The ability of disease and non-disease producing strains of Clostridium perfringens from chickens to adhere to extracellular matrix molecules and Caco-2 cells

Clostridium perfringens is a major enteric pathogen that is responsible for causing necrotic enteritis of poultry. The ability to adhere to the host’s intestinal epithelium and to extracellular matrix molecules (ECMM) in the gut, are strategies used by numerous bacterial enteropathogens, however, C. perfringens has received comparatively little attention in this respect. The present study investigated sixteen type A C. perfringens isolates from chickens, with varying disease producing ability with respect to necrotic enteritis in chickens, for their ability to adhere to nine different extracellular matrix molecules (ECMM) and to the intestinal epithelial cell line Caco-2. C. perfringens strains were able to bind to ECMMs and there was strain variation. Strains of C. perfringens that produced severe disease, were capable of binding to collagen type III, IV and V, fibrinogen, laminin and vitronectin at higher levels than less severe disease producing strains, suggesting that the ability to adhere to ECMMs might enhance virulence with respect to induction of necrotic enteritis. In addition, severe disease producing strains also bound better to collagen type III and IV and fibrinogen, than non-disease producing strains. The present study also showed that some strains of C. perfringens possessed the ability to adhere to Caco-2 cells; however no relationship was found between the ability to adhere to Caco-2 cells and disease producing ability.     

Polymorphism in a flea beetle for the ability to use an atypical host plant

The flea beetle Phyllotreta nemorum L. (Coleoptera: Chrysomelidae: Alticinae) is polymorphic for its ability to use Barbarea vulgaris R. Br. (Brassicaceae) as a host plant. The genetic factors influencing this ability show both sex-linked and autosomal inheritance. Evidence was found for the presence of major genes such as those found in earlier studies, but also of genes with a smaller effect which have not previously been found. Although the ability to survive on B. vulgaris exists in most populations in eastern Denmark, it is usually at a low frequency. Beetles collected on B. vulgaris, however, usually produced larvae that survived on this plant. The inheritance and the abundance of the ability to use B. vulgaris are discussed in the context of the evolution of the interaction between P. nemorum and its atypical host plant.     

Tat PTD-Endostatin-RGD: A novel protein with anti-angiogenesis effect in retina via eye drops

BackgroundDiabetic retinopathy is a leading cause of blindness. The objective was to design a novel fusion protein, Tat PTD-Endostatin-RGD, to treat retinal neovascularization via eye drops instead of traditional intravitreal injection trepapeutical methods.MethodThe anti-angiogenesis ability was evaluated in vitro by chick embryo chorioallantoic membrane assay, wound healing assay and tube formation assay. Corneal barrier and blood-retina barrier were constructed in vitro to investigate the penetration ability of Tat PTD-Endostatin-RGD. Western blot was used to detect the integrin α_vβ₃ expression level in rat retina microvascular endothelial cells which was stimulated by S-nitroso-N-acetylpenicillamine. The binding affinity of Tat PTD-Endostatin-RGD to integrin α_vβ₃ was investigated by evaluating the penetration ability on blood-retina barriers treated with S-nitroso-N-acetylpenicillamine. The pharmacodynamics and efficacy analysis were further carried out in the oxygen-induced retinopathy model in vivo. In addition, the pharmacokinetic profile via eye drops was studied on a C57BL/6 mice model.ResultTat PTD-Endostatin-RGD showed high anti-angiogenesis activity and high ability to penetrate these two barriers in vitro. The Western blot results indicated S-nitroso-N-acetylpenicillamine upregulated the expression level of integrin α_vβ₃ in a dose-dependent manner. Tat PTD-Endostatin-RGD showed a high affinity to rat retina microvascular endothelial cells treated with S-nitroso-N-acetylpenicillamine. The results showed that Tat PTD-Endostatin-RGD could inhibit abnormal angiogenesis in retina via eye drops.ConclusionTat PTD-Endostatin-RGD showed high penetration ability through ocular barriers, bound specifically to integrin α_vβ₃ and effectively inhibited the abnormal angiogenesis.General significanceTat PTD-Endostatin-RGD represents a potent novel drug applied via eye drops for fundus oculi neovascularization diseases.     

Live/dead state is not the factor influencing adhesion ability of Bifidobacterium animalis KLDS2.0603

Two essential requirements for probiotic bifidobacteria are that they be “live” and have “colonization” ability, following FAO/WHO guideline recommendations. The amount of research on the adhesion ability of bifidobacteria compares poorly with that of other probiotic bacteria, such as lactobacilli. The aim of the present study was to determine how gastrointestinal conditions affect the adhesion ability of bifidobacteria, and to investigate the relationship between the adhesion ability and the live/dead state of bifidobacteria. The adhesion ability of Bifidobacterium animalis KLDS2.0603 that had been subjected to the digestive enzymes, pepsin, trypsin, and proteinase K, was decreased significantly, but these treatments did not significantly change the strain’s survival rates, which were 98.78%, 97.60%, and 97.63% respectively. B. animalis KLDS2.0603 subjected to LiCl retained its adhesion ability but had a lower survival rate (59.28%) than the control group (P<0.01). B. animalis KLDS 2.0603 subjected to sodium metaperiodate exhibited higher adhesion ability than the control group (P<0.01), but the bacterial cells were killed totally. The results of transmission electron microscopy and laser scanning confocal microscopy showed that live/dead state of bifidobacteria was not one of the main factors that affected the adhesion ability of bifidobacteira, and that the substances affecting the adhesion ability of bifidobacteria were on the outer surface layer of the bifidobacterial cells. Our results also indicated that the substances related to the adhesion ability of bifidobacteria are proteinaceous. The above results will help us to understand the adhesion and colonization processes of bifidobacteria in the human gastrointestinal tract.     

Iron-Chelating Compounds Produced by Soil Pseudomonads: Correlation with Fungal Growth Inhibition

Strains of Pseudomonas putida, Pseudomonas sp., and Pseudomonas aeruginosa were examined for their ability to grow in the presence of the iron chelator, ethylenediamine-di-(o-hydroxyphenylacetic acid). In vitro fungal inhibition assays showed that the isolates varied in their ability to inhibit the growth of representative fungal plant pathogens. Fungal inhibition in vitro was superior to that of previously reported Pseudomonas sp. Studies with Fusarium oxysporum forma sp. lycopersici and a susceptible tomato cultivar demonstrated that Pseudomonas putida PPU3.1 was able to significantly reduce wilt disease.     

Cloning of Escherichia coli lacZ and lacY Genes and Their Expression in Gluconobacter oxydans and Acetobacter liquefaciens

An efficient transformation protocol for Gluconobacter oxydans and Acetobacter liquefaciens strains was developed by preparation of electrocompetent cells grown on yeast extract-ethanol medium. Plasmid pBBR122 was used as broad-host-range vector to clone the Escherichia coli lacZY genes in G. oxydans and A. liquefaciens. Although both lac genes were functionally expressed in both acetic acid bacteria, only a few transformants were able to grow on lactose. However, this ability strictly depended on the presence of a plasmid expressing both lac genes. Mutations in the plasmids and/or in the chromosome were excluded as the cause of growth ability on lactose.     

Role of Baculovirus IE2 and Its RING Finger in Cell Cycle Arrest

The ie2 gene of Autographa californica nuclear polyhedrosis virus (AcMNPV) is known to transactivate transient expression from viral promoters in a host cell-specific manner. We report that transfection of Spodoptera frugiperda (SF-21) cells with ie2 was sufficient to arrest the cell cycle, resulting in the accumulation of enlarged cells with abnormally high DNA contents. By 72 h posttransfection, more than 50% of ie2-transfected cells had DNA contents greater than 4N. There was no evidence of mitotic spindle formation in these cells, and expression of ie2 appeared to block cell cycle progression in S phase. Several ie2 mutants were analyzed to further define the region of IE2 responsible for arresting the cell cycle. Analysis of these mutants showed that deletion of the RING finger motif eliminated the ability of IE2 to arrest the cell cycle but did not affect its ability to transactivate the ie1 promoter. Moreover, mutation of a single conserved cysteine (C251) of the RING finger motif abolished the ability of IE2 to block cell cycle progression but had no apparent effect on its trans-regulatory activity. In contrast, a mutant of IE2 containing a deletion of residues 94 to 173 was able to block cell division but lacked trans-regulatory activity. Thus, the ability of IE2 to arrest the cell cycle depended on the integrity of the RING finger motif and was distinct from and independent of its ability to trans-activate the ie1 promoter. IE2 also arrested the division of cells derived from other insect species, Trichoplusia ni (TN-368 and BTI-TN-5B1-4) and Helicoverpa zea (Hz-AM1).     

Isolation and characterization of BTEX tolerant and degrading Pseudomonas putida BCNU 106

Bacterial strains growing in river sediments were screened to identify an organic solvent-tolerant strain of Pseudomonas. Using this screen, Pseudomonas sp. BCNU 106 was isolated on the basis of its ability to grow on benzene, toluene, ethylbenzene, and three xylene isomers, o-, m- and p-xylene, as its sole carbon source. BCNU 106 was identified as a gram-negative, rod-shaped aerobic and mesophilic bacterium, which grew in liquid media containing high concentrations of organic solvents. 16S rDNA analysis classified BCNU 106 as a new member of the genus Pseudomonas. BCNU 106 was distinguishable from other Pseudomonas strains that are tolerant to organic solvents in that the isolate had the ability to utilize all three xylene isomers as well as benzene, toluene and ethylbenzene. The unique properties of the isolate such as solvent-tolerance and the ability to degrade xylene isomers may have important implications for the efficient treatment of solvent wastes.     

Culture-attenuated pathogenic Leptospira lose the ability to survive to complement-mediated-killing due to lower expression of factor H binding proteins

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.     

Biofilm-Forming Abilities of Shiga Toxin-Producing Escherichia coli Isolates Associated with Human Infections

Forming biofilms may be a survival strategy of Shiga toxin-producing Escherichia coli to enable it to persist in the environment and the food industry. Here, we evaluate and characterize the biofilm-forming ability of 39 isolates of Shiga toxin-producing Escherichia coli isolates recovered from human infection and belonging to seropathotypes A, B, or C. The presence and/or production of biofilm factors such as curli, cellulose, autotransporter, and fimbriae were investigated. The polymeric matrix of these biofilms was analyzed by confocal microscopy and by enzymatic digestion. Cell viability and matrix integrity were examined after sanitizer treatments. Isolates of the seropathotype A (O157:H7 and O157:NM), which have the highest relative incidence of human infection, had a greater ability to form biofilms than isolates of seropathotype B or C. Seropathotype A isolates were unique in their ability to produce cellulose and poly-N-acetylglucosamine. The integrity of the biofilms was dependent on proteins. Two autotransporter genes, ehaB and espP, and two fimbrial genes, z1538 and lpf2, were identified as potential genetic determinants for biofilm formation. Interestingly, the ability of several isolates from seropathotype A to form biofilms was associated with their ability to agglutinate yeast in a mannose-independent manner. We consider this an unidentified biofilm-associated factor produced by those isolates. Treatment with sanitizers reduced the viability of Shiga toxin-producing Escherichia coli but did not completely remove the biofilm matrix. Overall, our data indicate that biofilm formation could contribute to the persistence of Shiga toxin-producing Escherichia coli and specifically seropathotype A isolates in the environment.     

Characterization and differentiation of 59 strains of Moraxella urethralis from clinical specimens

The biochemical characteristics of 59 strains of Moraxella urethralis from clinical specimens, primarily from urine and the female genital tract, were studied. The characteristics included (i) the inability to acidify carbohydrate substrates, (ii) the ability to produce phenylalanine deaminase, (iii) the ability to reduce nitrite, (iv) the lack of urease activity, and (v) the ability of most strains to alkalinize citrate. A means of differentiating M. urethralis from Moraxella osloensis and Moraxella phenylpyruvica was determined.     

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Reproductive and genetic roles of the maternal progenitor in the origin of common wheat (Triticum aestivum L.)

Common wheat (Triticum aestivum L., AABBDD genome) is thought to have emerged through natural hybridization between Triticum turgidum L. (AABB genome) and Aegilops tauschii Coss. (DD genome). Hybridization barriers and doubling of the trihaploid F₁ hybrids’ genome (ABD) via unreduced gamete fusion had key roles in the process. However, how T. turgidum, the maternal progenitor, was involved in these mechanisms remains unknown. An artificial cross‐experiment using 46 cultivated and 31 wild T. turgidum accessions and a single Ae. tauschii tester with a very short genetic distance to the common wheat D genome was conducted. Cytological and quantitative trait locus analyses of F₁ hybrid genome doubling were performed. The crossability and ability to cause hybrid inviability did not greatly differ between the cultivars and wild accessions. The ability to cause hybrid genome doubling was higher in the cultivars. Three novel T. turgidum loci for hybrid genome doubling, which influenced unreduced gamete production in F₁ hybrids, were identified. Cultivated T. turgidum might have increased the probability of the emergence of common wheat through its enhanced ability to cause genome doubling in F₁ hybrids with Ae. tauschii. The ability enhancement might have involved alterations at a relatively small number of loci.     

Role of the Flagellar Basal-Body Protein,FlgC, in the Binding of Salmonella enterica Serovar Enteritidis to Host Cells

Salmonella enterica serovar Enteritidis (SE) infection in humans is often associated with the consumption of contaminated poultry products. Binding of the bacterium to the intestinal mucosa is a major pathogenic mechanism of Salmonella in poultry. Transposon mutagenesis identified flgC as a potential binding mutant of SE. Therefore, we hypothesize FlgC which plays a significant role in the binding ability of SE to the intestinal mucosa of poultry. To test our hypothesis, we created a mutant of SE in which flgC was deleted. We then tested the in vitro and in vivo binding ability of ?flgC when compared to the wild-type SE strain. Our data showed a significant decrease in the binding ability of ?flgC to intestinal epithelial cells as well as in the small intestine and cecum of poultry. Furthermore, the decrease in binding correlated to a defect in invasion as shown by a cell culture model using intestinal epithelial cells and bacterial recovery from the livers and spleens of chickens. Overall, these studies indicate FlgC is a major factor in the binding ability of Salmonella to the intestinal mucosa of poultry.     

Cloning of the phosphonoacetate hydrolase gene from Pseudomonas fluorescens 23F encoding a new type of carbon–phosphorus bond cleaving enzyme and its expression in Escherichia coli and Pseudomonas putida

The phnA gene encoding a novel carbon–phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida. It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only. The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.     

Synthesis of P 1-(11-phenoxyundecyl)-P 2-(��-D-galactopyranosyl) diphosphate and P 1-(11-phenoxyundecyl)-P 2-(��-D-glucopyranosyl) diphosphate and investigation on their acceptor properties in the reaction of mannosyl residue transfer catalyzed by mannosyltransferase from Salmonella newport

P ¹-(11-phenoxyundecyl)-P ²-(??-D-galactopyranosyl) diphosphate and P ¹-(11-phenoxyundecyl)-P ²-(??-D-glucopyranosyl) diphosphate have been synthesized for the first time, and their ability to serve as a mannosyl residue substrate-acceptors in the enzymatic reaction, catalyzed by mannosyltransferase membrane preparation from Salmonella newport cells, was investigated. It was demonstrated that the derivative containing galactopyranose residue is able to accept the mannosyl residue from GDP-Man, while the derivative containing glucopyranose residue does not have such an ability.     

Host-Specific Pathogenicity and Genome Differences between Inbred Strains of Meloidogyne hapla

Five isolates of M. hapla originating from the Netherlands and California were inbred by sequential transfer of single egg masses to produce six strains. Cytological examination showed that oocytes of these strains underwent meiosis and had n = 16 chromosomes. Strains were tested for ability to infect and to develop on several hosts by in vitro assays. The two strains from California infected tomato roots at a higher rate than those from the Netherlands, but no difference among strains was seen for ability to develop on tomato with or without the resistance gene Mi-1. All strains developed on the common bean cultivar Kentucky Wonder, but strains differed in ability to develop on the nematode-resistant cultivar NemaSnap. Strain-specific differences were also seen in ability to infect and to develop on Solanum bulbocastanum clone SB-22. Strain VW13, derived from nematodes treated with the mutagen EMS, was defective in ability to infect tomato and potato roots in vitro. Comparison of DNA using AFLP markers showed an average of 4% of the bands were polymorphic across the six strains, but no correlation was observed between the geographical origin or virulence and DNA polymorphism pattern.     

Bioremediation of DDT contaminated soil using brown-rot fungi

The ability of brown-rot fungi (BRF) to eliminate DDT in artificially and historically contaminated soil was investigated to determine whether the BRF would be suitable for the bioremediation of DDT in soil. Gloeophyllum trabeum, Fomitopsis pinicola and Daedalea dickinsii showed an ability to eliminate DDT in artificially contaminated sterilized (SL) and un-sterilized (USL) soils. The addition of Fe²⁺ to the soil system enhanced the ability of some BRF to eliminate DDT. In the contaminated SL soil, the DDT was eliminated by approximately 41%, 9% and 15% by G. trabeum, F. pinicola and D. dickinsii, respectively. Compared with the controls, in the USL soil approximately 43%, 29% and 32% of DDT was eliminated and approximately 20%, 9% and 26% of DDD (1,1-dichloro-2,2-bis (4-chlorophenyl) ethane) was detected as a metabolic product with G. trabeum, F. pinicola and D. dickinsii, respectively. Of the BRF, G. trabeum demonstrated the greatest ability to eliminate DDT both in the SL and USL soils. G. trabeum was applied to a historically contaminated soil which had a DDT concentration more than three times the artificially contaminated soil. G. trabeum remediated about 64% of the initial DDT with the addition of Fe²⁺. There were no significant differences in the results with or without the addition of Fe²⁺, indicating that G. trabeum can be used directly for the degradation of DDT in soil without any other additional treatment. This study identified that G. trabeum is the most promising BRF for use in the bioremediation of DDT contaminated soil.     

Promotion of tomato (Lycopersicon esculentum Mill.) plant growth by rhizosphere competent 1-aminocyclopropane-1-carboxylic acid deaminase-producing streptomycete actinomycetes

The ability of streptomycete actinomycetes to promote growth of tomato through the production of 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase was evaluated under gnotobiotic and greenhouse conditions. To achieve this, 64 isolates of Streptomyces spp. obtained from a tomato rhizosphere in the United Arab Emirates were initially selected for their ability to produce ACC deaminase as well as indole-3-acetic acid (IAA) and subsequently for their rhizosphere competence as root colonizers. Of the two selected ACC deaminase-producing isolates showing exceptional rhizosphere competence, S. filipinensis no. 15 produced both ACC deaminase and IAA, whilst S. atrovirens no. 26 did not produce IAA. Under greenhouse conditions, the application of S. filipinensis no. 15 or S. atrovirens no. 26 resulted in the reduction of the endogenous levels of ACC, the immediate precursor of ethylene, in both roots and shoots and increased plant growth. Plant growth promotion was most pronounced in the presence of S. filipinensis no. 15 compared to S. atrovirens no. 26. This relative superiority in performance shows the advantage conferred to S. filipinensis no. 15 due to its ability to produce both IAA and ACC deaminase. In comparison, an ACC deaminase-producing isolate of S. albovinaceus no. 41 which was neither rhizosphere-competent nor capable of producing IAA, failed to promote plant growth compared to S. filipinensis no. 15 or S. atrovirens no. 26 although the growth promotion obtained by S. albovinaceus no. 41 was significant compared to control. The application of S. globosus no. 8, which was not rhizosphere-competent and did not produce detectable levels of ACC deaminase or IAA did not promote plant growth. These results indicate the importance of rhizosphere competence. In conclusion I report the production of ACC deaminase by streptomycete actinomycetes and its ability to enhance plant growth through reduction in the in planta levels of endogenous ACC and the consequent lowering of endogenous ethylene levels in plant tissues.     

Shift in competitive ability mediated by soil biota in an invasive plant

Understanding the shifts in competitive ability and its driving forces is key to predict the future of plant invasion. Changes in the competition environment and soil biota are two selective forces that impose remarkable influences on competitive ability. By far, evidence of the interactive effects of competition environment and soil biota on competitive ability of invasive species is rare. Here, we investigated their interactive effects using an invasive perennial vine, Mikania micrantha. The competitive performance of seven M. micrantha populations varying in their conspecific and heterospecific abundance were monitored in a greenhouse experiment, by manipulating soil biota (live and sterilized) and competition conditions (competition‐free, intraspecific, and interspecific competition). Our results showed that with increasing conspecific abundance and decreasing heterospecific abundance, (1) M. micrantha increased intraspecific competition tolerance and intra‐ vs. interspecific competitive ability but decreased interspecific competition tolerance; (2) M. micrantha increased tolerance of the negative soil biota effect; and (3) interspecific competition tolerance of M. micrantha was increasingly suppressed by the presence of soil biota, but intraspecific competition tolerance was less affected. These results highlight the importance of the soil biota effect on the evolution of competitive ability during the invasion process. To better control M. micrantha invasion, our results imply that introduction of competition‐tolerant native plants that align with conservation priorities may be effective where M. micrantha populations are long‐established and inferior in inter‐ vs. intraspecific competitive ability, whereas eradication may be effective where populations are newly invaded and fast‐growing.