Laccase activity in melanin-producing strains of Sinorhizobium meliloti

Laccase-like activity was detected in melanin-producing strains of Sinorhizobium meliloti mainly in cells at the stationary growth phase when copper was added to the medium. The laccase showed both syringaldazine and ABTS (2,2'-azino-bis-ethylbenzthiazoline-6-sulfonic acid) oxidase activities and was activated by the addition of 1.7 mM sodium dodecyl sulfate. Activity was totally inhibited by the addition of 1.0 mM EDTA, suggesting that the enzyme is a metal-dependent one. The enzyme was found to be cytosolic having an optimum pH of 5.0, an estimated molecular mass of 95 kDa and a K(m) of 4 microM for syringaldazine. Both laccase and tyrosinase activities were detected in melanin-producing S. meliloti strains. Plant growth-promoting (PGP) effect in rice by a laccase-producing S. meliloti strain when co-inoculated with Azospirillum brasilense Cd was observed. PGP effect by co-inoculation significantly increased plant yield compared to A. brasilense by itself. To the best of our knowledge this is the first report on laccase production in rhizobia and cooperation between Azospirillum and Sinorhizobium in rice.     

Population dynamics of a motile and a non-motile Azospirillum lipoferum strain during rice root colonization and motility variation in the rhizosphere

Abstract: Azospirillum lipoferum 4B and non-motile A. lipoferum 4T have been simultaneously isolated from rice rhizosphere at the same frequency. A. lipoferum 4T showed stable morphological and metabolic traits which are atypical for A. lipoferum species such as lack of motility, carbohydrate metabolism and laccase activity. Inoculation experiments showed that A. lipoferum 4T, but not A. lipoferum 4B, needed rice roots to stabilize in sterile soil. Both strains were able to colonize efficiently rice roots (10⁸ cfu g⁻¹ fresh roots) but motile form 4B remained dominant. In spite of their phenotypical differences, A. lipoferum 4B and 4T co-existed without exclusion in sterile soil (planted or not) and rice rhizosphere. Inoculation of rice roots with A. lipoferum 4B showed that rice rhizosphere enhanced the frequency of appearance of stable non-motile forms (40%). This percentage was weaker in plantlet growth medium (4%). However, these non-motile bacteria kept the same biochemical traits than the motile parental strain 4B (carbohydrates metabolism, laccase activity).     

Evaluation of an rRNA-targeted oligonucleotide probe for the detection of mosquitocidal strains of Bacillus sphaericus in soils: characterization of novel strains lacking toxin genes

Abstract: Colonies of Bacillus sphaericus on primary isolation have been identified using an oligonucleotide probe targeted to a specific region of the 16S rRNA. Of 3440 colonies from soil samples from Brazil, 57 hybridized to the probe in colony blots but when purified DNA was used in slot-blots the probe was more specific and only 27 isolates hybridized. Of these, 20 strains were confirmed as members of DNA homology group IIA (potential mosquitocidal strains) by ribotyping and isoenzyme analysis. However, none of these strains was toxic to Anopheles or Culex larvae, nor did they contain recognized toxin genes. This is the first demonstration of such non-pathogenic strains of B. sphaericus DNA homology group IIA and their common occurrence suggests that pathogenicity is not an important contribution to the success of these bacteria in the environment. Similar screening of strains from Scottish soils indicated that B. sphaericus DNA homology group IIA strains were less common in this habitat and none were recovered on this occasion.     

Phylogenetic analysis of and oligonucleotide probe development for eikelboom type 021N filamentous bacteria isolated from bulking activated sludge

Fifteen filamentous strains, morphologically classified as Eikelboom type 021N bacteria, were isolated from bulking activated sludges. Based on comparative 16S ribosomal DNA (rDNA) sequence analysis, all strains form a monophyletic cluster together with all recognized Thiothrix species (88.3 to 98.7% 16S rDNA sequence similarity) within the gamma-subclass of Proteobacteria. The investigated Eikelboom type 021N isolates were subdivided into three distinct groups (I to III) demonstrating a previously unrecognized genetic diversity hidden behind the uniform morphology of the filaments. For in situ detection of these bacteria, 16S rRNA-targeted oligonucleotide probes specific for the entire Eikelboom type 021N-Thiothrix cluster and the Eikelboom type 021N groups I, II, and III, respectively, were designed, evaluated, and successfully applied in activated sludge.     

彩绒革盖菌固体发酵生产木质素酶工艺优化研究*

采用均匀设计U15(5^8)和双温度培养法进行彩绒革盖菌固体发酵生产木质素酶,对漆酶、愈创木酚酶、多酚氧化酶的活力进行回归分析。结果表明：应用双温度培养法进行彩绒革盖菌固体发酵生产木质素酶时,在自然补给氧气,培养基pH自然(约6．5),并保持环境湿度约60％的条件下,20d是适宜的发酵周期;玉米浆、麸皮、(NH4)2SO4、水分适宜作为固体培养基的成分;少量的Tween-80有利于木质素酶的生产。     

Phylogenetic analysis of transformable strains of thermophilic Bacillus species

Few strains of thermophilic Bacillus spp are readily transformable with plasmid DNA. Given the considerable phylogenetic and phenotypic diversity amongst thermophilic bacilli, we have examined whether transformability is a trait associated with a particular phylogenetic group, by sequencing the 16S ribosomal RNA genes from transformable strains NUB3621, K1041, and NRRL1174. Although all of these strains were described in the literature as B. stearothermophilus, only NRRL1174 is closely related to the type strain of this species. Based on its 16S rDNA sequence and physiological data K1041 appeared to belong to the species B. thermodenitrificans, while NUB3621 showed a slightly closer relationship to B. thermoglucosidasius than to B. stearothermophilus. Therefore we conclude that the trait of transformability, though possibly strain-specific, is not limited to a single species of thermophilic Bacillus.     

Phylogenetic relationships of coconut phytoplasmas and the development of specific oligonucleotide PCR primers

Using the polymerase chain reaction the 16S rRNA genes and the 16S-23S spacer regions of phytoplasmas associated with lethal decline diseases of coconut palm (Cocos nucifera), were amplified from infected plants from Florida and the Yucatan region in Mexico and from east and west Africa. Following sequencing of the rDNA products, phylogenetic analysis confirmed that these coconut phytoplasmas form a separate cluster within the phytoplasma clade and that the pathogen causing diseases in west Africa formed a new sub-clade within this cluster. Analysis of the 16S-23S intergenic spacer regions confirmed the sequence diversity of this region and enabled two primers to be designed which were specific for the diseases found in east and west Africa. None of these specific primers, when paired with a universal primer, produced PCR amplification products from healthy coconut DNA, infected coconut DNA from the Caribbean or DNA from a variety of periwinkle (Catharanthus roseus)-maintained phytoplasmas. These specific primers can serve as effective tools for identifying particular coconut phytoplasmas in field samples.     

Isolation and identification of Bacillus sphaericus strains pathogenic for mosquito larvae

Three selective media for the isolation of Bacillus sphaericus have been compared. BATS medium and a formulation employing adenosine as the principal carbon source were the most effective for the recovery of spores of strain 1593. Anthranilic acid as the principal carbon source was less efficient. Eighty-four strains were isolated from mud samples using these media and were identified by computer. Identifications were confirmed for representative strains using DNA sequence homology. Most were B. sphaericus sensu stricto or members of an unnamed group. However, one strain (BSE 18) was identified as the DNA homology group IIB and this organism was found to be highly toxic toward larvae of Culex pipiens. Southern hybridization of BSE 18 DNA to a probe prepared from the cloned toxin gene from strain 1593 revealed that BSE 18 contained a typical gene for the 41.9-kDa toxin.     

Plasmid deoxyribonucleic acid in strains of Bacillus sphaericus and in Bacillus moritai

Bacillus moritai and six strains of Bacillus sphaericus pathogenic to dipteran larvae were examined for the presence of covalently closed circular (CCC) DNA. The plasmid profiles of the bacteria were analyzed using a cleared lysate electrophoresis technique. Four of the six strains of B. sphaericus examined contained CCC DNA. Strain SSII-1 contained two plasmids (pKA1, pKA2) having molecular weights of about 8.4 and 2.0 megadaltons (MDa). Strains 1404 and 1881 each contained one plasmid, pKA3 and pKA4, respectively. pKA3 had a molecular weight of about 8.2 MDa. pKA4 had a relatively large plasmid with a molecular weight of about 33.5 MDa. Strain K contained five size classes of CCC DNA. The plasmids pKA5, pKA6, pKA7, pKA8, and pKA9 had molecular weights of about 11.4, 10.9, 7.4, 7.0, and 6.4 MDa, respectively. Strains 1593-4 and 1691 were plasmidless and could not be distinguished from each other based on their plasmid profiles. B. moritai ATCC 21042 contained two size classes of CCC duplex DNA; pRF100 had a molecular weight of about 4.6 MDa and pRF101 had a molecular weight of about 2.1 MDa. No phenotype association with any of the isolated plasmids has been determined.     

Characterization of endogenous and recombinant forms of laccase-2, a multicopper oxidase from the tobacco hornworm, Manduca sexta

Laccases belong to the group of multicopper oxidases that exhibit wide substrate specificity for polyphenols and aromatic amines. They are found in plants, fungi, bacteria, and insects. In insects the only known role for laccase is in cuticle sclerotization. However, extracting laccase from the insect's cuticle requires proteolysis, resulting in an enzyme that is missing its amino-terminus. To circumvent this problem, we expressed and purified full-length and amino-terminally truncated recombinant forms of laccase-2 from the tobacco hornworm, Manduca sexta. We also purified the endogenous enzyme from the pharate pupal cuticle and used peptide mass fingerprinting analysis to confirm that it is laccase-2. All three enzymes had pH optima between 5 and 5.5 when using N-acetyldopamine (NADA) or N-β-alanyldopamine-alanyldopamine (NBAD) as substrates. The laccases exhibited typical Michaelis–Menten kinetics when NADA was used as a substrate, with K_m values of 0.46 mM, 0.43 mM, and 0.63 mM, respectively, for the full-length recombinant, truncated recombinant, and cuticular laccases; the apparent k_cat values were 100 min⁻¹, 80 min⁻¹, and 290 min⁻¹. The similarity in activity of the two recombinant laccases suggests that laccase-2 is expressed in an active form rather than as a zymogen, as had been previously proposed. This conclusion is consistent with the detection of activity in untanned pupal wing cuticle using the laccase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Immunoblot analysis of proteins extracted from both tanned and untanned cuticle detected only a single protein of 84 kDa, consistent with the full-length enzyme. With NBAD as substrate, the full-length recombinant and cuticular laccases showed kinetics indicative of substrate inhibition, with K_m values of 1.9 mM and 0.47 mM, respectively, and apparent k_cat values of 200 min⁻¹ and 180 min⁻¹. These results enhance our understanding of cuticle sclerotization, and may aid in the design of insecticides targeting insect laccases.     

The development and assessment of DNA and oligonucleotide probes for the specific detection of Bacillus anthracis

Two DNA probes and a number of oligonucleotide probes were designed from the virulence factor genes of Bacillus anthracis. These probes were tested for specificity against 52 B. anthracis strains and 233 Bacillus strains encompassing 23 other species. A rapid slot blotting technique was used for screening the large numbers of isolates involved. All probes tested appeared to be specific for B. anthracis under high stringency conditions. These probes could differentiate between virulent and avirulent strains. The probes were also applied to the detection of B. anthracis in routine environmental and clinical samples. A non-radioactive hybridization and detection system based on digoxigenin-11-dUTP was developed.     

Supplement comprising of laccase and citric acid as an alternative for antibiotics: In vitro triggers of melanin production

An indiscriminate use of antibiotics in humans and animals has led to the widespread selection of antibiotic‐resistance, thus constricting the use of antibiotics. A possible solution to counter this problem could be to develop alternatives that can boost the host immunity, thus reducing the quantity and frequency of antibiotic use. In this work, for the first time, citric acid and laccase were used as extracellular inducers of melanin production in yeast cells and human cell lines. It is proposed that the formulation of laccase and citric acid together could further promote melatonin‐stimulated, melanocyte‐derived melanin production. Melanization as a probe of immunity described in this study, is an easy and a rapid test compared to other immunity tests and it allows performing statistical analyses. The results showed the synergistic effect of citric acid and laccase on melanin production by yeast cells, with significant statistical differences compared to all other tested conditions (p: 0.0005–0.005). Laccase and citric acid together boosted melanin production after 8 days of incubation. An increase in melanin production by two human colon cells lines (Cacao‐2/15 and HT‐29) was observed on supplementation with both laccase and citric acid in the cell growth medium. Produced melanin showed antimicrobial properties similar to antibiotics. Therefore, a formulation with citric acid and laccase may prove to be an excellent alternative to reduce the antibiotic use in human and animal subjects.     

Phylogenetic analysis and heterologous expression of surface layer protein SlpC of Bacillus sphaericus C3-41

The surface layer protein encoding genes from five mosquito-pathogenic Bacillus sphaericus isolates were amplified and sequenced. Negative staining of the S-layer protein extracted from the cell wall of wild-type B. sphaericus C3-41 was prepared. It showed a flat-sheet crystal lattice structure. Two genes encoding the entire and N-terminally truncated S-layer protein (slpC and DeltaslpC respectively), were ligated into plasmid pET28a and expressed in Escherichia coli. SDS-PAGE revealed that about 130 KD and 110 KD proteins could be expressed in the cytoplasm of recombinant E. coli BL21(pET28a/slpC) and E. coli BL21(pET28a/DeltaslpC) respectively. Furthermore, an intracellular sheet-like or fingerprint-shape structure was investigated in two recombinant strains, which expressed SlpC and DeltaSlpC protein respectively, by ultrathin microscopy study, but bioassay results suggested that the S-layer protein of wild B. sphaericus C3-41 and recombinant E. coli BL21 (pET28a/slpC) have no direct toxicity against mosquito larvae. These results should provide information for further understanding of the function of S-layer protein of pathogenic B. sphaericus.     

Cellular fatty acid analysis as a potential tool for predicting mosquitocidal activity of Bacillus sphaericus strains.

Gas-liquid chromatography of fatty acid methyl esters and numerical analysis were carried out with 114 Bacillus sphaericus strains. Since only two clusters harbored mosquitocidal strains, this technique could be developed in screening programs to limit bioassays on mosquito larvae. It also allows differentiation of highly homologous strains.     

Cellular fatty acid analysis as a potential tool for predicting mosquitocidal activity of Bacillus sphaericus strains.

Gas-liquid chromatography of fatty acid methyl esters and numerical analysis were carried out with 114 Bacillus sphaericus strains. Since only two clusters harbored mosquitocidal strains, this technique could be developed in screening programs to limit bioassays on mosquito larvae. It also allows differentiation of highly homologous strains.     

Interpreting the SDS-PAGE protein patterns with self-organizing maps: application for the characterization of mosquito-pathogenic Bacillus strains

Aims: To present the pairwise comparison of potential mosquito‐pathogenic Bacillus strains based on their SDS‐PAGE protein patterns and to evaluate their characteristic toxicity patterns. Methods and Results: In this work, 20 Bacillus strains were subjected to qualitative toxicity tests against Aedes aegypti and Culex quinquefasciatus larvae. The selected strains were then characterized by SDS‐PAGE protein profiles. The highly heterogeneous multiple protein components of protein patterns were analysed using self‐organizing map (SOM), a ‘visualization and clustering’ tool. Members of mosquitocidal Bacillus species were classified in four distinct clusters, and then toxicity patterns were examined. Cluster (1, 1) comprised of three highly toxic strains of Bacillus sphaericus: SPH88, 1593 and KSD‐4; cluster (1, 2) consisted of two B. sphaericus strains: SSII‐1 and Bsp‐R that showed weak larvicidal activity; cluster (2, 1) constituted two B. sphaericus strains: WHO2297 and ISPC‐5 that possessed moderate toxicity; and cluster (2, 2) contained four B. thuringiensis ssp. israelensis strains: ONR‐60A, HD500, IPS70 and IPS82 belonging to serotype H14 but exhibited moderate to high mosquito larvicidal toxicity. Conclusions: SOM served as a colour‐coded alternate for easy visualization of similarities or dissimilarities between the strains even at the infra subspecies level. Furthermore, characteristic toxicity patterns of Bacillus strains of different clusters were determined. Significance and Impact of the Study: Analysis of electrophoretic protein patterns using SOM provides a better insight into the inter‐relationships of bacterial strains through similarity‐based clustering and pairwise comparison of two strains.     

Numerical classification and identification of Bacillus sphaericus including some strains pathogenic for mosquito larvae

Ninety-one strains of Bacillus sphaericus, including representatives of all the established DNA homology groups, related round-spored and oval-spored species, and six strains pathogenic for mosquito larvae, were examined for 155 characters. Numerical analyses (Jaccard coefficient/average linkage clustering) based on the 88 variable features revealed 14 clusters at the 79% similarity level that contained more than one strain and 17 single member clusters. All insect pathogenic strains were recovered in a single cluster and the classification was in accord with an established classification based on DNA sequence homology. Two frequency matrices for probabilistic identification were constructed and tested. A comprehensive matrix comprising 14 mesophilic, round-spored taxa and 27 tests gave good results for identification of hypothetical median organisms, cluster overlap and identifications of representative strains (based on data generated in the classification study). Reference strains for the 14 taxa and eight additional insect pathogenic strains were examined for the 27 tests and were correctly identified with high scores using this matrix. A second matrix comprising seven taxa and 13 tests also performed well in the theoretical evaluation and correctly identified the reference strains and insect pathogenic strains.     

Polycrystalline bismuth oxide films for development of amperometric biosensor for phenolic compounds

An attractive biocomposite based on polycrystalline bismuth oxide (BiO_x) film and polyphenol oxidase (PPO) was proposed for the construction of a mediator-free amperometric biosensor for phenolic compounds in environmental water samples. The phenolic biosensor could be easily achieved by casting the biocomposite on the surface of glassy carbon electrode (GCE) via the cross-linking step by glutaraldehyde. The laboratory-prepared bismuth oxide semiconductor was polymorphism. Its hydrophilicity provided a favorable microenvironment for retaining the biological activity of the immobilized protein. The parameters of the fabrication process and the various experimental variables for the enzyme electrode were optimized. The proposed PPO/BiO_x biosensor provided a linear response to catechol over a concentration range of 4 × 10⁻⁹ M to 1.5 × 10⁻⁵ M with a dramatically developed sensitivity of 11.3 A M⁻¹ cm⁻² and a detection limit of 1 × 10⁻⁹ M based on S/N = 3. In addition, the PPO/BiO_x biocomposite was characterized by scanning electron microscope (SEM), Fourier transform infrared spectra (FTIR) and rotating disk electrode voltammetry.     

Screening of Brazilian Bacillus sphaericus strains for high toxicity against Culex quinquefasciatus and Aedes aegypti

Abstract:  In this work, 246 Bacillus sphaericus strains were evaluated against Aedes aegypti and Culex quinquefasciatus larvae to select the most effective ones to be used as the basis of a national product. All strains were isolated from different regions of Brazil and they are stored in a Bacillus spp. collection at Embrapa Genetic Resources and Biotechnology. The selected strains were characterized by biochemical and molecular methods. Based on selective bioassays, 87 strains were identified as toxic to one or both target species. All of these strains contain genes that encode the 42, 51 kDa proteins that constitute the binary toxin and the 100 kDa Mtx1 toxin. All toxic strains presented a very high LC₅₀ against A. aegypti , so, a product based on any of these B. sphaericus strains would not be recommended for use in programmes to control A. aegypti . S201 had highest activity against C. quinquefasciatus , presenting the lowest LC₅₀ and LC₉₀ in bioassays.