Extracellular Degradative Enzymes Research Articles

Notopterol Suppresses IL-17-Induced Proliferation and Invasion of A549 Lung Adenocarcinoma Cells via Modulation of STAT3, NF-κB, and AP-1 Activation.

Interleukine-17 is a proinflammatory cytokine that promotes lung cancer growth and progression though the activation of the STAT3, NF-κB, and AP-1 signaling pathways. Therefore, blocking the IL-17-induced oncogenic pathway is a new strategy for the treatment of lung cancer. Notopterol, a furanocoumarin, has demonstrated anti-tumor effects in several types of tumors. However, its molecular function in relation to the IL-17-induced proliferation and invasion of A549 lung adenocarcinoma cells remains unknown. Here, notopterol exhibited an inhibitory effect on IL-17-promoted A549 cell proliferation and induced G0/G1 cell cycle arrest. Western blot analysis revealed that notopterol inhibited the expression of cell-cycle-regulatory proteins, including cyclin D1, cyclin E, CDK4, and E2F. Moreover, notopterol blocked IL-17-induced A549 cell migration and invasion by regulating the epithelial-mesenchymal transition (EMT) and reducing the expression of extracellular degradation enzymes. At the molecular level, notopterol treatment significantly down-regulated the IL-17-activated phosphorylation of Akt, JNK, ERK1/2, and STAT3, leading to a reduced level of transcriptional activity of NF-κB and AP-1. Collectively, our results suggest that notopterol blocks IL-17-induced A549 cell proliferation and invasion through the suppression of the MAPK, Akt, STAT3, AP-1, and NF-κB signaling pathways, as well as modulating EMT.

DegQ is an important policing link between quorum sensing and regulated adaptative traits in Bacillus subtilis.

Quorum sensing (QS) is a widespread bacterial communication system that controls important adaptive traits in a cell density-dependent manner. However, mechanisms by which QS-regulated traits are linked within the cell and mechanisms by which these links affect adaptation are not well understood. In this study, Bacillus subtilis was used as a model bacterium to investigate the link between the ComQXPA QS system, DegQ, surfactin and protease production in planktonic and biofilm cultures. The work tests two alternative hypotheses predicting that hypersensitivity of the QS signal-deficient mutant (comQ::kan) to exogenously added ComX, resulting in increased surfactin production, is linked to an additional genetic locus, or alternatively, to overexpression of the ComX receptor ComP. Results are in agreement with the first hypothesis and show that the P srfAA hypersensitivity of the comQ::kan mutant is linked to a 168 strain-specific mutation in the P degQ region. Hence, the markerless ΔcomQ mutant lacking this mutation is not overresponsive to ComX. Such hyper-responsiveness is specific for the P srfAA and not detected in another ComX-regulated promoter, the P aprE , which is under the positive control by DegQ. Our results suggest that DegQ by exerting differential effect on P srfAA and P aprE acts as a policing mechanism and the intracellular link, which guards the cell from an overinvestment into surfactin production. IMPORTANCE DegQ levels are known to regulate surfactin synthesis and extracellular protease production, and DegQ is under the control of the ComX-dependent QS. DegQ also serves as an important policing link between these QS-regulated processes, preventing overinvestment in these costly processes. This work highlights the importance of DegQ, which acts as the intracellular link between ComX production and the response by regulating extracellular degradative enzyme synthesis and surfactin production.

Effect of Curcuma longa extract on serum inflammatory markers and MRI-based synovitis in knee osteoarthritis: secondary analyses from the CurKOA randomised trial

BackgroundCurcuma longa (CL) extract is modestly effective for relieving knee symptoms in knee osteoarthritis (OA) patients; however, its mechanism of action is unclear. PurposeWe aimed to determine the effects of CL treatment on serum inflammatory markers over 12 weeks and to explore its potential effects on synovitis assessed by contrast-enhanced magnetic resonance imaging (CE-MRI) of the knee. MethodsSecondary analyses were conducted on the CL for knee OA (CurKOA) trial, which compared CL (n = 36) and placebo (n = 34) over 12 weeks for the treatment of knee OA. Systemic inflammatory markers (TNFα, IL6, and hsCRP) and a cartilage extracellular matrix degradative enzyme (MMP-3) were measured. A subgroup of participants (CL, n = 7; placebo, n = 5) underwent CE-MRI at baseline and a 12-week follow-up. ResultsOver 12 weeks, there were no between-group differences in change in hsCRP, IL-6, and TNFα levels. MMP-3 levels decreased in both CL (-1.31 ng/ml [95%CI: -1.89 to -0.73]) and placebo (-2.34 ng/ml [95%CI: -2.95 to -1.73]) groups, with the placebo group having a slightly greater decrease (1.03 ng/ml [95%CI: 0.19 to 1.88]). Most (10 of 12) sub-study participants had normal synovial thickness scores at baseline. One participant had mild synovitis in each of the placebo and CL groups. Synovitis status was stable for all except two participants, one each in the CL and placebo group, whose synovitis score increased. ConclusionThis is the first study that explored the effect of CL treatment on local and systemic inflammation using biochemical markers and CE-MRI outcomes on knee OA patients. Secondary analyses from this pilot study suggest that CL is unlikely to have clinically significant effects on systemic (inflammatory and cartilage) or local synovitis (CE-MRI) biomarkers compared to placebo. The mechanism of action for CL effect on pain remains unclear.

Mechanism of Phanerochaete chrysosporium-mediated tetracycline hydrochloride removal promoted by low-dose silver nanoparticles

The influence of silver nanoparticles (AgNPs) on environmental microorganisms has been extensively reported, however, there is currently limited understanding of effects of stress-induced by AgNPs on other pollutant degradation. In this study, Phanerochaete chrysosporium was stimulated by different concentrations of AgNPs to explore its effect on P. chrysosporium-mediated degradation of tetracycline hydrochloride (TCH). The results indicate that treatment with AgNPs at low concentration resulted in a considerable increase in TCH degradation, TCH degradation reached a maximum of 97% with 0.1 mg L−1 AgNPs treated after 3 d, and the viability of P. chrysosporium increased 1.48 times compared with the control group. The largest O2 influx of − 17 pmol cm−2 s−1 under 0.1 mg L−1 AgNPs treatment reveal the enhancement of P. chrysosporium respiration. In addition, low concentrations of AgNPs could induce the overexpression of cytochrome P450 and extracellular degradation enzymes MnP-related genes in stimulated cells, which further induce the production of extracellular degrading enzymes (51.0 U L−1 MnP and 23.7 U L−1 LiP) to accelerate the degradation of TCH. Together, our combined results uncovered the enhanced degradation mechanisms of P. chrysosporium induced by which AgNPs under sublethal dosage.

Impact of Soil Chemical Properties on the Growth Promotion Ability of Trichoderma ghanense, T. tomentosum and Their Complex on Rye in Different Land-Use Systems.

Microbial-based biostimulants that increase plant performance and ensure sustainable restoration of degraded soils are of great importance. The aim of the present study was to evaluate the growth promotion ability of indigenous Trichoderma ghanense, T. tomentosum and their complex on early rye seedlings in sustained grassland and arable soil. The impact of soil chemical properties on the ability of selected Trichoderma strains and their complex to promote plant growth was determined by the evaluation of the rye (Secale cereale L.) early seedling growth—measuring the length of shoots and roots as well as their dry weight. Trichoderma species were tested for their ability to produce extracellular degradative enzymes on solid media. Furthermore, the soil properties and CM-cellulase activity of soil were estimated. The indigenous Trichoderma strains possess the capacity to produce enzymes such as peroxidase, laccase, tyrosinase, and endoglucanase. The results indicated a significant (p < 0.05) increase in plant growth and the improvement of some soil chemical properties (total N, mobile humic and fulvic acids, exchangeable K2O, soil CM-cellulase activity) in inoculated soils when compared to the control. The growth of the roots of rye seedlings in sustained grassland was enhanced when T. tomentosum was applied (p = 0.005). There was an increase in total weight and shoot weight of rye seedlings when T. ghanense was used in the arable soil (p = 0.014 and p = 0.024). The expected beneficial effect of Trichoderma spp. complex on rye growth promotion was not observed in any tested soil. The results could find application in the development of new and efficient biostimulants, since not only do physiological characteristics of fungi play an important role but also the quality of the soil has an impact.

How do droppings of wintering waterbird accelerate decomposition of Carex cinerascens Kükenth litter in seasonal floodplain Ramsar Site?

The decomposition of wetland plant litter is pivotal in the functioning of wetland ecosystems in terms of organic matter mineralization progress and nutrient cycling. Aiming to investigate the effects of the droppings of wintering herbivorous waterbirds on the decomposition of Carex cinerascens Kukenth litter a newly designated Ramsar Site, decomposition patterns of three types of samples were characterized and compared by waterbird dropping addition and litterbag in situ experiment. The three types of samples are OCL (only C. cinerascens litter), OWD (only waterbird droppings) and CMD (C. cinerascens litter mixed with waterbird droppings), respectively. A total of 105 decomposition bags were placed and the experiment lasted 150 days. The results showed that the average decay rate of C. cinerascens cellulose increased by approximately 32.12% by the addition of bird droppings, while the C. cinerascens lignin decay rate increased by approximately 83.05%. And the release of carbon, nitrogen and phosphorous of C. cinerascens increased by approximately 18.3%, 61.09%, and 66.71%, respectively. We concluded that the addition of waterbird droppings significantly accelerated the decomposition of C. cinerascens litter and the release of carbon, nitrogen, and phosphorus. We have a preliminary deduction that waterbird droppings (1) altered the original stoichiometric ratio of the litter, (2) increased the available nutrient content, (3) increased the colonization ability of the microorganisms, and (4) stimulated the production of a series of extracellular degradation enzymes. This study provides a thinking that the important role of waterbirds should be given full attention in wetland restoration planning and management.

Physiological Responses of &lt;i&gt;Microcystis aeruginosa&lt;/i&gt; to Extracellular Degradative Enzymes and Algicidal Substance from Heterotrophic Bacteria

Physiological Responses of &lt;i&gt;Microcystis aeruginosa&lt;/i&gt; to Extracellular Degradative Enzymes and Algicidal Substance from Heterotrophic Bacteria

Virulence factors analysis of native isolates of Xanthomonas albilineans and Xanthomonas sacchari from Tucumán, Argentina, reveals differences in pathogenic strategies

AbstractXanthomonas albilineans (Xa) and X. sacchari (Xs) are both sugarcane pathogens. Xa is the causal agent of leaf scald disease, but there is limited information about the pathogenicity of Xs. The aim of this work was to study virulence factors of native strains of Xa (Xa32, Xa33, and XaM6) and Xs (Xs14 and Xs15) previously isolated from sugarcane with leaf scald symptoms, to gain insight into the biology of each microorganism. We analysed epiphytic survival, sensitivity to oxidative stress, extracellular degradative enzymes, cell motilities, exopolysaccharide (EPS) characteristics, cell adhesion, biofilm development, and control of stomatal regulation of the five strains. We observed that each species presented similar phenotypes for every factor analysed. Xa strains appeared to be more sensitive to oxidative stress and presented lower epiphytic survival than Xs. All strains presented endoglucanase activity; however, we could only detect protease and amylase activities in Xs strains. Swimming and sliding were higher in Xs, but twitching was variable among species. We also observed that only Xs strains produced a xanthan‐like EPS, presented a strong cell adhesion, and structured biofilm. We detected some intraspecific variations showing that higher amounts of EPS produced by Xs14 correlated with its higher sliding motility and its homogenous and more adhesive biofilm. In addition, EPSs of Xs14 and Xs15 presented differences in strand height and acetyl percentage. Finally, we found that strains of both species were able to interfere with stomatal aperture mechanism. All these differences could influence the colonization strategies and/or disease progression in each species.

Biosynthesis of Silver Nanoparticles by Pathogenic and Nonpathogenic Strains of Fusarium oxysporum f. sp. lycopersici

N ANOTECHNOLOGY, a highly promising discipline in science and technology, is the emerging and novel trend that will redesign the future of several existing know how which will change every aspect of our lives and leads to the generation of uniqueness in all the streams of technology. Biological synthesis of metal nanoparticles using microbes is gaining momentum due to the ecofriendly nature of the organisms which reduce toxic chemicals. Plant pathogenic fungi produce extracellular degradative enzymes that may be important in pathogenicity. Biosynthesis of silver nanoparticles was investigated using pathogenic Fusarium oxysporum f. sp. lycopersici and nonpathogenic strains of the same fungal species. These silver nanoparticles were characterized by means of UV–vis spectroscopy, transmission electron microscopy (TEM). UV–visible spectrum of the aqueous medium containing silver ion showed a peak at 420 nm corresponding to the Plasmon absorbance of silver nanoparticles. Transmission electron microscopy (TEM) micrograph showed formation of well-dispersed spherical silver nanoparticles with mean sizes 11.20 nm and 15.38 nm for pathogenic and nonpathogenic strains, respectively.

CCK1, a PMK1-type MAP kinase is required for hyphal growth, pigmentation, conidiation, enzyme activity, osmotic stress response, and pathogenicity in Corynespora cassiicola

Corynespora cassiicola is a fungal pathogen causing Corynespora Leaf Fall disease (CLF) in rubber trees (Hevea brasiliensis). CLF has great economic impact in rubber-producing countries. However, the molecular mechanisms involved in the pathogenicity of C. cassiicola remain poorly understood. Mitogen-activated protein kinase (MAPK) is one of the major signaling pathways responsible for regulation of fungal development and virulence in many plant fungal pathogens. The C. cassiicola cck1 gene encodes a MAPK that is homologous to the rice pathogen Magnaporthe grisea MAPK Pmk1 protein. In this study, targeted cck1 gene disruption and complementation experiments were carried out in C. cassiicola. Compared to the wild-type and CΔcck1 complement, the Δcck1 mutant had more pigmentation and exhibited dramatically reduced aerial mycelial mass and mycelial growth rates, along with the inability to produce conidia or conidiophores. In addition, the Δcck1 hyphae were darker in color and had less branches and septae with visibly condensed protoplasm. The Δcck1 mutant was more resistant to hyperosmotic stress, and was able to produce greater amount of laccase and lesser cellulase in vitro; however, the production of laccase was delayed. Importantly, Δcck1 mycelia and crude toxin were less virulent upon inoculation of rubber tree leaves. These findings indicate that the C. cassiicola CCK1 MAP kinase is a versatile regulator involved in nearly every aspect of plant fungal pathogenicity including mycelial growth, development, differentiation, pigmentation, conidiation, production of extracellular degradation enzymes, osmoregulation, and virulence.

English

Twenty one moderately halophilic bacterial strains were isolated from seawater and sediment in Alexandria Eastern Harbour, Egypt. The isolates were screened for the production of four extracellular degradative enzymes. The majority of isolates (57.1%) possessed significant enzyme activities, 43% of them have potentiality to produce amylase enzyme. The most active isolate for the production of amylase enzyme was identified by using a 16S rRNA sequence analysis as Bacillus lichineformis AH214. Optimization of the fermentation medium components and environmental factors using One Variable at a Time Approach and Plackett-Burman design was applied to enhance the amylase production by Bacillus lichineformis AH214. The maximum microbial amylase production could be achieved using an optimized medium of the following composition (g/l): 1.0 g yeast extract, 0.05 g K2HPO4, 0.25 g FeCl3, 15.0 g starch, 30.0 g NaCl, 0.75 g MgSO4.7H2O and inoculums size of 1.5 ml/50 ml and incubated at optimum conditions of pH 7, agitation speed 160 rpm, time 30 h and temperature 40&deg;C. On applying optimized medium in the fermentation process, an enzyme productivity of 13.44 U/mg protein was achieved with two fold increase compared to the basal one. The crude amylase produced by Bacillus lichineformis was stable up to 40&deg;C, pH 7.5 and 1.5 M NaCl. Key words: Halophiles, amylase, Bacillus lichineformis, Plackett-Burman, optimization.

Physicochemical Properties Analysis and Secretome of Aspergillus niger in Fermented Rapeseed Meal.

The nutrient digestibility and feeding value of rapeseed meal (RSM) for non-ruminant animals is poor due to the presence of anti-nutritional substances such as glucosinolate, phytic acid, crude fiber etc. In the present study, a solid state fermentation (SSF) using Aspergillus niger was carried out with the purpose of improving the nutritional quality of RSM. The chemical composition and physicochemical properties of RSM before and after fermentation were compared. To further understand possible mechanism of solid state fermentation, the composition of extracellular enzymes secreted by Aspergillus niger during fermentation was analysed using two-dimentional difference gel electrophoresis (2D-DIGE) combined with matrix assisted laser desorption ionization—time of flight—mass spectrometer (MALDI-TOF-MS). Results of the present study indicated that SSF had significant effects on chemical composition of RSM. The fermented rapeseed meal (FRSM) contained more crude protein (CP) and amino acid (AA) (except His) than unfermented RSM. Notably, the small peptide in FRSM was 2.26 time larger than that in unfermented RSM. Concentrations of anti-nutritional substrates in FRSM including neutral detergent fiber (NDF), glucosinolates, isothiocyanate, oxazolidithione, and phytic acid declined (P < 0.05) by 13.47, 43.07, 55.64, 44.68 and 86.09%, respectively, compared with unfermented RSM. A. niger fermentation disrupted the surface structure, changed macromolecular organic compounds, and reduced the protein molecular weights of RSM substrate. Total proteins of raw RSM and FRSM were separated and 51 protein spots were selected for mass spectrometry according to 2D-DIGE map. In identified proteins, there were 15 extracellular hydrolases secreted by A. niger including glucoamylase, acid protease, beta-glucanase, arabinofuranosidase, xylanase, and phytase. Some antioxidant related enzymes also were identified. These findings suggested that A. niger is able to secrete many extracellular degradation enzymes (especially lignocellulosic hydrolyzing enzymes, acid proteases and phytase) during fermentation of RSM, thus altering chemical composition and physicochemical properties of RSM.

Biotransformation of β-hexachlorocyclohexane by the saprotrophic soil fungus Penicillium griseofulvum

β-Hexachlorocyclohexane (β-HCH) is a persistent organic pollutant (POP) of global concern with potentially toxic effects on humans and ecosystems. Fungal tolerance and biotransformation of toxic substances hold considerable promise in environmental remediation technologies as many fungi can tolerate extreme environmental conditions and possess efficient extracellular degradative enzymes with relatively non-specific activities. In this research, we have investigated the potential of a saprotrophic soil fungus, Penicillium griseofulvum Dierckx, isolated from soils with high concentrations of isomers of hexachlorocyclohexane, to biotransform β-HCH, the most recalcitrant isomer to microbial activity. The growth kinetics of the fungus were characterized after growth in stirred liquid Czapek-Dox medium. It was found that P. griseofulvum was able to grow in the presence of 1mgL−1 β-HCH and in stressful nutritional conditions at different concentrations of sucrose in the medium (0 and 5gL−1). The effects of β-HCH and the toluene, used as a solvent for β-HCH addition, on P. griseofulvum were investigated by means of a Phenotype MicroArray™ technique, which suggested the activation of certain metabolic pathways as a response to oxidative stress due to the presence of the xenobiotics. Gas chromatographic analysis of β-HCH concentration confirmed biodegradation of the isomer with a minimum value of β-HCH residual concentration of 18.6%. The formation of benzoic acid derivatives as dead-end products of β-HCH biotransformation was observed and this could arise from a possible biodegradation pathway for β-HCH with important connections to fungal secondary metabolism.

Strain-dependent response to Cu(2+) in the expression of laccase in Pycnoporus coccineus.

The effects of Cu(2+) on the activity and expression of laccase were investigated in seven different strains of Pycnoporus coccineus collected from different regions in Korea. Cu(2+) was toxic to mycelial growth at concentrations greater than 0.5 mM CuSO4 and showed complete growth inhibition at 1 mM in the liquid culture. However, Cu(2+) significantly upregulated the extracellular laccase activity at 0.2 mM in five strains of P. coccineus, IUM4209, IUM0032, IUM0450, IUM0470, and IUM4093, whereas two strains, IUM0253 and IUM0049, did not respond to Cu(2+), despite being closely related to the other five strains. Subsequent RT-PCR analysis also showed that the laccase mRNA was highly expressed only in the former five strains in the presence of Cu(2+). Taken together, these results indicate that Cu(2+) regulates expression of the laccase gene in a strain-dependent manner. The five strains commonly produced a single predominant laccase protein with a molecular weight of 68 kDa. Peptide sequencing revealed that the laccase was a homolog of Lcc1 of P. coccineus, which was isolated in China. The Cu(2+)-induced culture supernatants exhibited high degradation of polycyclic aromatic hydrocarbons, indicating that the 68-kDa laccase is the primary extracellular degradative enzyme in P. coccineus.

The ComP-ComA Quorum System Is Essential For “Trojan horse” Like Pathogenesis in Bacillus nematocida

Bacillus nematocida B16 has been shown to use “Trojan horse” mechanism in pathogenesis that has characteristics of “social” behavior. The ComP-ComA system, a conserved quorum sensing system in the genus Bacillus, functions in many physiological processes including competence development, lipopeptide antibiotic surfactin production, degradative enzyme production and even some unknown functions. Here we investigated the requirement of ComP-ComA system in B. nematocida B16 for its pathogenicity against nematodes. The ΔcomP mutant displayed deficiencies in attracting and killing nematodes, due to the absence of attractive signal molecules and the decreased expressions of virulence factors, respectively. Contrarily, a complemented comP mutant at least partially resumed its pathogenicity. Our data from transcriptional analysis further confirmed that this signaling system directly or indirectly regulated the expressions of two major virulence proteases in the infection of B. nematocida B16. Bioinformatics analyses from comparative genomics also suggested that the potential target genes of transcription factor ComA were involved in the processes such as the synthesis of attractants, production of extracellular degradative enzymes and sortase, secondary metabolites biosynthesis, regulation of transcription factors, mobility, as well as transporters, most of which were different from a saprophytic relative B. subtilis 168. Therefore, our investigation firstly revealed that the participation and necessity of ComP-ComA signaling system in bacterial pathogenesis.

Ecological role and biotechnological potential of mangrove fungi: a review

Mangroves are inhabited by large number of fungal communities, known as manglicolous fungi. They include mostly marine fungi and small group of terrestrial fungi and can be categorized into saprophytic, parasitic, and symbiotic fungi. Fungi in mangrove environment play an important ecological role in decomposition of organic matter by production of variety of extracellular degradative enzymes such as cellulase, xylanase, pectinase, amylase, and so on. Such enzymes can be isolated from the mangrove fungi and harnessed for several biotechnological applications. Several bioactive metabolites are derived from mangrove fungi, specially from mangrove endophytic fungi, which are used in pharmaceutical and nutraceutical industries to produce antimicrobial, anticancer, antioxidant, antidiabetic, and other therapeutic agents. Besides, certain mangrove fungi also contribute toward production of biopesticides useful in control of plant diseases, while others produce microbial lipids used as potential feed stock for biodiesel production. In spite of immense ecological role and biotechnological potentials, mangrove fungi are not extensively studied. The present review provides information on diversity and ecological role of mangrove fungi along with their biotechnological potentials as source of novel drugs, enzymes, biodiesel, biopesticides, and many other applications.

Crystal structure of the novel PaiB transcriptional regulator from Geobacillus stearothermophilus

The pai operon is transcriptionally regulated by PaiB and has been found to be essential for cell growth and involved in the negative control of sporulation and degradative enzyme production. The pai operon was initially cloned as a fragment of the Bacillus subtilis chromosome consisting of two genes paiA and paiB.1 The presence of both of these genes on a multicopy vector revealed that both decrease the levels of extracellular degradative enzymes such as subtilisin, neutral protease, levansucrase, α-amylase, and alkaline phosphatases and also greatly reduce the frequency of sporulation.2 Based on the study by Strauch (1993), paiB appears to encode a regulator belonging to the same family as the AbrB, Hpr, and Sin regulators. AbrB, Hpr, Sin, and PaiB control the synthesis of degradative enzymes that occurs during the transition between vegetative growth and the onset of the stationary phase and sporulation in B. subtilis.2 Known sporulation genes have been identified as targets for direct control by AbrB (spo0E, spo0H, and spoVG) and Sin (spoIIA, spoIID, spoIIE, and spoIIG).3 Hpr and PaiB may also regulate spo genes directly as evidenced by the Spo phenotype of strains carrying multicopy plasmids that overexpress these two genes.1 Preliminary bioinformatic data suggested that PaiB might be a split barrel flavin-binding regulator. Herein, we report the first crystal structure of the Pai transcriptional regulator: PaiB from Geobacillus stearothermophilus. The structure of PaiB demonstrates that it is a homodimer and that each monomer consists of three domains: large, intermediate, and C-terminal. The large and intermediate domains fold into a structure similar to pyridoxine 5′-phosphate oxidase (PNPO) and form an analogous flavin mononucleotide (FMN)-binding cavity. The small C-terminal domain of PaiB possesses a helix-turn-helix (HTH) DNA-binding fold. The comparisons of sequence and structure show that PaiB is a representative of a new sequence family of transcriptional regulators. Detailed analysis of the protein structure provides insights into the potential biological function.

Proteome analysis of fungal and bacterial involvement in leaf litter decomposition

Fungi and bacteria are key players in the decomposition of leaf litter, but their individual contributions to the process and their interactions are still poorly known. We combined semi-quantitative proteome analyses (1-D PAGE-LC-MS/MS) with qualitative and quantitative analyses of extracellular degradative enzyme activities to unravel the respective roles of a fungus and a bacterium during litter decomposition. Two model organisms, a mesophilic Gram-negative bacterium (Pectobacterium carotovorum) and an ascomycete (Aspergillus nidulans), were grown in both, pure culture and co-culture on minimal medium containing either glucose or beech leaf litter as sole carbon source. P. carotovorum grew best in co-culture with the fungus, whereas growth of A. nidulans was significantly reduced when the bacterium was present. This observation suggests that P. carotovorum has only limited capabilities to degrade leaf litter and profits from the degradation products of A. nidulans at the expense of fungal growth. In accordance with this interpretation, our proteome analysis revealed that most of the extracellular biodegradative enzymes (i.e. proteases, pectinases, and cellulases) in the cultures with beech litter were expressed by the fungus, the bacterium producing only low levels of pectinases.

Complete Genome Sequence of Citrus Huanglongbing Bacterium, ‘CandidatusLiberibacter asiaticus’ Obtained Through Metagenomics

Citrus huanglongbing is the most destructive disease of citrus worldwide. It is spread by citrus psyllids and is associated with a low-titer, phloem-limited infection by any of three uncultured species of alpha-Proteobacteria, 'Candidatus Liberibacter asiaticus', 'Ca. L. americanus', and 'Ca. L. africanus'. A complete circular 'Ca. L. asiaticus' genome has been obtained by metagenomics, using the DNA extracted from a single 'Ca. L. asiaticus'-infected psyllid. The 1.23-Mb genome has an average 36.5% GC content. Annotation revealed a high percentage of genes involved in both cell motility (4.5%) and active transport in general (8.0%), which may contribute to its virulence. 'Ca. L. asiaticus' appears to have a limited ability for aerobic respiration and is likely auxotrophic for at least five amino acids. Consistent with its intracellular nature, 'Ca. L. asiaticus' lacks type III and type IV secretion systems as well as typical free-living or plant-colonizing extracellular degradative enzymes. 'Ca. L. asiaticus' appears to have all type I secretion system genes needed for both multidrug efflux and toxin effector secretion. Multi-protein phylogenetic analysis confirmed 'Ca. L. asiaticus' as an early-branching and highly divergent member of the family Rhizobiaceae. This is the first genome sequence of an uncultured alpha-proteobacteria that is both an intracellular plant pathogen and insect symbiont.

Ligninolytic Enzymes Produced by Phanerohaete Chrysosporium 1038 and Biotransformation of Lignin

ABSTRACTThe influence of nitrogen and Mn2+ ions concentrations in growth medium on the activity of extracellular lignin degradation enzymes: lignin peroxidase (LiP—EC 1.11.1.14), manganese peroxidase (MnP—EC 1.11.1.13) and laccase (Lac—EC 1.10.3.2), produced by Phanerochaete chrysosporium strain 1038, was studied. Evidence for de lignification processes was provided by FTIR spectroscopy after 24 h incubation of lignin samples with concentrated extracellular fluids of activated strain 1038. FTIR spectra showed decrease of carboxylic groups and increase of hydroxylated and methoxylated aliphatic groups, which indicates an oxidative cleavage. The resulting modifications of the lignin structure are due to the low specificity of the ligninolytic enzyme system, consisting of extracellular LiP, MnP and Lac. The addition of 3.5mg/L Mn2+, 63 mg/L Cu2+ and 2.5mM veratryl alcohol is necessary for the activation of lignolytic complex and obtaining of optimal conditions for delignification process after incubation of lignin samples with fluids from the strain. The effect of growth conditions on the enzyme activities of Phanerochaete chrysosporium 1038 and efficiency of delignification procedure are discussed.