Usp is a novel E. coli genotoxin active against mammalian cells. Optimal in vivo activity of Usp requires Imu2. Infection with E. coli usp(+) imu1-3(+) induces a response characteristic of apoptosis.
Solar salterns operate only for short dry periods of the year in the north shore of the Adriatic Sea because of its relatively humid and cold Mediterranean climate. In a previous paper, we showed that the NaCl precipitation ponds (crystallizers) of Northern Adriatic Secovlje salterns have different haloarchaeal populations from those typically found in dry and hot climates such as Southern Spain. To check whether there is a common pattern of haloarchaeal diversity in these less extreme conditions, diversity in crystallizers of other Adriatic solar salterns in Ston, Croatia was ascertained by molecular and culture methods. In addition, the cultivation approach was used to further describe haloarchaeal diversity in both salterns. Over the period of two solar salt collection seasons, isolates related to species of the genera Haloferax, Haloarcula, and Haloterrigena were recovered from both salterns. Within the same sampling effort, relatives of the genus Halorubrum and a Natrinema-like isolate were cultivated from Slovenian Secovlje salterns while Halobacterium related isolates were obtained from the Croatian Ston salterns. Concurrent with our previous findings, a library of Croatian saltern crystallizer PCR-amplified 16S rRNA genes was dominated by sequences related to the genus Halorubrum. The microbial community structure was similar in both salterns but diversity indices showed greater values in Slovenian salterns when compared with Croatian salterns.
Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.
Bacteria that colonise the extreme environment of household dishwasher rubber seals were investigated using cultivation-dependent and metagenomic approaches. All 30 dishwashers investigated were colonised by various bacteria. Cultivation approaches resulted in 632 bacterial isolates in total, belonging to four phyla, eight classes, 40 genera and 74 species. The majority were Gram-positive, as solely Firmicutes and Actinobacteria. Bacilli represented half of the Gram-positive isolates, and were dominated by the Bacillus cereus group. Gammaproteobacteria were primarily represented by Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Escherichia coli. All isolates were tested for resistance to seven selected antibiotics. Metagenomic assessment of the bacterial biodiversity of the dishwasher rubber seals confirmed the predominance of Gram-positive bacteria, as primarily Actinobacteria dominated by Gordonia, followed by Proteobacteria dominated by Gammaproteobacteria, and by pathogenic species such as Escherichia sp., Acinetobacter baumannii, Pseudomonas sp., Stenotrophomonas maltophilia, and Enterobacter sp.. Metagenomic assessment of bacterial biodiversity in the tap water connected to dishwashers revealed predominance of Gram-negative bacteria, and in particular Proteobacteria, dominated by Betaproteobacteria, mainly represented by Tepidimonas sp.. Both Actinobacteria and Firmicutes showed low numbers, while there were markedly more Alphaproteobacteria and Betaproteobacteria in the tap water. These data indicate that colonisation of dishwasher rubber seals depends primarily on the bacterial input from the dirty vessels, and much less on the bacteria in the tap water.
BackgroundThe Escherichia coli uropathogenic-specific protein (Usp) is a bacteriocin-like genotoxin, active against mammalian cells and associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia. Usp is encoded by a small pathogenicity island with three downstream small open reading frames (Imu1-3) that are believed to provide immunity to the producer. To prevent host suicide, colicins, bacteriocins of E. coli, form tight complexes with their cognate immunity proteins. Colicin – immunity protein complexes are among the strongest protein complexes known. Here, the Usp associated immunity protein 3 (Imu3) was partially characterized to gain insight into its role and mechanism of activity.ResultsIsolation and partial characterisation of the Usp-associated immunity protein-3 (Imu3) revealed that, while Usp and Imu3 do not form a high affinity complex, Imu3 exhibits DNA and RNA binding activity. Imu3 was also shown to protect DNA against degradation by colicin E7.ConclusionsOur data infer that nonspecific DNA binding of the Imu3 immunity protein, prevents suicide of E. coli producing the genotoxin Usp.
Revival studies of Aeropyrum pernix show that the viability of cells and cell recovery after heat treatment depends on the temperature of treatment. Differential scanning calorimetry (DSC) is used to analyze the relative thermal stabilities of cellular components of A. pernix and to identify the cellular components responsible for the observed lag phase and reduced maximum growth following a heat treatment. DSC thermograms show 5 visible endothermic transitions with 2 major transitions. DSC analysis of isolated crude ribosomes aids the assignment of the 2 major peaks observed in whole-cell thermograms to denaturation of ribosomal structures. A comparison of partial and immediate full rescan thermograms of A. pernix whole cells indicates that both major peaks represent irreversible thermal transitions. A DNA peak is also identified in the whole-cell thermogram by comparison with the optical data of isolated pure DNA. DNA melting is shown to be irreversible in dilute solution, whereas it is partially reversible in whole cells, owing at least in part, to restricted volume effects. In contrast to mesophilic organisms, hyperthermophilic A. pernix ribosomes are more thermally stable than DNA, but in both organisms, irreversible changes leading to cell death occur owing to ribosomal denaturation.
Bacteria that colonise the extreme environment of household dishwasher rubber seals were investigated using cultivation-dependent and metagenomic approaches. All 30 dishwashers investigated were colonised by various bacteria. Cultivation approaches resulted in 632 bacterial isolates in total, belonging to four phyla, eight classes, 40 genera and 74 species. The majority were Gram-positive, as solely Firmicutes (dominated by the Bacillus cereus group) and Actinobacteria. Gammaproteobacteria were primarily represented by Stenotrophomonas maltophilia, Pseudomonas aeruginosa and Escherichia coli. All isolates were tested for resistance to seven selected antibiotics. Metagenomic assessment of the bacterial biodiversity of the dishwasher rubber seals confirmed the predominance of Gram-positive bacteria, as primarily Actinobacteria, followed by Proteobacteria dominated by Gammaproteobacteria, and by pathogenic species such as Escherichia sp., Acinetobacter baumannii, Pseudomonas sp., Stenotrophomonas maltophilia, and Enterobacter sp.. Metagenomic assessment of bacterial biodiversity in the tap water connected to dishwashers revealed predominance of Gram-negative bacteria, in particular Proteobacteria, mainly represented by Tepidimonas sp.. Both Actinobacteria and Firmicutes showed low numbers in the tap water. These data indicate that colonisation of dishwasher rubber seals probably depends primarily on the bacterial input from the dirty vessels, and much less on the bacteria in the tap water. Based on the antibiotic resistance data, the dishwasher rubber seal bacterial isolates do not represent a serious threat for the spread of antibiotic resistance into the household environment. Nevertheless dishwashers cannot be ignored as potential sources of human infections, in particular for immuno-compromised individuals.
BackgroundArchitectural proteins have important roles in compacting and organising chromosomal DNA. There are two potential histone counterpart peptide sequences (Alba1 and Alba2) in the Aeropyrum pernix genome (APE1832.1 and APE1823).Methodology/Principal FindingsThese two peptides were expressed and their interactions with various DNAs were studied using a combination of various experimental techniques: surface plasmon resonance, UV spectrophotometry, circular dichroism–spectropolarimetry, gel-shift assays, and isothermal titration calorimetry.Conclusions/SignificanceOur data indicate that there are significant differences in the properties of the Alba1 and Alba2 proteins. Both of these Alba proteins can thermally stabilise DNA polynucleotides, as seen from UV melting curves. Alba2 and equimolar mixtures of Alba1/Alba2 have greater effects on the thermal stability of poly(dA-dT).poly(dA-dT). Surface plasmon resonance sensorgrams for binding of Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 to DNA oligonucleotides show different binding patterns. Circular dichroism indicates that Alba2 has a less-ordered secondary structure than Alba1. The secondary structures of the Alba proteins are not significantly influenced by DNA binding, even at high temperatures. Based on these data, we conclude that Alba1, Alba2, and equimolar mixtures of Alba1/Alba2 show different properties in their binding to various DNAs.
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