Ginger Essential Oils-Loaded Nanoemulsions: Potential Strategy to Manage Bacterial Leaf Blight Disease and Enhanced Rice Yield.

The bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is one of the most serious rice diseases, causing huge yield losses worldwide. Several technologies and approaches have been opted to reduce the damage; however, these have had limited success. Recently, scientists have been focusing their efforts on developing efficient and environmentally friendly nanobactericides for controlling bacterial diseases in rice fields. In the present study, a scanning electron microscope (SEM), transmission electron microscope (TEM), and a confocal laser scanning microscope (CLSM) were utilized to investigate the mode of actions of ginger EOs on the cell structure of Xoo. The ginger EOs caused the cells to grow abnormally, resulting in an irregular form with hollow layers, whereas the dimethylsulfoxide (DMSO) treatment showed a typical rod shape for the Xoo cell. Ginger EOs restricted the growth and production of biofilms by reducing the number of biofilms generated as indicated by CLSM. Due to the instability, poor solubility, and durability of ginger EOs, a nanoemulsions approach was used, and a glasshouse trial was performed to assess their efficacy on BLB disease control. The in vitro antibacterial activity of the developed nanobactericides was promising at different concentration (50-125 µL/mL) tested. The efficacy was concentration-dependent. There was significant antibacterial activity recorded at higher concentrations. A glasshouse trial revealed that developed nanobactericides managed to suppress BLB disease severity effectively. Treatment at a concentration of 125 µL/mL was the best based on the suppression of disease severity index, AUDPC value, disease reduction (DR), and protection index (PI). Furthermore, findings on plant growth, physiological features, and yield parameters were significantly enhanced compared to the positive control treatment. In conclusion, the results indicated that ginger essential oils loaded-nanoemulsions are a promising alternative to synthetic antibiotics in suppressing Xoo growth, regulating the BLB disease, and enhancing rice yield under a glasshouse trial.

Single- or double-membrane-bound vesicles and P, Ca, and Fe-containing granules in Xanthomonas citri cultured on a solid medium.

The organelle-like structures of Xanthomonas citri, a bacterial pathogen that causes citrus canker, were investigated using an analytical transmission electron microscope. After high-pressure freezing, the bacteria were then freeze-substituted for imaging and element analysis. Miniscule electron-dense structures of varying shapes without a membrane enclosure were frequently observed near the cell poles in a 3-day culture. The bacteria formed cytoplasmic electron-dense spherical structures measuring approximately 50 nm in diameter. Furthermore, X. citri produced electron-dense or translucent ellipsoidal intracellular or extracellular granules. Single- or double-membrane-bound vesicles, including outer-inner membrane vesicles, were observed both inside and outside the cells. Most cells had been lysed in the 3-week X. citri culture, but they harbored one or two electron-dense spherical structures. Contrast-inverted scanning transmission electron microscopy images revealed distinct white spherical structures within the cytoplasm of X. citri. Likewise, energy-dispersive X-ray spectrometry showed the spatial heterogeneity and co-localization of phosphorus, oxygen, calcium, and iron only in the cytoplasmic electron-dense spherical structures, thus corroborating the nature of polyphosphate granules.

Synthesis and Antibacterial Evaluation of Novel 1,3,4-Oxadiazole Derivatives Containing Sulfonate/Carboxylate Moiety.

Abstract: In order to discover new lead compounds with high antibacterial activity, a series of new derivatives were designed and synthesized by introducing a sulfonate or carboxylate moiety into the 1,3,4-oxadiazole structure. Antibacterial activity against two phytopathogens, Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas axonopodis pv. citri (Xac), was assayed in vitro. The preliminary results indicated that ten compounds including 4a-1-4a-4 and 4a-11-4a-16 had good antibacterial activity against Xoo, with EC50 values ranging from 50.1-112.5 µM, which was better than those of Bismerthiazol (253.5 µM) and Thiodiazole copper (467.4 µM). Meanwhile, 4a-1, 4a-2, 4a-3 and 4a-4 demonstrated good inhibitory effect against Xanthomonas axonopodis pv. citri with EC50 values around 95.8-155.2 µM which were better than those of bismerthiazol (274.3 µM) and thiodiazole copper (406.3 µM). In addition, in vivo protection activity of compound 4a-2 and 4a-3 against rice bacterial leaf blight was 68.6% and 62.3%, respectively, which were better than bismerthiazol (49.6%) and thiodiazole copper (42.2%). Curative activity of compound 4a-2 and 4a-3 against rice bacterial leaf blight was 62.3% and 56.0%, which were better than bismerthiazol (42.9%) and thiodiazole copper (36.1%). Through scanning electron microscopy (SEM) analysis, it was observed that compound 4a-2 caused the cell membrane of Xanthomonas oryzae pv. oryzae ruptured or deformed. The present results indicated novel derivatives of 5-phenyl sulfonate methyl 1,3,4-oxadiazole might be potential antibacterial agents.

Xanthomonas citri subsp. citri and Xanthomonas arboricola pv. pruni: Comparative analysis of two pathogens producing similar symptoms in different host plants.

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.

Antibacterial effects of volatiles produced by Bacillus strain D13 against Xanthomonas oryzae pv. oryzae.

Recent investigations have demonstrated that bacteria employ the volatile compounds they produce during interactions with other organisms, such as plants, fungi, nematodes and bacteria. However, studies focused on the antibacterial activity of plant growth-promoting rhizobacteria (PGPR) volatiles against bacterial phytopathogens are still rare. In this study, Bacillus strain D13, which is antagonistic to Xanthomonas oryzae pv. oryzae (Xoo), was isolated and screened. Volatile compounds emitted from strain D13 reduced the colony diameter and cell motility of Xoo cultured in divided Petri plates. Transmission electron micrograph analysis showed concentration in cytoplasm and altered surface morphology in the majority of Xanthomonas cells after co-cultivation with strain D13. Transcriptional expression of virulence-associated genes in Xoo was repressed. Based on gas chromatography/mass spectrometry (GC/MS) analysis, 12 volatile compounds specifically produced by strain D13 were identified. Among them, decyl alcohol and 3,5,5-trimethylhexanol inhibited the growth of Xoo at minimum inhibitory amounts of 0.48 and 2.4 mg, respectively. Furthermore, transcriptional expression of virulence-associated genes was also repressed by decyl alcohol and 3,5,5-trimethylhexanol. These results are useful for a better understanding of the biocontrol mechanisms of Bacillus.

The Effect of Citrus Essential Oils and Their Constituents on Growth of Xanthomonas citri subsp. citri.

Citrus bacterial canker (CBC) caused by Xanthomonas citri subsp. citri (Xcc), is the most devastating of the citrus diseases worldwide. During our study, we found that Essential oils (EOs) of some citrus cultivars are effective on Xcc. Therefore, it prompted us to determine the plant metabolites responsible for the antibacterial properties. We obtained EOs from some locally cultivated citrus by using a Clevenger apparatus and their major constituents were identified by gas chromatography/mass spectrometry (GC-MS). The effect of Citrus aurantium, C. aurantifolia, Fortunella sp. EOs and their major constituents were evaluated against Xcc-KVXCC1 using a disk diffusion assay. Minimal inhibitory and bactericidal concentration of the EOs and their constituents were determined using the broth microdilution method. C. aurantium, C. aurantifolia Eos, and their major constituents including citral, linalool, citronellal, geraniol, α-terpineol, and linalyl acetate indicated antibacterial effects against Xcc. The C. aurantifolia EO and citral showed the highest antibacterial activity among the tested EOs and constituents with inhibition zones of 15 ± 0.33 mm and 16.67 ± 0.88 mm, respectively. Synergistic effects of the constituents were observed between α-terpineol-citral, citral-citronellal, citral-geraniol, and citronellal-geraniol by using a microdilution checkerboard assay. Transmission electron microscopy revealed that exposure of Xcc cells to citral caused cell wall damage and altered cytoplasmic density. We introduced C. aurantifolia and C. aurantium EOs, and their constituents citral, α-terpineol, citronellal, geraniol, and linalool as possible control agents for CBC.

Surface polysaccharides and quorum sensing are involved in the attachment and survival of Xanthomonas albilineans on sugarcane leaves.

Xanthomonas albilineans, the causal agent of sugarcane leaf scald, is a bacterial plant pathogen that is mainly spread by infected cuttings and contaminated harvesting tools. However, some strains of this pathogen are known to be spread by aerial means and are able to colonize the phyllosphere of sugarcane before entering the host plant and causing disease. The objective of this study was to identify the molecular factors involved in the survival or growth of X. albilineans on sugarcane leaves. We developed a bioassay to test for the attachment of X. albilineans on sugarcane leaves using tissue-cultured plantlets grown in vitro. Six mutants of strain XaFL07-1 affected in surface polysaccharide production completely lost their capacity to survive on the sugarcane leaf surface. These mutants produced more biofilm in vitro and accumulated more cellular poly-ß-hydroxybutyrate than the wild-type strain. A mutant affected in the production of small molecules (including potential biosurfactants) synthesized by non-ribosomal peptide synthetases (NRPSs) attached to the sugarcane leaves as well as the wild-type strain. Surprisingly, the attachment of bacteria on sugarcane leaves varied among mutants of the rpf gene cluster involved in bacterial quorum sensing. Therefore, quorum sensing may affect polysaccharide production, or both polysaccharides and quorum sensing may be involved in the survival or growth of X. albilineans on sugarcane leaves.

Xanthomonas albilineans OmpA1 appears to be functionally modular and both the OMC and C-like domains are necessary for leaf scald disease of sugarcane.

Several EZ-Tn5 insertions in gene locus XALc_0557 (OmpA1) of the sugarcane leaf scald pathogen Xanthomonas albilineans XaFL07-1 were previously found to strongly affect pathogenicity and endophytic stalk colonization. XALc_0557 has a predicted OmpA N-terminal outer membrane channel (OMC) domain and an OmpA C-like domain. Further analysis of mutant M468, with an EZ-Tn5 insertion in the upstream OMC domain coding region, revealed impaired epiphytic and endophytic leaf survival, impaired resistance to sodium dodecyl sulfate (SDS), structural defects in the outer membrane (OM), and hyperproduction of OM vesicles. Cloned full-length XALc_0557 complemented M468 for all phenotypes tested, including pathogenicity, resistance to SDS, and ability to survive both endophytically and epiphytically. Another construct, pCT47.3, which expressed only the C-like domain of XALc_0557, restored resistance to SDS in M468 but failed to complement any other mutant phenotype, indicating that the C-like domain functioned independently of the OMC domain to help maintain OM integrity. pCT47.3 also complemented pathogenicity, resistance to SDS, and stalk colonization in mutant M1152, which carries an EZ-Tn5 insert in the C-like coding region, indicating that both predicted domains are modular and necessary but neither is sufficient for X. albilineans pathogenicity, endophytic survival in, and epiphytic survival on sugarcane.

Action of chitosan against Xanthomonas pathogenic bacteria isolated from Euphorbia pulcherrima.

The antibacterial activity and mechanism of two kinds of chitosan were investigated against twelve Xanthomonas strains recovered from Euphorbia pulcherrima. Results indicated that both chitosans markedly inhibited bacterial growth based on OD loss. Furthermore, the release of DNA and RNA from three selected strains was increased by both chitosans. However, the release of intracellular proteins was inhibited by both chitosans at different concentration and incubation times, except chitosan A at 0.1 mg/mL for 0.5 h incubation and 0.2 mg/mL for 2.0 h incubation increased the release of proteins, indicating the complexity of the interaction and cell membranes, which was affected by incubation time, bacterial species, chitosan type and concentration. Transmission electron microscopic observations revealed that chitosan caused changes in protoplast concentration and surface morphology. In some cells, the membranes and walls were badly distorted and disrupted, while other cells were enveloped by a thick and compact ribbon-like layer. The contrary influence on cell morphology may explain the differential effect in the release of material. In addition, scanning electron microscope and biofilm formation test revealed that both chitosans removed biofilm biomass. Overall, this study showed that membrane and biofilm play an important role in the antibacterial mechanism of chitosan.

The wxacO gene of Xanthomonas citri ssp. citri encodes a protein with a role in lipopolysaccharide biosynthesis, biofilm formation, stress tolerance and virulence.

Xanthomonas citri ssp. citri (Xcc) causes citrus canker, one of the most economically damaging diseases affecting citrus worldwide. Biofilm formation is important for the pathogen to survive epiphytically in planta prior to the induction of canker symptoms. In this study, two EZ-Tn5 transposon mutants of Xcc strain 306, affected in biofilm formation, were isolated; subsequent analyses led to the identification of a novel gene locus XAC3596 (designated as wxacO), encoding a putative transmembrane protein, and the rfbC gene, encoding a truncated O-antigen biosynthesis protein. Sodium dodecylsulphate-polyacrylamide gel electrophoresis revealed that lipopolysaccharide (LPS) biosynthesis was affected in both wxacO and rfbC mutants. The wxacO mutant was impaired in the formation of a structured biofilm on glass or host plant leaves, as shown in confocal laser scanning microscopy analysis of strains containing a plasmid expressing the green fluorescent protein. Both wxacO and rfbC mutants were more sensitive than the wild-type strain to different environmental stresses, and more susceptible to the antimicrobial peptide polymyxin B. The two mutants were attenuated in swimming motility, but not in flagellar formation. The mutants also showed reduced virulence and decreased growth on host leaves when spray inoculated. The affected phenotypes of the wxacO and rfbC mutants were complemented to wild-type levels by the intact wxacO and rfbC genes, respectively. This report identifies a new gene influencing LPS production by Xcc. In addition, our results suggest that a structurally intact LPS is critical for survival in the phyllosphere and for the virulence of Xcc.

[Correlation between endogenous hydrogen peroxide and mesosome formation in cellular injured bacteria].

OBJECTIVE: Ultrastructural alteration and hydrogen peroxide (H2O2) localization were examined in Xanthomonas under cellular injury using transmission electron microscopy. METHODS: Histochemical methods were used in the present study. RESULTS: Intriguingly, the injury led to presence of an additional location of H2O2 accumulation within the cells. There was an association between the frequency and size of the additional location of H2O2 accumulation and the degrees of injury. Furthermore, an additional ultrastructure, mesosomes, was also present in injured cells. The frequency and size of mesosomes also increased with the increasing degrees of injury. CONCLUSION: Result of multiple linear regression showed that the size of mesosome plays as a key factor in the quantity of excess H2O2 accumulation in bacteria under cellular injury. Linear correlation was confirmed between quantity of excess H2O2 accumulation and the size of mesosome in injured cells. This finding intensely indicated that mesosomes are just the additional location of H2O2 accumulation in cells under cellular injury. The excess H2O2 accumulation in mesosomes should be a positive regulatory mechanism in bacteria under cellular injury.

Low temperature and anhydrous electron microscopy techniques to observe the infection process of the bacterial pathogen Xanthomonas fragariae on strawberry leaves.

Preserving the structural arrangement of the components of a bacterial infection process within a plant for microscopy study is a technical challenge because of the different requirements of each component for optimal preservation and visualization. We used low temperature scanning electron microscopy (cryo-SEM), anhydrous fixation at ambient temperature and freeze-substitution for transmission electron microscopy to examine fractured and sectioned strawberry leaves infected with Xanthomonas fragariae. Cryo-SEM images of fractured samples showed the bacterial colonization of mesophyll air spaces in the leaf, limited by the vascular bundles and the orientation and packing of bacteria in extracellular polysaccharide. Transmission electron microscopy of samples fixed using osmium tetroxide dissolved in FC-72 solvent at ambient temperature showed that the entire plant/bacteria/extracellular polysaccharide system was preserved in situ, and showed plasmolysis of mesophyll cells and disruption of organelles. In freeze-substitution samples, osmium tetroxide in FC-72 solvent gave superior preservation of the extracellular polysaccharide as compared to a conventional cocktail. In addition, strands believed to be xanthan were preferentially contrasted to show their density and orientation around the bacterial cells. We conclude that anhydrous fixation using osmium tetroxide in FC-72 at ambient temperature gave the best preservation of the entire system, and freeze-substitution using this same fixative enhanced the visualization of strands in the biofilm.

Renal and antibacterial effects induced by myotoxin I and II isolated from Bothrops jararacussu venom.

Bothrops jararacussu myotoxin I (BthTx-I; Lys 49) and II (BthTX-II; Asp 49) were purified by ion-exchange chromatography and reverse phase HPLC. In this work we used the isolated perfused rat kidney method to evaluate the renal effects of B. jararacussu myotoxins I (Lys49 PLA2) and II (Asp49 PLA2) and their possible blockage by indomethacin. BthTX-I (5 microg/ml) and BthTX-II (5 microg/ml) increased perfusion pressure (PP; ct120=110.28+/-3.70 mmHg; BthTX I=171.28+/-6.30*mmHg; BthTX II=175.50+/-7.20*mmHg), renal vascular resistance (RVR; ct120=5.49+/-0.54 mmHg/ml.g(-1)min(-1); BthTX I=8.62+/-0.37*mmHg/ml g(-1)min(-1); BthTX II=8.9+/-0.36*mmHg/ml g(-1)min(-1)), urinary flow (UF; ct(120)=0.14+/-0.01ml g(-1)min(-1); BthTX I=0.32+/-0.05*ml g(-1)min(-1); BthTX II=0.37+/-0.01*ml g(-1)min(-1)) and glomerular filtration rate (GFR; ct120=0.72+/-0.10 ml g(-1)min(-1); BthTX I=0.85+/-0.13*ml g(-1)min(-1); BthTX II=1.22+/-0.28*ml g(-1)min(-1)). In contrast decreased the percent of sodium tubular transport (%TNa(+); ct(120)=79,76+/-0.56; BthTX I=62.23+/-4.12*; BthTX II=70.96+/-2.93*) and percent of potassium tubular transport (%TK(+);ct120=66.80+/-3.69; BthTX I=55.76+/-5.57*; BthTX II=50.86+/-6.16*). Indomethacin antagonized the vascular, glomerular and tubular effects promoted by BthTX I and it's partially blocked the effects of BthTX II. In this work also evaluated the antibacterial effects of BthTx-I and BthTx-II against Xanthomonas axonopodis. pv. passiflorae (Gram-negative bacteria) and we observed that both PLA2 showed antibacterial activity. Also we observed that proteins Also we observed that proteins chemically modified with 4-bromophenacyl bromide (rho-BPB) decrease significantly the antibacterial effect of both PLA2. In conclusion, BthTx I and BthTX II caused renal alteration and presented activity antimicrobial. The indomethacin was able to antagonize totally the renal effects induced by BthTx I and partially the effects promoted by BthTx II, suggesting involvement of inflammatory mediators in the renal effects caused by myotoxins. In the other hand, other effects could be independently of the enzymatic activity of the BthTX II and the C-terminal domain could be involved in both effects promoted for PLA2.

Pair-dependent co-aggregation behavior of non-flocculating sludge bacteria.

Two strains of non-flocculating sewage sludge bacteria (Xanthomonas sp. S53 and Microbacterium esteraromaticum S51) showed 91% and 77% co-aggregation, respectively, with Acinetobacter johnsonii S35 using a spectrophometric assay. The co-aggregates in case of Xanthomonas sp. S53 and A. johnsonii S35 were above 100 microm and stable against EDTA (2 mM) and a commercial protease (0.2 mg ml(-1)). Protease/periodate pretreatment of the partners did not affect this co-aggregation. On the other hand, co-aggregates of M. esteraromaticum S51 and A. johnsonii S35 (50-70 microm) were deflocculated by EDTA or protease. Protease pretreatment of M. esteraromaticum S51 and periodate pretreatment of A. johnsonii S35 prevented their co-aggregation with respective untreated partners. The potential co-aggregation mechanisms of A. johnsonii S35 varied depending upon the other partner involved.

Gluconacetobacter diazotrophicus, a sugar cane endosymbiont, produces a bacteriocin against Xanthomonas albilineans, a sugar cane pathogen.

Gluconacetobacter diazotrophicus in liquid culture secretes proteins into the medium. Both medium containing Gluconacetobacter protein and a solution of this protein after acetone precipitation appeared to inhibit the growth of Xanthomonas albilineans in solid culture. This apparent inhibition of bacterial growth has, in fact, been revealed to be lysis of bacterial cells, as demonstrated by transmission electron microscopy. Fractionation of the Gluconacetobacter protein mixture in size-exclusion chromatography reveals a main fraction with lysozyme-like activity which produces lysis of both living bacteria and isolated cell walls.

Growth temperature-dependent variation of cell envelope lipids and antibiotic susceptibility in Stenotrophomonas (Xanthomonas) maltophilia.

Clinical isolates of Stenotrophomonas (Xanthomonas) maltophilia showed growth temperature-dependent variation in susceptibility (TDVS) to aminoglycoside antibiotics between 30 degrees C and 37 degrees C, but little or no TDVS effect for polymixin B, colistin, ceftazidime, chloramphenicol and piperacillin. When phenylethanol was added at sub-inhibitory concentrations, the TDVS effect was eliminated. Gas liquid chromatography showed that 13-methyl tetradecanoate (i-15;0), was the predominant fatty acid, and was present in lower proportions in cells grown at 30 degrees C than 37 degrees C, by contrast to the unsaturated acids, which were found in increased proportions in cells grown at 30 degrees C. However, the extent of these shifts in composition did not correlate with the extent of the TDVS effect in individual strains. Membrane analysis by spin label-electron spin resonance spectroscopy showed that strains exhibiting TDVS had significantly decreased membrane fluidity compared with susceptible strains at 30 degrees C. Furthermore, analysis of the outer and cytoplasmic membranes from the strains with TDVS revealed that in organisms grown at 30 degrees C, the outer membrane remained in a more rigid conformation than the cytoplasmic membrane. We conclude that resistance of S. maltophilia to aminoglycoside antibiotics at 30 degrees C correlates with changes in the conformation of the outer membrane so that binding and/or uptake of the antibiotic is inhibited.

AVRXa10 protein is in the cytoplasm of Xanthomonas oryzae pv. oryzae.

AVRXa10 from Xanthomonas oryzae pv. oryzae was tagged with a unique hydrophilic octapeptide (FLAG) to permit antibody-mediated identification and purification of the gene product. X. o. pv. oryzae that produced tagged AVRXa10 elicited a hypersensitive response (HR) on rice cultivars containing the resistance gene Xa-10, but not on cultivars lacking Xa-10. The tagged AVRXa10 protein purified from Escherichia coli or X. o. pv. oryzae did not elicit a hypersensitive response in rice with the Xa-10 resistance gene. Anti-FLAG monoclonal antibodies reacted with a 119-kDa protein in both E. coli and X. o. pv. oryzae cells expressing the tagged avrXa10 gene. Polyclonal antibodies raised against purified AVRXa10 protein reacted with the 119-kDa protein and several additional proteins from X. o. pv. oryzae, which probably are the products of genes related to avrXa10. Biochemical fractionation and immunoelectronmicroscopy analysis was used to demonstrate that AVRXa10 was located in the cytoplasm of X. o. pv. oryzae cells when grown in planta or in culture medium.

Differential binding capacity and internalisation of bacterial substrates as factors in growth rate of Acanthamoeba spp.

Acanthamoeba spp. are free-living predators that selectively feed on bacteria. Adherence of the bacterial food source to the trophozoite membrane is followed by internalisation and digestion. Through co-cultivation of A. castellanii and A. polyphaga, individually, with Xanthomonas maltophilia, Escherichia coli, Staphylococcus epidermidis and Pseudomonas aeruginosa (despite the amoebicidal properties of the latter organism), specificity with regard to the preferred bacterial substrate was judged. X. maltophilia and P. aeruginosa adhered almost immediately forming a multilayered mantle of bacilli around trophozoites of both species of amoebae. E. coli adhered to fewer trophozoites and in smaller numbers. X. maltophilia was readily internalised after co-cultivation for 8 h, whereas P. aeruginosa, E. coli and S. epidermidis were not internalised even after co-cultivation for 24 h. These data suggest that the suitability of a bacterial food source for the Acanthamoeba spp. studied is associated not only with the proclivity with which the bacterial species binds to the trophozoite surface, but also with the rate of its internalisation.

Coagulation autolysis in microorganisms and its relation to coagulase production.

The phenomenon of coagulation autolysis was observed in two model microorganisms, i.e., a bacterial culture and an imperfect fungus. It was characterized by impairment of the cell membranes, followed by condensation and dehydration of the cytoplasm and long-term preservation of the cells in the form of coagulated cytoplasm. In this respect, it was similar to coagulation necrosis of human tissues. The autolysis in the microorganisms was accompanied by increase of their coagulase activity, the substrate specificity of the enzyme rather broad. The coagulase activity of the microorganisms was detected during the culture period between the lag-phase and the exponential growth phase, i.e., the phase of their active growth. It served as a signal to induce biosynthesis of peptidohydrolase and cleavage of proteins. We believe that the phenomenon of coagulation autolysis in these microorganisms is rather typical and can be considered as an adaptative reaction, inducing a cascade of events from synthesis of coagulase to overproduction of peptidohydrolases with proteolytic activity.

Immunoelectron microscopic demonstration of an esterase on the outer membrane of Xanthomonas maltophilia.

Xanthomonas maltophilia (later synonym of Pseudomonas maltophilia), an ubiquitous species, is known to show proteolytic and lipolytic activities. A cell-bound esterase which hydrolyzes beta-naphthyl acetate during growth has been extracted from a strain isolated from soil. Because of its strongly hydrophobic character, the enzyme could be efficiently solubilized only by Triton X-100. This nonionic detergent must be added in polyacrylamide gels to permit migration. Polyclonal rabbit antibodies raised against the Triton-soluble esterase complex were used to localize the enzyme at the ultrastructural level. Electron microscopy of cell sections of this organism and immunogold labeling demonstrated that the enzyme was located on the outer membrane. Such an envelope-bound esterase may produce assimilable substrates for X. maltophilia which can grow in various environments.