Xanthomonas arboricola pv. pruni (Xap) causes bacterial spot of stone fruit and almond, an important plant disease with a high economic impact. Biofilm formation is one of the mechanisms that ...microbial communities use to adapt to environmental changes and to survive and colonize plants. Herein, biofilm formation by Xap was analyzed on abiotic and biotic surfaces using different microscopy techniques which allowed characterization of the different biofilm stages compared to the planktonic condition. All Xap strains assayed were able to form real biofilms creating organized structures comprised by viable cells. Xap in biofilms differentiated from free-living bacteria forming complex matrix-encased multicellular structures which become surrounded by a network of extracellular polymeric substances (EPS). Moreover, nutrient content of the environment and bacterial growth have been shown as key factors for biofilm formation and its development. Besides, this is the first work where different cell structures involved in bacterial attachment and aggregation have been identified during Xap biofilm progression. Our findings provide insights regarding different aspects of the biofilm formation of Xap which improve our understanding of the bacterial infection process occurred in Prunus spp and that may help in future disease control approaches.
Comparative studies in Xanthomonas have provided a vast amount of data that enabled to deepen in the knowledge of those factors associated with virulence and Xanthomonas plant interaction. The ...species of this genus present a wide range of host plants and a large number of studies have been focused to elucidate which mechanism are involved in this characteristic. In this study, comparative genomic and phenotypic analysis were performed between X. citri subsp. citri (Xcc), one of the most studied pathogens within Xanthomonas, and X. arboricola pv. pruni (Xap), a pathogen which has aroused great interest in recent time. The work was aimed to find those elements that contribute to their host divergence despite the convergence in the symptoms that each species cause on Citrus spp. and Prunus spp., respectively. This study reveals a set of genes that could be putatively associated with the adaptation of these pathogens to their hosts, being the most remarkable those involved in environmental sensing systems such as the case of the TonB-dependent transporters, the sensors of the two-component system and the methyl accepting chemotaxis proteins. Other important variants were found in processes related to the decomposition of the cell wall as could be appreciated by their dissimilar set of cell-wall degrading enzymes. Type three effectors, as one of the most important factors in delineating the host specificity in Xanthomonas, also showed a different array when comparing both species, being some of them unique to each pathogen. On the other hand, only small variations could be connected to other features such as the motility appendages and surface adhesion proteins, but these differences were accompanied by a dissimilar capacity to attach on host and non-host leaf surface. The molecular factors found in this work provide the basis to perform a more in-depth functional analyses that unveil those actual factors associated with pathogenesis and host specificity in Xcc and Xap.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The preparation of immobilized derivatives of lipases that may be useful to develop industrial processes of organic synthesis is an exciting field of research in which three main features have to be ...simultaneously considered: (a) immobilized derivatives have to be compatible with very different reaction requirements (e.g. continuos adjustment of pH with concentrated alkali, use of aqueous media or organic solvents, etc.); (b) Sometimes, some activity/stability properties of lipases should be improved during immobilization; and (c) because of a complex mechanism of action, lipases are poorly active in the absence of hydrophobic interfaces. In this paper, we will review different approaches for lipase immobilization mainly related to the further use of immobilized derivatives to carry out enantio and regioselective hydrolysis in high water-activity systems. Special emphasis is paid to the selective adsorption of lipases on tailor-made strongly hydrophobic support surfaces. This new immobilization procedure is based on the assumption that the large hydrophobic area that surrounds the active site of lipases is the one mainly involved in their adsorption on strongly hydrophobic solid surfaces. Thus, lipases recognize these surfaces similarly to those of their natural substrates and they suffer interfacial activation during immobilization. This immobilization method permits: (a) promote a dramatic hyper-activation of most of lipases after their immobilization. That is, adsorbed lipases show very enhanced esterase activity in the absence of additional hydrophobic interfaces; (b) promote highly selective adsorption of lipases, at very low ionic strength, from impure protein extracts. That is, we can associate immobilization and purification of lipases; (c) promote interesting improvements of enantioselectivity after immobilization; and (d) promote a strong but reversible immobilization that enables us to recover these expensive supports after inactivation of immobilized lipases.
Fluorescent proteins have been used to track plant pathogens to understand their host interactions. To be useful, the transgenic pathogens must present similar behaviour than the wild-type isolates. ...Herein, a GFP marker was used to transform two plant pathogenic bacteria,
Agrobacterium
and
Xanthomonas
, to localize and track the bacteria during infection. The transgenic bacteria were evaluated to determine whether they showed the same fitness than the wild-type strains or whether the expression of the GFP protein interfered in the bacterial activity. In
Agrobacterium
, the plasmid used for transformation was stable in the bacteria and the strain kept the virulence, while
Xanthomonas
was not able to conserve the plasmid and transformed strains showed virulence variations compared to wild-type strains. Although marking bacteria with GFP to track infection in plants is a common issue, works to validate the transgenic strains and corroborate their fitness are not usual. Results, presented here, confirm the importance of proper fitness tests on the marked strains before performing localization assays, to avoid underestimation of the microbe population or possible artificial effects in its interaction with the plant.
Three different approaches are proposed to increase the resistance of enzymes against hydrogen peroxide. (a)
Multipoint covalent immobilization. Through this technique, enzyme rigidity would be ...greatly increased and hence, any conformational change on the enzyme structure involved before or after oxidation with hydrogen peroxide becomes greatly prevented. (b)
Oriented immobilization on supports having large internal surfaces. The immobilization of enzymes, through different areas of their surface on solid supports with internal morphology composed by large surfaces, promotes a certain masking of the enzyme areas that are very close to the support surface. In this way, the accessibility of hydrogen peroxide to such protein areas becomes greatly restricted. (c)
Additional chemical modification of immobilized enzyme derivatives with polymers. By adding thick barriers surrounding the whole enzyme molecule, the effective concentration of hydrogen peroxide in the proximity of the most sensitive residues may be strongly reduced. Multipoint covalently immobilized
d-amino acid oxidase (DAAO) from
Rhodotorula gracilis on glyoxyl-agarose is 11-fold more stable than native enzyme against the deleterious effect of hydrogen peroxide. On the other hand, DAAO from
Trigonopsis variabilis was not stabilized by rigidification but it could be highly stabilized by an adequate combination of the best orientation on the support plus an additional modification with poly-aldehyde polymers.
The crude porcine pancreas lipase (PPL) extract is a mixture of several proteins (mainly lipases and esterases). In order to develop enzymatic catalysts with good catalytic properties for hydrolytic ...enantioselective reactions in aqueous homogeneous medium, we studied the immobilization of the different enzymes contained in the crude PPL extracts by selective sequential adsorption on hydrophobic supports bearing octyl, octadecyl and phenyl groups. Some minor proteins were selectively adsorbed on octyl and octadecyl supports while the most abundant lipase was adsorbed on the support bearing phenyl groups. The enantioselectivity of the different lipase derivatives were tested considering the hydrolysis of esters of 1,2-epoxi-1-propanol (glycidol). The most abundant lipase contained in the commercial crude PPL extract resulted almost inactive while some lipases contained in low concentrations displayed high activities and enantioselectivities. The most interesting results were obtained with a 28-kDa protein selectively adsorbed on octyl-agarose. With this enzyme derivative, the residual butyric ester of glycidol was recovered with 96% enantiomeric excess at 55% conversion.
Lipases contained in commercial samples of lipase extracts from
Rhizopus niveus (RNL) and
Candida rugosa (CRL) have been selectively adsorbed on hydrophobic supports at very low ionic strength. Under ...these conditions, adsorption of other proteins (including some esterases) is almost negligible. More interestingly, these lipases could be separated in several active fractions as a function of a different rate or a different intensity of adsorption on supports activated with different hydrophobic groups (butyl-, phenyl- and octyl-agarose). Thus, although RNL seemed to be a homogeneous sample by SDS-PAGE, it could be separated, via sequential adsorption on the different supports, into three different fractions with very different thermal stability and substrate specificity. For example, one fraction hydrolyzed more rapidly ethyl acetate than ethyl butyrate, while another hydrolyzed the acetate ester 7-fold slower than the butyrate. Similar results were obtained with samples of CRL. Again, we could obtain three different fractions showing very different properties. For example, enantioselectivity for the hydrolysis of (
R,
S) 2-hydroxy-4-phenylbutanoic acid ethyl ester ranged from 1.2 to 12 for different CRL fractions. It seems that very slight structural differences may promote a quite different interfacial adsorption of lipases on hydrophobic supports as well as a quite different catalytic behavior. In this way, this new ‘interfacial affinity chromatography’ seems to be very suitable for an easy separation of such slightly different lipase forms.
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