Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene

Aims: Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. Methods and Results: Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in the rough variants of the same strain. Temperature significantly increased the adhesion of rhizoid variants up to 20°C. Modification of bacterial surface suggested that adhesion molecules contain both carbohydrates and proteins. LPS did not differ between the variants of the same strain. Conclusions: The results suggest that in Fl. columnare both rhizoid colony morphology and high chondroitinase activity are needed for virulence and that temperature may promote the adhesion of the virulent variants to surfaces at fish farms. Significance and Impact of the Study: New information is produced on the virulence mechanisms of Fl. columnare and the reasons behind the survival of the bacterium at fish farms.     

Genetic and virulence characterization of Flavobacterium columnare from channel catfish (Ictalurus punctatus)

Aim: To develop a method for conducting pulsed-field gel electrophoresis (PFGE) on Flavobacterium columnare, to use PFGE to characterize F. columnare channel catfish isolates, and to determine whether variation in pathogenic potential exists in F. columnare isolates from channel catfish. Methods and Results: On the basis of PFGE-derived profiles, similarity dendrograms constructed for more than 30 F. columnare isolates showed two major genetic groups with more than 60% similarity. Channel catfish fingerlings challenged with PFGE group A isolates by bath immersion had significantly higher average mortalities (>60%) than fish challenged with PFGE group B isolates (<9%). However, abrasion and skin mucus removal made channel catfish fingerlings susceptible to disease caused by group B isolates following immersion exposure. Conclusion: Our results suggest that two genetic divisions of F. columnare channel catfish isolates exist, and that isolates in PFGE group A isolates tend to be more pathogenic to immunocompetent channel catfish fingerlings than PFGE group B isolates. Significance and Impact of the Study: PFGE is a potentially useful tool for determining whether F. columnare isolates are more likely to be primary or secondary pathogens. Pathogenesis research for columnaris disease in catfish should focus on pathogenic isolates from PFGE group A.     

Sensitive and rapid detection of Flavobacterium columnare in channel catfish Ictalurus punctatus by a loop-mediated isothermal amplification method

AIMS: To evaluate the loop-mediated isothermal amplification method (LAMP) for rapid detection of Flavobacterium columnare and determine the suitability of LAMP for rapid diagnosis of columnaris infection in channel catfish, Ictalurus punctatus. METHODS AND RESULTS: A set of four primers, two outer and two inner, were designed specifically to recognize 16S ribosomal RNA gene of this pathogen. Bacterial genomic DNA templates were prepared by hot lysis in a lysis buffer. Amplification of the specific gene segments was carried out at 65 degrees C for 1 h. The amplified gene products were analysed by agarose gel electrophoresis and detected by staining gels with ethidium bromide. A PCR assay was also included in this study. Our results demonstrate that the ladder-like pattern of bands from 204 bp specific to the Fl. columnare 16S ribosomal RNA gene was amplified. The detection limit of the LAMP assay was comparable to that of PCR in prepared genomic DNA reactions. In addition, this optimized LAMP assay was able to detect the Fl. columnare 16S ribosomal RNA gene in experimentally infected channel catfish. CONCLUSIONS: The LAMP assay for Fl. columnare detection in channel catfish was established. SIGNIFICANCE AND IMPACT OF THE STUDY: Because LAMP assay is a rapid, sensitive, specific, simple and cost-effective assay for Fl. columnare detection in channel catfish, it is useful for rapid diagnosis of Fl. columnare in fish hatcheries and the field.     

Activities of Wogonin Analogs and Other Flavones against Flavobacterium columnare

In our on‐going pursuit to discover natural products and natural product‐based compounds to control the bacterial species Flavobacterium columnare, which causes columnaris disease in channel catfish (Ictalurus punctatus), we synthesized flavone and chalcone analogs, and evaluated these compounds, along with flavonoids from natural sources, for their antibacterial activities against two isolates of F. columnare (ALM‐00‐173 and BioMed) using a rapid bioassay. The flavonoids chrysin ( 1a ), 5,7‐dihydroxy‐4′‐methoxyflavone ( 11 ), isorhamnetin ( 26 ), luteolin ( 27 ), and biochanin A ( 29 ), and chalcone derivative 8b showed strong antibacterial activities against F. columnare ALM‐00‐173 based on minimum inhibition concentration (MIC) results. Flavonoids 1a, 8, 11, 13 (5,4′‐dihydroxy‐7‐methoxyflavone), 26 , and 29 exhibited strong antibacterial activities against F. columnare BioMed based upon MIC results. The 24‐h 50% inhibition concentration (IC₅₀) results revealed that 27 and 29 were the most active compounds against F. columnare ALM‐00‐173 (IC₅₀ of 7.5 and 8.5 mg/l, resp.), while 26 and 29 were the most toxic compound against F. columnare BioMed (IC₅₀ of 9.2 and 3.5 mg/l, resp.). These IC₅₀ results were lower than those obtained for wogonin against F. columnare ALM‐00‐173 and F. columnare BioMed (28.4 and 5.4 mg/l, resp.). However, based on MIC results, none of the compounds evaluated in this study were as active as wogonin (MIC 0.3 mg/l for each F. columnare isolate). Further modification of the wogonin structure to enhance antibacterial is of interest.     

Molecular typing of isolates of the fish pathogen, Flavobacterium columnare, by single-strand conformation polymorphism analysis

Flavobacterium columnare intraspecies diversity was revealed by analyzing the 16S rRNA gene and the 16S-23S internal spacer region (ISR). Standard restriction fragment length polymorphism (RFLP) of these sequences was compared with single-strand conformation polymorphism (SSCP). Diversity indexes showed that both 16S-SSCP and ISR-SSCP improved resolution (D>or=0.9) when compared with standard RFLP. ISR-SSCP offered a simpler banding pattern than 16S-SSCP while providing high discrimination between isolates. SSCP analysis of rRNA genes proved to be a simple, rapid, and cost-effective method for routine fingerprinting of F. columnare.     

Molecular cloning, overexpression and biochemical characterization of hypothetical beta-lactamases of Mycobacterium tuberculosis H37Rv

Aim:  Molecular cloning, overexpression and biochemical characterization of the genes from the Mycobacterium tuberculosis H37Rv genome having hypothetical β-lactamases activity.
Methods and Results:  Analysis of the M. tuberculosis H37Rv genome revealed that Rv 2068c , Rv 0406 c and Rv 3677 c gene products were predicted to exhibit β-lactamases activity. All the three genes were cloned in pET28a vector and overexpressed in C41 (DE3) Escherichia coli cells. The His-tagged recombinant proteins were confirmed by immunoblotting and were shown to have β-lactamase activity by the hydrolysis of nitrocefin and other β-lactams. Catalytic parameters for all the recombinant proteins were derived followed by the enzyme inhibition studies. Antibiotic susceptibility studies using the recombinant strains showed an increased resistance against different classes of β-lactam antibiotics.
Conclusion:  The study revealed the possibility of more than one gene in M. tuberculosis , encoding proteins having β-lactamase or β-lactamase-like activity, giving wide spectrum of resistance against β-lactams.
Significance and Impact of the Study:  Systematic study of hypothetical β-lactamases of M. tuberculosis and related species and their correlation with β-lactam and inhibitor susceptibility profile might be useful in developing new antibiotic regime for the treatment of tuberculosis caused by multiple drug resistant (MDR) strains.     

Purification and characterization of an exo-beta-1,3-glucanase produced by Trichoderma asperellum

Trichoderma asperellum produces at least two extracellular beta-1,3-glucanases upon induction with cell walls from Rhizoctonia solani. A beta-1,3-glucanase was purified by gel filtration and ion exchange chromatography. A typical procedure provided 35.7-fold purification with 9.5% yield. The molecular mass of the purified exo-beta-1,3-glucanases was 83.1 kDa as estimated using a 12% (w/v) SDS-electrophoresis slab gel. The enzyme was only active toward glucans containing beta-1,3-linkages and hydrolyzed laminarin in an exo-like fashion to form glucose. The K(m) and V(max) values for exo-beta-1,3-glucanase, using laminarin as substrate, were 0.087 mg ml(-1) and 0.246 U min(-1), respectively. The pH optimum for the enzyme was pH 5.1 and maximum activity was obtained at 55 degrees C. Hg(2+) strongly inhibited the purified enzyme.     

High level expression in Escherichia coli of isopenicillin N synthase genes from Flavobacterium and Streptomyces, and recovery of active enzyme from inclusion bodies

A T7 promoter-based vector was used to express the isopenicillin N synthase (IPNS) genes of Flavobacterium sp. 12,154 and Streptomyces jumonjinensis in Escherichia coli. Most of the IPNS synthesized at 37 degrees C, and representing some 22% and 51% of the total cell protein respectively, occurred in an insoluble, enzymatically inactive form. Active IPNS was recovered in a rapid and simple two-step procedure in which the insoluble material was first denatured in 5 M urea and then refolded by passing the solubilized IPNS through a G-25 Sephadex sizing column. Further chromatography on DEAE-Sepharose resulted in highly active IPNS preparations. This procedure was found to be well suited for scaling up to produce large amounts of IPNS.     

Association patterns of Pseudomonas aeruginosa clinical isolates as revealed by virulence traits, antibiotic resistance, serotype and genotype

The aims of this study were to assess the association patterns of 96 clinical isolates of Pseudomonas aeruginosa using hierarchical cluster analysis from data obtained from the measurement of the physicochemical cell surface properties, adhesion and initial biofilm formation abilities, to investigate any correspondence with source, serotype, beta-lactam pattern, motility and M13-PCR genogroup or clonal lineage, as well as to select clinical isolates that could act as representatives of the genotypic and phenotypic diversity of this P. aeruginosa population from a Portuguese Central Hospital. The isolates were phenotypically characterized by their ability to adhere and form biofilms on polystyrene surfaces, their affinity to hexadecane and silicone, their swimming and twitching abilities, their antibiotic susceptibility patterns and their serotypes. No particular phenotypic cluster associated with the same source, serotype, beta-lactam pattern, motility and M13-PCR genogroup and clonal lineage was found. Nevertheless, five representative strains of the P. aeruginosa population from this Hospital, selected on the basis of low genetic similarity, were also found to be dispersed among the phenotypic clusters.     

Production and characterization of a thermostable protease produced by an asporogenous mutant ofBacillus stearothermophilus

Summary An asporogenous mutant ofBacillus stearothermophilus (TPM-8) which produces 4-fold higher levels of a thermostable neutral protease than does wild-type strain 308-1 was obtained by mutagenesis with ethyl methanesulfonate. The protease produced by both the mutant and wild-type strain is a metalloprotease requiring Zn²⁺ and Ca²⁺ for activity and thermostability, respectively. It has a temperature optimum of 80°C at pH 7.0 and is highly thermostable, retaining 60% of its activity after 60 min at 85°C. The properties of the enzyme are similar to those of thermolysin.     

Production and partial characterization of a beta-amylase by Xanthophyllomyces dendrorhous

AIMS: The characterization of a beta-amylase produced by Xanthophyllomyces dendrorhous. METHODS AND RESULTS: Growth in different culture media showed that X. dendrorhous produces an amylase whose synthesis is repressed by the carbon source and induced by starch and maltose. Enzymatic assays using substrates with different degrees of polymerization together with viscosity experiments revealed that the enzyme was beta-amylase. According to the biochemical characterization, the enzyme has a molecular weight of 240 kDa and a Km of 1.35 mg ml-1. The optimum pH and temperature were 5.5 and 50 degrees C, respectively. Using different inhibitors of the enzymatic activity it was shown that cysteine, tryptophan and serine are essential amino acids for catalysis. CONCLUSIONS: Xanthophyllomyces dendrorhous CECT1690 synthesizes and secretes beta-amylase that could be a by-product, in addition to carotenoid pigments, in the fermentation downstream. SIGNIFICANCE AND IMPACT OF THE STUDY: The beta-amylase produced by X. dendrorhous may have certain industrial applications.     

Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.     

Induction studies showing evidence of the similarities between an inducible intracellular and extracellular β-d-glucosidase produced by a species of Monilia

Abstract A Monilia sp. produced an inducible intracellular β- d -glucosidase (IG-2) which is the nascent form of the extracellular enzyme (EG-1) prior to its secretion into the extracellular medium. The other intracellular β- d -glucosidase (IG-1) produced was a constitutive enzyme. Highest yields of the inducible β- d -glucosidase resulted when Monilia sp. was grown on insoluble cellulose. Cellobiose and d -glucose appeared to repress β- d -glucosidase formation at high substrate levels and synthesis occurred only once the levels of these sugars in the medium were nearly depleted.     

Isolation, characterization and physiological properties of an autolytic-deficient mutant of Streptococcus pneumoniae

Summary A spontaneous mutation in the gene lyt encoding the pneumococcal autolysin has been characterized. This mutation, named lyt-32, which behaves as a high-efficiency marker in pneumococcal transformation, is a single base pair GC deletion causing the appearance of two consecutive termination codons in the amino terminal part of the sequence of the autolysin gene. The mutant lyt gene did not code for a polypeptide of relative molecular mass corresponding to the pneumococcal E form amidase in Escherichia coli maxicells. Pneumococcal cells containing the lyt-32 mutation (M32) were fully transformable, multiplied at a normal growth rate forming small chains and showed a tolerant response when treated with beta-lactam antibiotics. Strain M32 represents the first example of a mutant of Streptococcus pneumoniae completely lacking amidase as a consequence of an alteration in the structural gene coding for the pneumococcal autolysin.     

Purification and characterization of a novel esterase (beta-hydroxypalmitate methyl ester hydrolase) and prevention of the expression of virulence by Ralstonia solanacearum

AIMS: To screen novel micro-organisms and enzymes capable of degrading 3-hydroxypalmitic acid methyl ester (3-OH PAME), the quorum-sensing signal molecule (quormone), which regulates the virulence of Ralstonia solanacearum. METHODS AND RESULTS: Ideonella sp. 0-0013, a betaproteobacterium isolated from soil using the selective-enrichment culture method, was grown on plates containing 3-OH PAME as its main carbon source. beta-Hydroxypalmitate methyl ester hydrolase (betaHPMEH) purified from the supernatant of the Ideonella sp. 0-0013 culture exhibited high hydrolysing activity towards the ester bond of 3-OH PAME and eliminated the 3-OH PAME activity, thereby reducing the virulence of R. solanacearum. An Escherichia coli transformant of the betahpmeh gene expression vector degraded 3-OH PAME, and the crude enzyme from the transformant inhibited in vitro production of the R. solanacearum exopolysaccharide (EPS). CONCLUSIONS: The ability of betaHPMEH to hydrolyse 3-OH PAME inhibited the production of EPS by the R. solanacearum wild-type strain, indicating that betaHPMEH inhibits the effects of activation of virulence genes. This ability will be potentially useful for pest control of the wilt disease caused by this bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: This enzyme is the first protein that has been found to degrade a quormone other than N-acyl homoserine lactone.     

Molecular characterization and localization of the RIC-3 protein, an effector of nicotinic acetylcholine receptor expression

The RIC-3 protein acts as a regulator of acetylcholine nicotinic receptor (nAChR) expression. In Xenopus laevis oocytes the human RIC-3 (hRIC-3) protein enhances expression of α7 receptors and abolishes expression of α4β2 receptors. In vitro translation of hRIC-3 evidenced its membrane insertion but not the role as signal peptide of its first transmembrane domain (TMD). When the TMDs of hRIC-3 were substituted, its effects on nAChR expression were attenuated. A certain linker length between the TMDs was also needed for α7 expression enhancement but not for α4β2 inhibition. A combination of increased α7 receptor steady state levels, facilitated transport and reduced receptor internalization appears to be responsible for the increase in α7 membrane expression induced by hRIC-3. Antibodies against hRIC-3 showed its expression in SH-SY5Y and PC12 cells and its induction upon differentiation. Immunohistochemistry demonstrated the presence of RIC-3 in rat brain localized, in general, in places where α7 nAChRs were found.     

Production, purification and structural characterization of an exopolysaccharide produced by a probiotic Lactobacillus plantarum MTCC 9510

Exopolysaccharides (EPS) from lactic acid bacteria contribute to specific rheology and texture of fermented milk products and finds applications even in non-dairy foods and in therapeutics. Box-Behnken model of response surface methodology (RSM) was employed to formulate the production medium for exopolysaccharide (EPS). FT-IR spectral analysis of the purified EPS from Lactobacillus plantarum MTCC 9510 revealed prominent characteristic groups corresponding to polyhydric alcohols. The degradation temperature (Td) of the polysaccharide was found to be 260°C with the help of thermo gravimetric analysis (TGA). Structure elucidation of the EPS showed that it consists of a trisaccharide repeating unit of α-d-glucose, β-d-glucose and α-d-mannose.     

Production, purification and characterization of chitosanase produced by Gongronella sp. JG

AIMS: To optimize the production condition of chitosanases of Gongronella sp. JG and to characterize the major chitosanase. METHODS AND RESULTS: In the optimized medium and culturing condition, strain JG produced 800 micromol min(-1) l(-1) chitosanase activity at 72 h. The major chitosanase - csn1 was purified through three chromatography steps: CM (carboxymethyl)-Sepharose fast flow (FF), Sephacryl S200, SP (sulfopropyl)-Sepharose FF. The molecular weight and the pI value of csn1 were about 90,000 Da and 5 x 8, respectively. Its specific activity was 82 micromol min(-1) mg(-1). The optimal reaction pH for csn1 was between 4 x 6 and 4 x 8. The optimal reaction temperature was 50 degrees C. The half-life of csn1 at 50 degrees C was estimated to be about 65 min. Mn(2+) was a strong stimulator of csn1 activity, both at 1 and 10 mmol l(-1). csn1 showed its highest activity with chitosan of 85% degree of deacetylation, but did not hydrolyse colloidal chitin and carboxylmethyl cellulose. In 20 mmol l(-1) sodium acetate buffer (pH 4 x 8) and at 50 degrees C, the K(m) of csn1 was calculated to be 4 x 5 mg ml(-1). CONCLUSIONS: The production condition of chitosanases by Gongronella JG was optimized and the major chitosanase, csn1, was characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work for the first time reported the production, purification and characterization of chitosanases produced by fungus of Gongronella sp. These results provided us more information on fungal chitosanases.