetic variability was observed to various extents. Complete nucleotide sequence of a plasmid was determined that contained probably a new rep gene only distantly related to known selenomonas Rep proteins but at noncoding regions shared high homology with already known plasmids. Our results document considerable structural instability and sequence variability of analysed rep gene cassettes and suggest a modular structure of S. ruminantium plasmids potentially accessible for rep gene module exchanges.

ß-Fructofuranosidase and sucrose phosphorylase of rumen bacterium Pseudobutyrivibrio ruminis strain 3.

The subject of this study was the fructan and sucrose degrading enzymes of bacterium Pseudobutyrivibrio ruminis strain 3. It was stated that cell extract from bacteria growing on inulin contained ß-fructofuranosidase (EC 3.2.1.80 and/or EC 3.2.1.26) and sucrose phosphorylase (EC 2.4.1.7), while the bacteria maintained on sucrose showed only phosphorylase. Partially purified ß-fructofuranosidase digested inulooligosaccharides and sucrose to fructose or fructose and glucose, respectively, but was unable to degrade the long chain polymers of commercial inulin and Timothy grass fructan. Digestion rate of inulooligosaccharides fit Michaelis-Menten kinetics with V(max) 5.64 µM/mg/min and K(m) 1.274%, respectively, while that of sucrose was linear. Partially purified sucrose phosphorylase digested only sucrose. The digestion products were fructose, glucose-1P and free glucose. The reaction was in agreement with Michaelis-Menten kinetics. The V(max) were 0.599 and 0.584 µM/mg/min, while K(m) were 0.190 and 0.202% for fructose release and glucose-1P formation, respectively, when bacteria grew on inulin. The V(max) were, however, 1.37 and 1.023 µM/mg/min, while K(m) were 0.264 and 0.156%, if bacteria were grown on sucrose. The free glucose was hardly detectable for the enzyme originated from inulin grown bacteria, but glucose levels ranged from 0.05 to 0.25 µM/mg/min, when cell extract from bacteria grown on sucrose was used. Release of free glucose was observed when no inorganic phosphate was present in reaction mixture.

Peptidoglycan hydrolase enterolysin a recognizes lipoteichoic acid chains in the cell walls of sensitive bacteria.

C-terminal domain of peptidoglycan hydrolase enterolysin A (EnlA) is involved in specific recognition and binding to the target cell envelopes and represents true cell wall binding (CWB) domain. Sensitivity/resistance to EnlA is dependent on binding ability/disability of its CWB domain. We assume that main mechanism of resistance against EnlA is absence of the specific receptor on the cell surface, which is necessary for binding of the enzyme molecule. Using competitive and enzymatic assays we have uncovered the chemical nature of the EnlA receptor, which is a lipoteichoic acid.

Morphological and phylogenetical studies on a new soil hypotrich ciliate: Kahliella matisi spec. nov. (Hypotrichia, Kahliellidae).

The morphology, ontogenesis, encystment, and 18S rRNA gene sequence of a new soil hypotrich ciliate, Kahliella matisi, were studied. Main characteristics of K. matisi are: (1) two short and six longitudinal cirral rows right of the adoral zone of membranelles and four longitudinal rows left of it; (2) three dorsal kineties, of which kinety 1 extends along the left cell margin, kinety 2 runs in a slightly sigmoidal line, and kinety 3 is distinctly shortened posteriorly. Ontogenesis is similar to that in congeners, especially in the development of the marginal rows and long dorsal kineties, the preservation of some old cirral rows after division, and the direction of the neokinetal wave. However, there are some peculiarities: (1) reorganization of the proximal parental adoral membranelles; (2) splitting of opisthe's anlage II into the cirral streak II and III; and (3) formation of the parental cirral row R3 from anlagen IV and V. During encystment, the body diminishes and becomes globular, the nuclear apparatus is reorganized, and the ciliature is resorbed. In our molecular phylogenies, the family Kahliellidae is polyphyletic and the position of K. matisi is rather poorly resolved, indicating a relationship with oxytrichids.

Evaluation of human telomeric g-quadruplexes: the influence of overhanging sequences on quadruplex stability and folding.

To date, various G-quadruplex structures have been reported in human telomeric sequences. Human telomeric repeats can form many topological structures depending on conditions and on base modification; parallel, antiparallel, and hybrid forms. The effect of salts and some specific ligands on conformational switches between different conformers is known, but the influence of protruding sequences has rarely been discussed. In this paper, we analyze different quadruplex-forming oligomers derived from human telomeric sequences which contain 3'- and 5'-protruding nucleotides, not usually associated with the G-quadruplex motif. The study was performed using electrophoresis, CD, and UV spectroscopies. The major findings are (i) protruding nucleotides destabilize the G-quadruplex structure, and (ii) overhanging sequences influence the folding of the quadruplex.

Structural instability of small rolling circle replication plasmids from Selenomonas ruminantium.

The complete nucleotide sequence of pSRD192 plasmid from Selenomonas ruminantium 19D has been obtained and analyzed. The plasmid, 2334bp in length, was shown to replicate by rolling circle replication mechanism. By PCR method variability of pSRD192-like plasmids was investigated and another variant of pSRD192-like plasmid; the pSRM22 plasmid 2338bp in length; was detected and characterized. Both pSRD192 and pSRM22 plasmids share an identical rep gene and origins of replication to that of another S. ruminantium pONE429 plasmid. Other than that there are additional regions of sequence similarity between the three plasmids, interspersed with divergent regions. The sequence comparisons suggest structural instability of pSRD192-like rolling circle replication plasmids.

Treponema zioleckii sp. nov., a novel fructan-utilizing species of rumen treponemes.

During studies on fructan degradation in the rumen, a Treponema-like bacterium able to utilize Timothy grass fructan, commercial inulin and sucrose as the sole carbon source was recovered from sheep rumen. At least two different fructanolytic enzymes were identified in cell-free extracts of the isolated bacterium. Characterization of the strain by a polyphasic approach indicated that it can be regarded as a representative of a new bacterial species within the genus Treponema. Electron microscopy showed that the bacterium exhibited all of the features typical of spirochetes. The helical cells measured 5.4-11.5 microm x 0.42-0.51 microm and possessed up to seven regular coils. The bacterium utilized various plant mono- and disaccharides as fermentable substrates. Formate, acetate and ethanol in a molar ratio of 16 : 10 : 1 were the end products of glucose fermentation. The major cellular fatty acids were C(13:0), C(14:0), C(14:1), C(15:0), C(15:1) and C(16:0). The nearly complete 16S rRNA gene sequence was obtained, and phylogenetic analysis of the 16S rRNA gene showed the highest similarity to rumen Treponema strain CA. We propose the name Treponema zioleckii sp. nov. for this novel rumen spirochete with strain kT as the type strain.

Heterologous expression of functionally active enterolysin A, class III bacteriocin from Enterococcus faecalis, in Escherichia coli.

The heterologous expression of enterolysin A (EnlA), heat-labile class III bacteriocin from Enterococcus faecalis II/1 with anti-listerial activity, was studied in Escherichia coli. The PCR amplified products of enterolysin A structural gene, N-terminal part of EnlA with endopeptidase-like activity and C-terminal part of EnlA similar to a lysis gene of bacteriophage, were cloned in prelinearized pQE-30UA expression vector. The expression of EnlA structural gene led to the synthesis and secretion of functional-active His-tagged enterolysin A protein, which was purified to homogeneity using His-Select Cartridge and was shown to be fully active against the indicator strain. The expression of N-terminal or C-terminal part of EnlA and deletion of last 58 amino acids from C-terminal domain of EnlA led to the synthesis of biologically non-active proteins.

A unique pair of GATC specific DNA methyltransferases in Mitsuokella multiacida.

Two GATC specific methylases together with Sau3AI isoschizomeric restriction endonuclease were partially characterized in Mitsuokella multiacida 46/5. This is the first report on the presence of solitary Dam methyltransferase alongside GATC specific restriction-modification system resulting in the unusual two-fold methylation of the GATC motifs.