Exogenous phage recombinase-independent inactivation of chromosomal genes in Yersinia enterocolitica

Characterization of newly identified genes is necessary to understand their functions. Phenotypic characterization of isogenic mutants provides good understanding of the functions of the genes in wild type strains. In the present study, we report the use of linear dsDNA as a substrate for homologous recombination in Yersinia enterocolitica. A double-stranded linear recombinant DNA (LRD) containing an antibiotic resistance gene flanked by homologous regions to the target gene was created. Transformation of this LRD into Y. enterocolitica led to the replacement of targeted loci with antibiotic resistance gene. Using this strategy, two chromosomal genes namely urease C (ureC) and hemophore A (hasA) were disrupted in three strains of Y. enterocolitica. These recombinations were independent of the EPR functions. This is the first report of EPR-independent inactivation of chromosomal genes in Y. enterocolitica strains.     

A simplified method for detecting pathogenic Yersinia enterocolitica in slaughtered pig tonsils

The aim of this study was to collect preliminary data on the carriage of pathogenic Yersinia enterocolitica in slaughtered pigs in France and to test a simplified method for detecting these strains from tonsils. From January to March 2009, 900 tonsil swabs were taken from pigs at one slaughterhouse in Brittany, France. The swabs were vortexed in 10 ml PSB broth, then 1 ml was added to 9 ml ITC broth. The media were incubated for 48 h at 25 °C. The PSB enrichment broth was streaked on CIN plates and the ITC enrichment broth on SSDC plates. In addition to the ISO 10273 method, we also streaked ITC enrichment broth on CIN plates. The plates were incubated for 24 h at 30 °C, and we then streaked a maximum of four typical colonies per plate onto a plate containing chromogenic medium (YeCM), for the isolation of pathogenic Y. enterocolitica isolates. In parallel, biochemical assays were carried out to confirm the identification of the isolates as Yersinia and to determine biotype.After passage on a YeCM plate and biochemical tests, 380 strains were confirmed to be pathogenic Y. enterocolitica. Finally, with the ISO 10273 method, 9.1% (CI_95% [5.8-12.4]) of tonsil swabs and 60% (CI_95% [45.4-74.6]) of the batches were positive. With the ITC-CIN method, 14.0% (CI_95% [10.7-17.3]) of the tonsil swabs and 68.9% (CI_95% [54.3-83.5]) of the batches were positive. Identification as pathogenic Y. enterocolitica was confirmed for 97.0% of the typical colonies obtained on the chromogenic medium, YeCM. The most prevalent biotype was biotype 4 (80.5% of the isolates), followed by biotype 3.This study demonstrates that the ITC-CIN method, followed by streaking on YeCM, may be an effective approach to the isolation of pathogenic Y. enterocolitica from tonsil swabs and the recovery of positive samples. This method is less time-consuming than the ISO 10273 method and reduces the number of biochemical tests required for the confirmation of Yersinia identification, through the use of YeCM.     

The reversible depletion and reconstitution of a copper ion in Coprinus cinereus laccase followed by spectroscopic techniques

The specific activities of crude and purified Coprinus cinereus laccase preparations could be enhanced by a factor of 10-12 by activation with copper ions. The copper to protein contents of purified non-activated laccase were 2.3 ± 0.1 compared to 3.3 ± 0.1 in purified activated laccase indicating that only a fraction of the laccase can be activated. Purified laccase not activated with copper ions shows in isoelectric focusing four bands in order of decreasing pI in a ratio 1/5/3/1 where only bands I and II had laccase activity. Purified activated laccase showed only three bands (I, II and III) in the ratio 5/4/1 all with some laccase activity. The pH profile of the activity for activated and non-activated laccase showed identical behavior indicating that the active forms were the same. The change in UV-Vis around 330 nm following the depletion and reconstitution of the enzyme combined with activity measurements supports the reversibility of the selective removal and insertion of copper ions at the type 2 site. The circular dichroism spectrum of activated purified laccase has characteristic changes around 350 nm relative to non-activated laccase indicative of changes at the type 2/type 3 sites. The difference between the electron paramagnetic resonance spectra of non-activated and activated C. cinereus laccase indicates that a fraction of the non-activated purified laccase contained a copper(II) signal with a coupling constant between a type 1 and a type 2 copper(II). This electron paramagnetic resonance signal could be explained by an induced asymmetry in the type 3 site due to a missing type 2 copper ion.     

Selective isolation of Yersinia pestis from plague-infected fleas

We evaluated Yersinia CIN agar for the isolation of Yersinia pestis from infected fleas. CIN media is effective for the differentiation of Y. pestis from flea commensal flora and is sufficiently inhibitory to other bacteria that typically outcompete Y. pestis after 48 h of growth using less selective media.     

Oxidation of anthracene by immobilized laccase from Trametes versicolor

The laccase of Trametes versicolor was immobilized on the functionalized nanoparticles SBA-15 with the average diameter less than 10 nm. Laccase mediated oxidations of anthracene (ANT) were investigated in the presence of two mediators, 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) and 1-hydroxybenzotriazole (HBT). Oxidation of ANT was more efficiently enhanced by adding 1 mM of HBT than that by adding ABTS. After 48 h oxidation HBT group significantly oxidized ANT with residue 58% relative to 88% in the ABTS group. HPLC and GC/MS analyses indicated the main product of ANT oxidation was anthraquinone (ANQ). The fluorescein diacetate (FDA) uptake of two human cell lines was used to assess the cytotoxicity and genotoxicity of ANT and ANQ. Treatments with ANT and ANQ at 5 and 10 μM exhibited significant cytotoxicity to the HaCaT cells and the A3 lymphocytes and no significant genotoxicity was observed. The results illustrated that ANQ is less toxic than ANT as well.     

Structure of the O-antigen of Yersinia pseudotuberculosis O:4a revised

The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Yersinia pseudotuberculosis O:4a and studied by NMR spectroscopy, including 2D ROESY and ¹H, ¹³C HMBC experiments. The following structure of the pentasaccharide repeating unit of the polysaccharide was established, which differs from the structure reported earlier [Gorshkova, R. P. et al., Bioorg. Khim. 1983, 9, 1401-1407] in the linkage modes between the monosaccharides: where Tyv stands for 3,6-dideoxy-d-arabino-hexose (tyvelose). The structure of the Y. pseudotuberculosis O:4a antigen resembles that of Y. pseudotuberculosis O:2c, which differs in the presence of abequose (3,6-dideoxy-d-xylo-hexose) in place of tyvelose only.     

Characterization of an aspartate-dependent acid survival system in Yersinia pseudotuberculosis

Enteric bacteria have developed various survival systems that protect against acid stress. In this study, an aspartate-dependent acid survival system is characterized in Yersinia pseudotuberculosis. The expression of aspartase (AspA) was confirmed to be increased at acidic pH by proteomic and lacZ fusion analyses. Addition of aspartate increased acid survival of the wild type but not the aspA knockout mutant. AspA increases acid survival by producing ammonia as demonstrated by mutation and in vitro enzyme activity analyses. This is the first demonstration that an enzyme involved in aspartate metabolism plays a role in acid survival in an enteric bacterium.     

Structural modelling of substrate binding and inhibition in penicillin V acylase from Pectobacterium atrosepticum

Penicillin V acylases (PVAs) and bile salt hydrolases (BSHs) have considerable sequence and structural similarity; however, they vary significantly in their substrate specificity. We have identified a PVA from a Gram-negative organism, Pectobacterium atrosepticum (PaPVA) that turned out to be a remote homolog of the PVAs and BSHs reported earlier. Even though the active site residues were conserved in PaPVA it showed high specificity towards penV and interestingly the penV acylase activity was inhibited by bile salts. Comparative modelling and docking studies were carried out to understand the structural differences of the binding site that confer this characteristic property. We show that PaPVA exhibits significant differences in structure, which are in contrast to those of known PVAs and such enzymes from Gram-negative bacteria require further investigation.     

Anthraquinone dye assisted the decolorization of azo dyes by a novel Trametes trogii laccase

A new Trametes trogii laccase was purified and its biochemical properties were subsequently characterized. After a survey of other T. trogii laccases, this laccase showed a lower isoelectric point, different N-terminal sequence and kinetic parameters. Recently most laccase-catalyzed decolorizations of synthetic dyes are single-solute studies with commercially available dyes as model pollutants and need the employment of redox mediators. In this study, to simulate the real industry wastewaters, experiments of laccase-catalyzed decolorization of mixed dyes constituted by azo and anthraquinone dyes were carried out. The results showed that anthraquinone dyes, playing the role of mediators, dramatically promoted the degradation of azo dyes when there was no exogenous mediator in the reaction mixture. This study represents the first attempt to decolorize the mixtures of azo and anthraquinone dyes by purified T. trogii laccase, suggesting great potential for laccase to decolorize textile industry wastewaters.     

Pre-steady state kinetic studies on the microsecond time scale of the laccase from Trametes versicolor

Laccase from Trametes versicolor reduces dioxygen to water. The enzyme is used in green chemistry applications such as the selective oxidation of alcohols in the presence of a suitable mediator (TEMPO) or in biofuel cells. We studied the catalytic mechanism of the enzyme by the stopped-flow and our newly developed rapid-mixing rapid sampling method, which has an experimental dead time of 75 ± 15 μs. Equilibrium and kinetic analyses yielded a reduction potential of 717 ± 5 mV for Type 1 copper center. EPR and low-temperature UV-Vis spectroscopy indicate that oxidation of the blue copper center and OO bond splitting occur within 100 μs, without detectable formation of a peroxide intermediate. These results indicate a rapid internal electron transfer between the various copper centers (>25.000/s) and rapid binding of O₂ (k_on > 5 × 10⁷ M⁻¹ s⁻¹). Mechanistic aspects of the catalytic cycle are shortly discussed.     

Biochemical insights into a novel thermo/organo tolerant bilirubin oxidase from Thermosediminibacter oceani and its application in dye decolorization

Higher activity and stability at neutral pH and tolerance toward anions have made bilirubin oxidases (BODs) proper candidates for industrial utilizations. A putative BOD from Thermosediminibacter oceani (ToBOD) exhibited high stability over a pH range of 3.5–10.0 after heterologous expression and purification. The optimal temperature for the enzyme activity was 75 °C. ToBOD displayed a high thermostability with a half-life of 180 min at 70 °C and 120 min at 80 °C, respectively. K_m and K_cat values were 126.5 μM and 130.9 S⁻¹ for ABTS, 19.6 μM and 72.5 S⁻¹ for SGZ, and 31.2 μM and 76.2 S⁻¹ for unconjugated bilirubin, respectively. ToBOD showed tolerance to 10% and 50% (v/v) of water-miscible organic solvents and Triton X-100 as a non-ionic surfactant. In the presence of ABTS as the mediator, ToBOD decolorized malachite green (MG) and Congo red (CR) dyes at a rate of about 63% and 71%, respectively in 2 h. Decolorization was improved within 4 h at a rate of 86% and 89% for MG and CR, respectively. Structural analyses of ToBOD showed that lower folding heat capacity, folding enthalpy, folding free energy, and side-chain hydrogen bonds have a correlation with in vitro biochemical properties.     

Inhibitors of polygalacturonase and laccase of Botrytis cinerea and their application to the control of this fungus

EDTA, calcium chloride, and two siderophores (rhodotorulic acid produced by Rhodotorula glutinis BNM 0524, and enterochelin from the bacterium Rahnella aquatilis BNM 0523) were evaluated as possible inhibitors of polygalacturonase (PG) and laccase (LC) from Botrytis cinerea. The aim was to apply them to the control of this pathogen, taking into account the fact that these enzymes are related to the invasion and installation of the fungus in the host. Two B. cinerea Pers.:Fr strains (BNM 0527 and BNM 0528) were used. Enzyme activities were measured in the supernatant of 7-day-old cultures. EDTA, calcium chloride, rhodotorulic acid, or enterochelin were added in the reaction mixture. Laccase activity from two strains was more affected by enterochelin (70-80% inhibition) than by the other compounds, while polygalacturonase was more inhibited (45% inhibition) by calcium chloride. The inhibitors were added to the growth medium and after 7 days of culture, the activities of the enzymes were measured in the supernatants. The production of PG and LC in both strains was lower when enterochelin or calcium chloride was added. In the third step, when the inhibitors were tested on apple, all them provided both effects, preventive and curative, against infections caused by B. cinerea, with EDTA and rhodotorulic acid exhibiting more preventive effects while calcium chloride and enterochelin provided more control of pre-existing infections (curative effect), coinciding with their ability to inhibit the production of polygalacturonase and laccase.     

The TonB protein of Yersinia enterocolitica and its interactions with TonB-box proteins

Summary The tonB gene is required for energy-dependent transport processes across the outer membrane of gram-negative bacteria. Using the antibiotics albomycin and ferrimycin, a tonB mutant of Yersinia enterocolitica was isolated. Comparison of the tonB mutant with the parent strain revealed that in Y. enterocolitica the uptake of ferrioxamine, ferrichrome, pesticin and heme is TonB-dependent. The tonB gene from Y. enterocolitica was sequenced and found to be similar to those of other Enterobacteria. The Y. enterocolitica tonB gene complemented a Y. enterocolitica tonB mutant. In contrast, some Tong functions of an Escherichia coli tonB mutant were not restored by the tonB gene of Y. enterocolitica. The observed differences in the ability to complement E. coli Tong functions correlated with the degree to which the Tong boxes of the receptors and colicins differed from the TonB box consensus sequence. Furthermore, the N-terminal membrane anchor of the TonB proteins and the TolA protein are likely to form an -helix with an identical sequence motif (SHLS) located at one face of the a-helix, suggesting this region to be involved in the functional cross-talk between the TonB-ExbBD-and TolABQR-dependent transport systems across the outer membrane.     

Development of an up-converting phosphor technology-based 10-channel lateral flow assay for profiling antibodies against Yersinia pestis

In this study, a 10-channel up-converting phosphor technology-based lateral flow (TC-UPT-LF) assay was developed to profile antibodies against Yersinia pestis. Ten expressed Y. pestis proteins were covalently conjugated with an up-converting phosphor particle to develop double-antigen sandwich immunochromatographic strips to detect corresponding antibodies. After optimization one by one, each strip was integrated into a TC-UPT-LF disc for simultaneously detection of different antibodies. A scanning biosensor was also developed to acquire the results. The performance of the TC-UPT-LF assay was evaluated by using standard samples and plague monkey serum samples. Fifty-one patient serum samples were detected by the TC-UPT-LF assay. The TC-UPT-LF disc could be stable for 10 days at 37 °C with an average CV of 10.3%. Its sensitivity and qualitative results are comparable to those of ELISA. Its linearity fitting coefficient of determination (R²) for different antibody detection is between 0.93 and 0.99. Besides F1 antibody, the LcrV and YopD antibodies also showed higher positive ratio than the other seven antibodies, as 100% (13/13) and 92% (12/13) in monkey sera and 86.3% (44/51) and 66.7% (34/51) in patient sera, respectively. It is suggested that the TC-UPT-LF assay has been successfully developed for multi-detection and LcrV and YopD can be the potential diagnostic markers of the plague.     

小肠结肠炎耶尔森菌对多粘菌素B的压力应答

【背景】小肠结肠炎耶尔森菌(Yersinia enterocolitica)是重要的人畜共患食源性病原菌。由于其生存环境与传染性生活方式,小肠结肠炎耶尔森菌暴露在各种生存压力中,而胞膜压力应答能力对维持其环境耐受性和毒力发挥着重要作用。【目的】探究小肠结肠炎耶尔森菌在胞膜压力应答中的调节机制。【方法】通过使用多粘菌素B破坏小肠结肠炎耶尔森菌细胞膜的稳定性,并从生长能力、运动能力、生物被膜形成能力以及相关基因表达的变化探讨Rcs (Regulator of Capsule Synthesis)系统对多粘菌素B产生的胞膜压力的应答。【结果】多粘菌素B引起的细胞胞膜压力抑制了小肠结肠炎耶尔森菌的运动和生物被膜形成能力;而阻断Rcs信号途径后,小肠结肠炎耶尔森菌的运动和生物被膜形成能力有所恢复。对flhC、hmsS、hmsT等关键下游表型基因的表达水平的分析结果表明Rcs双组分系统对由多粘菌素B诱导的胞膜压力作出响应,通过感知胞膜胁迫向胞内传递信号,积极地调控细菌增强对抗菌肽的抗性。【结论】明确了Rcs双组分系统在响应多粘菌素B压力胁迫中的特异性调控作用,加深了对小肠结肠炎耶尔森菌环境应答机制...     

Influence of NaCl and Na2SO4 on the kinetics and dye decolorization ability of crude laccase from Ganoderma lucidum

In a solid state medium using yellow passion fruit waste as substrate, the basidiomycete Ganoderma lucidum produced a laccase as the main ligninolytic enzyme. This crude enzyme presented Michaelian behavior with both substrates tested, namely 3-ethylbenzthiazoline-6-sulphonic acid (ABTS) and the anthraquinone dye remazol brilliant blue R (RBBR). The K_M’s for these substrates were, respectively, 0.232 × 10⁻³ and 0.602 × 10⁻³ M. The actions of NaCl and Na₂SO₄, two important salts usually found in textile wastewaters, were investigated. The enzyme was inhibited by NaCl, but not by Na₂SO₄. Inhibition by NaCl was of the mixed type with two different inhibition constants. The enzyme was able to completely decolorize RBBR in the presence of 1.0 M Na₂SO₄ and 50% decolorization was found in the presence of 0.1 M NaCl. Such properties certainly make the enzyme a good agent for textile dye effluent treatment considering the fact that wastewaters of this industry usually contain high concentrations of NaCl and Na₂SO₄.