<Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis> inhibits <Emphasis Type=Italic>in-vitro Candida</Emphasis> biofilm development

Background  

Elucidation of the communal behavior of microbes in mixed species biofilms may have a major impact on understanding infectious diseases and for the therapeutics. Although, the structure and the properties of monospecies biofilms and their role in disease have been extensively studied during the last decade, the interactions within mixed biofilms consisting of bacteria and fungi such as Candida spp. have not been illustrated in depth. Hence, the aim of this study was to evaluate the interspecies interactions of Pseudomonas aeruginosa and six different species of Candida comprising C. albicans, C. glabrata, C. krusei, C. tropicalis, C. parapsilosis, and C. dubliniensis in dual species biofilm development.     

Inhibition of biofilm formation by the antisense peptide nucleic acids targeted at the <Emphasis Type=Italic>motA</Emphasis> gene in <Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis> PAO1 strain

Pseudomonas aeruginosa is a ubiquitous bacterium which is able to attach to many abiotic and biotic surfaces and form biofilms resulting in infections. The motA gene was an essential gene in the early phase of biofilm formation of P. aeruginosa PAO1. In this study, antisense peptide nucleic acids (PNAs) and PNAs conjugated with the peptide (KFF)₃K were used to investigate whether they could mediate gene-specific antisense effects in P. aeruginosa PAO1. We found that antisense (KFF)₃K-PNA targeted at motA gene could inhibit biofilm formation in P. aeruginosa PAO1 in a dose-dependent manner. The minimal effective concentration of this antisense agent was 1 μmol l⁻¹, and the inhibited effect could last for at least 8 h. When compared with the control group, the value of OD570 of P. aeruginosa PAO1 reduced apparently when treated with (KFF)₃K-PNA. The expression of motA was sharply reduced when treated with (KFF)₃K-PNA, but reduced slightly when treated with PNA, and had no reduction when treated with (KFF)₃K. Our results demonstrated that the cell-penetrating peptide of (KFF)₃K improved significantly the antisense inhibition effect of PNA. The (KFF)₃K-PNA conjugates might be used as antisense agent for inhibition of the biofilm formation. This provides exciting possibility for developing new tool for microbial genetic treatment.     

Substrate specificity characterization of a thermostable keratinase from <Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis> KS-1

A feather-degrading strain of Pseudomonas aeruginosa KS-1 was used in the present study. Its crude cell-free fermentation broth completely degraded chicken feather within 12 h, in the absence of disulphide reductase activity. Keratinase from its extracellular broth was purified and characterized, assuming that it would be a potential β-keratin-degrading enzyme with prospective applications in degradation of β-plaques of prions. The keratinase was purified by using Q-Sepharose anion exchange chromatography and its molecular weight, as determined by SDS–PAGE analysis, was 45 kDa. It was an alkaline, serine protease with pH and temperature optima of 9 and 60°C, respectively. The enzyme was highly thermostable with a t _1/2 > 2 h at 80°C and had a very high K to C (keratinolytic to caseinolytic) ratio of 2.5. Besides feather keratin, it also hydrolyzed a variety of other complex substrates including fibrin, gelatin and meat protein. Its activity on synthetic substrates revealed that it efficiently cleaves them in the order phenylalanine > lysine > alanine > leucine p-nitroanilides. It also cleaved insulin B chain between Val¹²-Glu¹³, Ala¹⁴-Leu¹⁵, Gly²⁰-Glu²¹ and Arg²²-Gly²³ residues.     

Phototropic H<Subscript>2</Subscript> production by a newly isolated strain of <Emphasis Type=Italic>Rhodopseudomonas</Emphasis><Emphasis Type=Italic>palustris</Emphasis>

Rhodopseudomonas palustris TN1 was isolated from Songkhla Lake, Thailand. It phototrophically generates H₂ from the predominant volatile fatty acids (VFAs) produced from microbial dark-fermentations of palm oil milling effluent; yields from 20 mM butyrate, acetate and propionate were 4.7, 2.5, and 1.7 mol H₂ mol VFA⁻¹ with light efficiencies of 1.8, 1, and 0.2%, respectively. Optimum conditions were pH 7 and 3000 lux, although production was reduced by only 33% at 1000 lux. CO₂ evolution never exceeded 9 mmol mol VFA⁻¹.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Rapid biodegradation and decolorization of Direct Orange 39 (Orange TGLL) by an isolated bacterium <Emphasis Type=Italic>Pseudomonas aeruginosa</Emphasis> strain BCH

A newly isolated novel bacterium from sediments contaminated with dyestuff was identified as Pseudomonas aeruginosa strain BCH by 16S rRNA gene sequence analysis. The bacterium was extraordinarily active and operative over a wide rage of temperature (10–60°C) and salinity (5–6%), for decolorization of Direct Orange 39 (Orange TGLL) at optimum pH 7. This strain was capable of decolorizing Direct Orange 39; 50 mg l⁻¹ within 45 ± 5 min, with 93.06% decolorization, while maximally it could decolorize 1.5 g l⁻¹ of dye within 48 h with 60% decolorization. Analytical studies as, UV–Vis spectroscopy, FTIR, HPLC were employed to confirm the biodegradation of dye and formation of new metabolites. Induction in the activities of lignin peroxidases, DCIP reductase as well as tyrosinase was observed, indicating the significant role of these enzymes in biodegradation of Direct Orange 39. Toxicity studies with Phaseolus mungo and Triticum aestivum revealed the non-toxic nature of degraded metabolites.     

Biodegradation of ethanethiol in aqueous medium by a new <Emphasis Type=Italic>Lysinibacillus sphaericus</Emphasis> strain RG-1 isolated from activated sludge

In the present study, a new bacterial strain isolated from activated sludge has been identified as Lysinibacillus sphaericus based on its morphology, physiochemical properties, and the results of 16S ribosomal RNA (rRNA) gene sequence analysis. This new bacterial strain uses ethanethiol as both carbon source and energy source. The key factors for controlling the degradation efficiency of ethanethiol by this strain were found to be initial ethanethiol concentration, temperature, and pH value of solutions. Under the optimized conditions, as well as 4 mg l⁻¹ ethanethiol, 30°C, and pH = 7.0, almost 100% ethanethiol can be degraded within 96 h and sulfate as a final product was detected in aqueous medium. The degradation reaction of ethanethiol over this newly isolated strain can be described by pseudo first-order equation in which the maximum degradation rate constant K and the minimum half-life were respectively calculated to be 0.0308 h⁻¹ and 22.5 h under the optimal conditions.     

<Emphasis Type=Italic>Boletus kermesinus</Emphasis>, a new species of <Emphasis Type=Italic>Boletus</Emphasis> section <Emphasis Type=Italic>Luridi</Emphasis> from central Honshu,Japan

Boletus kermesinus, a new species of Boletus section Luridi, is fully described and illustrated based on the materials collected in subalpine coniferous forests of central Honshu, Japan. It has distinctive features of dark-red basidiomata having distinct viscidity in the pileus surface, usually unchanging flesh, discolorous red pores, and an entirely reticulate stipe becoming coarsely lacerate-rimose with age.     

Characterization of a multifunctional feather-degrading <Emphasis Type=Italic>Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

Characterization of polyhydroxyalkanoate and the <Emphasis Type=Italic>phaC</Emphasis> gene of <Emphasis Type=Italic>Paracoccus seriniphilus</Emphasis> E71 strain isolated from a polluted marine microbial mat

Polyhydroxyalakanote (PHA) was produced by the marine bacteria Paracoccus seriniphilus Strain E71. Three methods were used for screening PHA in this strain: (1) microscopic analysis, (2) specifically designed primers for amplify fragments of phaC gene from Gram negative bacteria, and (3) measurements using spectroscopy, calorimetry, thermogravimetry, and rheology. The polyhydroxyalkanoic acid synthase gene (phaC) sequence had 77% identity with the phaC gene of P. denitrificans PD1222 strain. Additionally, the translated sequence showed an 86% similarity with the amino acid sequence of the phaC gene N-terminal portion of the P. denitrificans PD1222 strain. Our phaC sequence was closely related to two phaC sequences that correspond to P. denitrificans; therefore, this is the first report of a sequence of phaC that codifies a poly-(3-hydroxyalkanoate) synthase class I, specifically a poly-beta-hydroxybutyrate polymerase from the marine bacteria Paracoccus seriniphilus. The polymer PHA of E71 melts at 167.86°C (T _m), which corresponded to the fusion of the crystalline polymer and thermally degrades at 296.52°C, indicating that the biopolymer has good thermal stability. Rheology showed that this polymer behaves as a nonNewtonian fluid. All these characteristics suggest that the E71 strain produces a PHA that corresponds to the crystalline thermoplastic polymer PHB type.     

Attenuation of fibrosis <Emphasis Type=Italic>in vitro</Emphasis> and <Emphasis Type=Italic>in vivo</Emphasis> with <Emphasis Type=Italic>SPARC</Emphasis> siRNA

Introduction  

SPARC is a matricellular protein, which, along with other extracellular matrix components including collagens, is commonly over-expressed in fibrotic diseases. The purpose of this study was to examine whether inhibition of SPARC can regulate collagen expression in vitro and in vivo, and subsequently attenuate fibrotic stimulation by bleomycin in mouse skin and lungs.     

Interaction of <Emphasis Type=Italic>Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type=Italic>Campylobacter jejuni</Emphasis> and <Emphasis Type=Italic>Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Suppression of disease in tomato infected by <Emphasis Type=Italic>Pythium ultimum</Emphasis> with a biosurfactant produced by <Emphasis Type=Italic>Pseudomonas koreensis</Emphasis>

The use of biosurfactants is a promising alternative in biological control of zoospore-producing oomycetes, which are a major plant pathogen world-wide in a wide variety of crops. Oomycetes are of particular concern in closed hydroponic cultivation systems. The present study investigated the efficacy of a biosurfactant produced by Pseudomonas koreensis and added as a crude extract against the oomycete Pythium ultimum in hydroponic tomato cultivation. A significant reduction in disease was observed. Biosurfactant addition did not affect the indigenous root microflora when evaluated as sole carbon source utilisation. Chemical analysis, using electrospray hybrid mass spectrometry (ESI-MSMS), of the biosurfactant indicated it to be lokisin, a cyclic lipopeptide. These results confirm that biosurfactants are important in developing sustainable biological control strategies for oomycetes.     

<Emphasis Type=Italic>In vitro</Emphasis> organogenesis of <Emphasis Type=Italic>Passiflora alata</Emphasis>

Passiflora alata in vitro organogenesis was studied based on explant type, culture medium composition, and incubation conditions. The results indicated that the morphogenic process occurred more efficiently when hypocotyl segment-derived explants were cultured in media supplemented with cytokinin and AgNO₃ incubated under a 16-h photoperiod. The shoot bud elongation and plant development were obtained by transferring the material to MSM culture medium supplemented with GA₃ and incubated in flasks with vented lids. Histological analyses of the process revealed that the difficulties in obtaining plants could be related to the development of protuberances and leaf primordia structures, which did not contain shoot apical meristem. Roots developed easily by transferring elongated shoots to 1/2 MSM culture medium. Plant acclimatization occurred successfully, and somaclonal variation was not visually detected. The efficiency of this organogenesis protocol will be evaluated for genetic transformation of this species to obtain transgenic plants expressing genes that can influence the resistance to Cowpea aphid borne mosaic virus.