Genome analysis of novel Apilactobacillus sp. isolate from butterfly (Pieris canidia) gut reveals occurrence of unique glucanogenic traits and probiotic potential.

This study was conducted with a perception that fructose-rich niches may inhabit novel species of lactic acid bacteria that are gaining importance as probiotics and for the production of exopolysaccharides that have applications in food and pharmaceuticals. Recently, some Lactobacillus species have been reclassified as fructophilic lactic acid bacteria due to their preference for fructose over glucose as a carbon source. These bacteria are likely to be found in fructose rich niches such as flower nectar and insects that feed on it. We explored the butterfly gut and acquired a new isolate, designated as F1, of fructophilic lactic acid bacteria, which produces a glucan-type exopolysaccharide. Whole genome sequencing and in silico analysis revealed that F1 has significantly lower average nucleotide identity and DNA-DNA hybridization values as compared to its closest Apilactobacillus neighbors in phylogenetic analysis. Therefore, we declare the isolate F1 as a novel Apilactobacillus species with the proposed name of Apilactobacillus iqraium F1. Genome mining further revealed that F1 harbors genes for exopolysaccharide synthesis and health-promoting attributes. To this end, F1 is the only Apilactobacillus species harboring three diverse α-glucan-synthesis genes that cluster with different types of dextransucrases in the dendrogram. Moreover, many nutritional marker genes, as well as genes for epithelial cell adhesion and antimicrobial synthesis, were also detected suggesting the probiotic attributes of F1. Overall analysis suggests A. iqraium sp. F1 be a potential candidate for various health beneficial and pharmaceutical applications.

Production of a high molecular weight levan by Bacillus paralicheniformis, an industrially and agriculturally important isolate from the buffalo grass rhizosphere.

A new exopolysaccharide (EPS) producing Gram-positive bacterium was isolated from the rhizosphere of Bouteloua dactyloides (buffalo grass) and its EPS product was structurally characterized. The isolate, designated as LB1-1A, was identified as Bacillus paralicheniformis based on 16S rRNA gene sequence and phylogenetic tree analysis. The EPS produced by LB1-1A was identified as a levan, having ß(2 → 6) linked backbone with ß(2 → 1) linkages at the branch points (4.66%). The isolate LB1-1A yielded large amount (~ 42 g/l) of levan having high weight average molecular weight (Mw) of 5.517 × 107 Da. The relatively low degree of branching and high molecular weight of this levan makes B. paralicheniformis LB1-1A a promising candidate for industrial applications.

Whole Genome Sequence Analysis of a Novel Apilactobacillus Species from Giant Honeybee (Apis dorsata) Gut Reveals Occurrence of Genetic Elements Coding Prebiotic and Probiotic Traits.

Apilactobacillus spp. are classified as obligate fructophilic lactic acid bacteria (FLAB) that inhabit fructose-rich niches such as honeybee gut. Lactic acid bacteria are an important component of the gut microbiome and play a crucial role in maintaining gut health. In this study, a new FLAB strain HBW1, capable of producing glucan-type exopolysaccharide, was isolated from giant honeybee (Apis dorsata) gut and subjected to whole genome sequencing (WHS) to determine its health-beneficial traits. The genome size of the isolate was 1.49 Mb with a GC content of 37.2%. For species level identity, 16S rDNA sequence similarity, genome to genome distance calculator (dDDH), and average nucleotide identity (ANI) values were calculated. Phylogenetic analysis showed that the isolate HBW1 belongs to the Apilactobacillus genus. The dDDH and ANI values in comparison with closely clustered Apilactobacillus kunkeei species were 52% and 93.10%, respectively. Based on these values, we concluded that HBW1 is a novel species of Apilactobacillus, and we propose the name Apilactobacillus waqarii HBW1 for it. Further, WHS data mining of HBW1 revealed that it harbors two glucosyltransferase genes for prebiotic glucan-type exopolysaccharide synthesis. Moreover, chaperon (clp) and methionine sulfoxide reductase (msrA, msrB, and msrC) genes as well as nutritional marker genes for folic acid (folD) and riboflavin biosynthesis (rib operon), important for conferring probiotic properties, were also detected. Occurrence of these genetic traits make HBW1 an excellent candidate for application to improve gut function.

Hidden Role of Gut Microbiome Dysbiosis in Schizophrenia: Antipsychotics or Psychobiotics as Therapeutics?

Schizophrenia is a chronic, heterogeneous neurodevelopmental disorder that has complex symptoms and uncertain etiology. Mounting evidence indicates the involvement of genetics and epigenetic disturbances, alteration in gut microbiome, immune system abnormalities, and environmental influence in the disease, but a single root cause and mechanism involved has yet to be conclusively determined. Consequently, the identification of diagnostic markers and the development of psychotic drugs for the treatment of schizophrenia faces a high failure rate. This article surveys the etiology of schizophrenia with a particular focus on gut microbiota regulation and the microbial signaling system that correlates with the brain through the vagus nerve, enteric nervous system, immune system, and production of postbiotics. Gut microbially produced molecules may lay the groundwork for further investigations into the role of gut microbiota dysbiosis and the pathophysiology of schizophrenia. Current treatment of schizophrenia is limited to psychotherapy and antipsychotic drugs that have significant side effects. Therefore, alternative therapeutic options merit exploration. The use of psychobiotics alone or in combination with antipsychotics may promote the development of novel therapeutic strategies. In view of the individual gut microbiome structure and personalized response to antipsychotic drugs, a tailored and targeted manipulation of gut microbial diversity naturally by novel prebiotics (non-digestible fiber) may be a successful alternative therapeutic for the treatment of schizophrenia patients.

Production of a broad spectrum streptothricin like antibiotic from halotolerant Streptomyces fimbriatus isolate G1 associated with marine sediments.

Streptomyces have been reported as a remarkable source for bioactive secondary metabolites with complex structural and functional diversity. In this study, 35 isolates of genus Streptomyces were purified from rhizospheric and marine soils collected from previously unexplored habitats and screened for antimicrobial activities. One of these isolates, G1, when tested in vitro, was found highly active against wide range of microbes including Gram-positive, Gram-negative bacteria, and different fungal pathogens. It was identified as mesophilic, alkaliphilic, and moderately halotolerant as it showed optimum growth at temperature 30 °C, pH 8.0 in casein-starch-peptone-yeast extract-malt extract medium supplemented with 5% NaCl. Sequence analysis of the 16S rRNA gene indicated 100% identity of this isolate to Streptomyces fimbriatus. Moreover, maximum antimicrobial activity was achieved in starch nitrate medium supplemented with 1% glycerol as carbon and 0.03% soy meal as nitrogen source. The antimicrobial compounds produced by this isolate were extracted in methanol. Bioassay-guided fractionation through thin layer chromatography of methanolic extract resulted in the separation of a most active fraction with an Rf value of 0.46. This active fraction was characterized by FTIR and LCMS analysis and found similar to streptothricin D like antibiotic with m/z 758.42.

Crystal structure of an inulosucrase from Halalkalicoccus jeotgali B3T, a halophilic archaeal strain.

Several archaea harbor genes that code for fructosyltransferase (FTF) enzymes. These enzymes have not been characterized yet at structure-function level, but are of extreme interest in view of their potential role in the synthesis of novel compounds for food, nutrition, and pharmaceutical applications. In this study, 3D structure of an inulin-type fructan producing enzyme, inulosucrase (InuHj), from the archaeon Halalkalicoccus jeotgali was resolved in its apo form and with bound substrate (sucrose) molecule and first transglycosylation product (1-kestose). This is the first crystal structure of an FTF from halophilic archaea. Its overall five-bladed ß-propeller fold is conserved with previously reported FTFs, but also shows some unique features. The InuHj structure is closer to those of Gram-negative bacteria, with exceptions such as residue E266, which is conserved in FTFs of Gram-positive bacteria and has possible role in fructan polymer synthesis in these bacteria as compared to fructooligosaccharide (FOS) production by FTFs of Gram-negative bacteria. Highly negative electrostatic surface potential of InuHj, due to a large amount of acidic residues, likely contributes to its halophilicity. The complex of InuHj with 1-kestose indicates that the residues D287 in the 4B-4C loop, Y330 in 4D-5A, and D361 in the unique α2 helix may interact with longer FOSs and facilitate the binding of longer FOS chains during synthesis. The outcome of this work will provide targets for future structure-function studies of FTF enzymes, particularly those from archaea.

Growth Inhibition of Common Enteric Pathogens in the Intestine of Broilers by Microbially Produced Dextran and Levan Exopolysaccharides.

Antibiotics are generally applied for treatment or as subtherapeutic agents to overcome diseases caused by pathogenic bacteria including Escherichia coli, Salmonella and Enterococcus species in poultry. However, due to their possible adverse effects on animal health and to maintain food safety, probiotics, prebiotics, and synbiotics have been proposed as alternatives to antibiotic growth promoters (AGPs) in poultry production. In this study, the effects of prebiotics on the augmentation of broiler's indigenous gut microbiology were studied. Day old 180 broilers chicks were divided into four treatment groups: G, L, C1, and C2. The groups G and L were fed with basal diet containing 3% dextran and 3% levan, respectively. Control groups were fed with basal diets without antibiotic (C1) and with antibiotics (C2). The experimental groups showed decreased mortality as compared to control groups. After 35 days, the chickens were euthanized and intestinal fluid was analyzed for enteric pathogens on chromogenic agar plates and by 16S rRNA gene sequencing. Inhibition of the growth of E. coli and Enterococcus was observed in groups G and L, respectively, whereas Salmonella was only present in group C1. Also, high populations of lactic acid bacteria were detected in the intestine of prebiotic fed birds as compared to controls. These results depict that dextran and levan have the potential to replace the use of antibiotics in poultry feed for inhibiting the growth of common enteric pathogens. To the best of our knowledge, this is the first study where effects of dextran and levan on intestinal microbiota of broilers have been reported.

Raman spectroscopy for the analysis of different exo-polysaccharides produced by bacteria.

In this study, Raman spectroscopy is employed for the characterization and comparison of two different classes of exo-polysaccharides including glucans and fructans which are produced by different bacteria. For this purpose, nine samples are used including five samples of glucans and four of fructans. Raman spectral results of all these polysaccharides show clear differences among various glucans as well as fructans showing the potential of this technique to identify the differences within the same class of the compounds. Moreover, these two classes are also compared on the basis of their Raman spectral data and can be differentiated on the basis of their unique Raman features. Multivariate data analysis techniques, Principle Component Analysis (PCA) is found very helpful for the comparison of the Raman spectral data of these classes of the carbohydrates.

Phylogenetic characterization and novelty of organic sulphur metabolizing genes of Rhodococcus spp. (Eu-32).

Rhodococcus spp. (Eu-32) has the unique ability to metabolize organic sulphur containing compounds like dibenzothiophene through an extended sulphur specific pathway (Akhtar et al., in FEMS Microbiol Lett 301:95-102, 2009). Efforts were made to isolate and characterize the presumed desulphurizing genes (dszABC) involved in the sulphur specific pathway of isolate Eu-32 by employing standard and degenerate polymerase chain reaction primers. The partial dszA gene sequence of isolate Eu-32 showed 92% sequence identity with a putative FMNH-2 dependent monooxygenase of Rhodococcus erythropolis PR4. The dszC gene sequence showed 99% homology with the dibenzothiophene monooxygenase desulphurizing enzyme of another Rhodococcus species. The dszB gene was not unambiguously identified. A phylogenetic analysis by maximum likelihood method of the 16S rRNA gene and deduced DszA and C amino acid sequences suggest that horizontal gene transfer events might have taken place during the evolution of desulphurizing genes of Rhodococcus spp. (Eu-32).

The role of conserved inulosucrase residues in the reaction and product specificity of Lactobacillus reuteri inulosucrase.

The probiotic bacterium Lactobacillus reuteri 121 produces two fructosyltransferase enzymes, a levansucrase and an inulosucrase. Although these two fructosyltransferase enzymes share high sequence similarity, they differ significantly in the type and size distribution of fructooligosaccharide products synthesized from sucrose, and in their activity levels. In order to examine the contribution of specific amino acids to such differences, 15 single and four multiple inulosucrase mutants were designed that affected residues that are conserved in inulosucrase enzymes, but not in levansucrase enzymes. The effects of the mutations were interpreted using the 3D structures of Bacillus subtilis levansucrase (SacB) and Lactobacillus johnsonii inulosucrase (InuJ). The wild-type inulosucrase synthesizes mostly fructooligosaccharides up to a degree of polymerization of 15 and relatively low amounts of inulin polymer. In contrast, wild-type levansucrase produces mainly levan polymer and fructooligosaccharides with a degree of polymerization < 5. Although most of the inulosucrase mutants in this study behaved similarly to the wild-type enzyme, the mutation G416E, at the rim of the active site pocket in loop 415-423, increased the hydrolytic activity twofold, without significantly changing the transglycosylation activity. The septuple mutant GM4 (T413K, K415R, G416E, A425P, S442N, W486L, P516L), which included two residues from the above-mentioned loop 415-423, synthesized 1-kestose only, but at low efficiency. Mutation A538S, located behind the general acid/base, increased the enzyme activity two to threefold. Mutation N543S, located adjacent to the +1/+2 sub-site residue R544, resulted in synthesis of not such a wide variety of fructooligosaccharides than the wild-type enzyme. The present study demonstrates that the product specificity of inulosucrase is easily altered by protein engineering, obtaining inulosucrase variants with higher transglycosylation specificity, higher catalytic rates and different fructooligosaccharide size distributions, without changing the ß(2-1) linkage type in the product.

Crystal structure of inulosucrase from Lactobacillus: insights into the substrate specificity and product specificity of GH68 fructansucrases.

Fructansucrases (FSs) catalyze a transfructosylation reaction with sucrose as substrate to produce fructo-oligosaccharides and fructan polymers that contain either ß-2,1 glycosidic linkages (inulin) or ß-2,6 linkages (levan). Levan-synthesizing FSs (levansucrases) have been most extensively investigated, while detailed information on inulosucrases is limited. Importantly, the molecular basis of the different product specificities of levansucrases and inulosucrases is poorly understood. We have elucidated the three-dimensional structure of a truncated active bacterial GH68 inulosucrase, InuJ of Lactobacillus johnsonii NCC533 (residues 145-708), in its apo form, with a bound substrate (sucrose), and with a transfructosylation product. The sucrose binding pocket and the sucrose binding mode are virtually identical with those of GH68 levansucrases, confirming that both enzyme types use the same fully conserved structural framework for the binding and cleavage of the donor substrate sucrose in the active site. The binding mode of the first transfructosylation product 1-kestose (Fru-ß(2-1)-Fru-α(2-1)-Glc, where Fru=fructose and Glc=glucose) in subsites -1 to +2 shows for the first time how inulin-type fructo-oligosaccharide bind in GH68 FS and how an inulin-type linkage can be formed. Surprisingly, observed interactions with the sugar in subsites +1 and +2 are provided by residues that are also present in levansucrases. The binding mode of 1-kestose and the presence of a more distant sucrose binding site suggest that residues beyond the +2 subsite, in particular residues from the nonconserved 1B-1C loop, determine product linkage type specificity in GH68 FSs.

Inulin and levan synthesis by probiotic Lactobacillus gasseri strains: characterization of three novel fructansucrase enzymes and their fructan products.

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. Here, we report an evaluation of fructan synthesis in three Lactobacillus gasseri strains, identification of the fructansucrase-encoding genes and characterization of the recombinant proteins and fructan (oligosaccharide) products. High-performance anion-exchange chromatography and nuclear magnetic resonance analysis of the fructo-oligosaccharides (FOS) and polymers produced by the L. gasseri strains and the recombinant enzymes revealed that, in situ, L. gasseri strains DSM 20604 and 20077 synthesize inulin (and oligosaccharides) and levan products, respectively. L. gasseri DSM 20604 is only the second Lactobacillus strain shown to produce inulin polymer and FOS in situ, and is unique in its distribution of FOS synthesized, ranging from DP2 to DP13. The probiotic bacterium L. gasseri DSM 20243 did not produce any fructan, although we identified a fructansucrase-encoding gene in its genome sequence. Further studies showed that this L. gasseri DSM 20243 gene was prematurely terminated by a stop codon. Exchanging the stop codon for a glutamine codon resulted in a recombinant enzyme producing inulin and FOS. The three recombinant fructansucrase enzymes characterized from three different L. gasseri strains have very similar primary protein structures, yet synthesize different fructan products. An interesting feature of the L. gasseri strains is that they were unable to ferment raffinose, whereas their respective recombinant enzymes converted raffinose into fructan and FOS.

Analysis of the dibenzothiophene metabolic pathway in a newly isolated Rhodococcus spp.

Out of 17 samples collected from diverse environments, 110 bacterial isolates of varied characteristics were screened for their dibenzothiophene-desulphurizing activity. A single isolate, Eu-32, originating from a soil sample taken from the roots of a eucalyptus tree, displayed dibenzothiophene-desulphurizing activity. This isolate metabolized dibenzothiophene to 2-hydroxybiphenyl (2-HBP), as detected by HPLC, and was also able to use other organic sulphur compounds as a sole sulphur source. Based on morphological, biochemical and molecular studies, it was found that the organism belongs to the genus Rhodococcus, with a maximum of 95% identity to species in this genus for the partial sequence of the 16S rRNA gene. Isolate Eu-32 could desulphurize 0.2 mM dibenzothiophene to 2-HBP in 72 h at a temperature of 30 degrees C and pH 7.0. The structure and molecular mass of metabolites produced from dibenzothiophene desulphurization were identified by GC-MS, and two sulphur-free products, 2-HBP and biphenyl, were detected in ethyl acetate extract. It was concluded that isolate Eu-32 is a unique desulphurizing biocatalyst that desulphurizes dibenzothiophene through an extended, sulphur-specific degradation pathway with the selective cleavage of C-S bonds.

The probiotic Lactobacillus johnsonii NCC 533 produces high-molecular-mass inulin from sucrose by using an inulosucrase enzyme.

Fructansucrase enzymes polymerize the fructose moiety of sucrose into levan or inulin fructans, with beta(2-6) and beta(2-1) linkages, respectively. The probiotic bacterium Lactobacillus johnsonii strain NCC 533 possesses a single fructansucrase gene (open reading frame AAS08734) annotated as a putative levansucrase precursor. However, (13)C nuclear magnetic resonance (NMR) analysis of the fructan product synthesized in situ revealed that this is of the inulin type. The ftf gene of L. johnsonii was cloned and expressed to elucidate its exact identity. The purified L. johnsonii protein was characterized as an inulosucrase enzyme, producing inulin from sucrose, as identified by (13)C NMR analysis. Thin-layer chromatographic analysis of the reaction products showed that InuJ synthesized, besides the inulin polymer, a broad range of fructose oligosaccharides. Maximum InuJ enzyme activity was observed in a pH range of 4.5 to 7.0, decreasing sharply at pH 7.5. InuJ exhibited the highest enzyme activity at 55 degrees C, with a drastic decrease at 60 degrees C. Calcium ions were found to have an important effect on enzyme activity and stability. Kinetic analysis showed that the transfructosylation reaction of the InuJ enzyme does not obey Michaelis-Menten kinetics. The non-Michaelian behavior of InuJ may be attributed to the oligosaccharides that were initially formed in the reaction and which may act as better acceptors than the growing polymer chain. This is only the second example of the isolation and characterization of an inulosucrase enzyme and its inulin (oligosaccharide) product from a Lactobacillus strain. Furthermore, this is the first Lactobacillus strain shown to produce inulin polymer in situ.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of Pakistan and their industrial importance

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99 percent 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97 percent, 98 percent and 99 percent 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.

Avaliou-se a biodiversidade e a importância industrial de bactérias indígenas da mina de sal Khewra, Paquistão. Efetuou-se a amplificação do 16S rDNA dos isolados por PCR empregando-se os iniciadores universais FD1 e rP1, e os produtos foram seqüenciados comercialmente. Essas seqüências de genes foram comparadas com outras seqüências disponíveis no GenBank a fim de encontrar seqüências relacionadas, construindo-se uma árvore filogenética para essas bactérias. Os genes foram depositados no GenBank obtendo-se os números de acesso. A maioria dos isolados pertenceu a diferentes espécies do gênero Bacillus, apresentando 92-99 por cento de identidade de 16S rDNA com a respectiva cepa de referencia. Outros isolados apresentaram alta similaridade com Escherichia coli, Staphylococcus arlettae e Staphylococcus gallinarum, com 97 por cento, 98 por cento e 99 por cento de similaridade de16S rDNA, respectivamente. A capacidade dos isolados produzirem enzimas industriais (amilase, carboximetilcelulase, xilanase, celulase e protease) foi verificada. Todos os isolados foram testados em placas quanto a degradação de amido, carboximetilcelulose, xilana, celulose e caseína. Os isolados BPT-5, 11, 18, 19 e 25 produziram grandes quantidades de enzimas degradadoras de carboidratos e proteínas. Conclui-se que a mina de Sal Khewra apresenta diferentes grupos de bactérias, que são fontes potenciais de enzimas industriais de aplicação comercial.

Biodiversity and phylogenetic analysis of culturable bacteria indigenous to Khewra salt mine of pakistan and their industrial importance.

Culturable bacterial biodiversity and industrial importance of the isolates indigenous to Khewra salt mine, Pakistan was assessed. PCR Amplification of 16S rDNA of isolates was carried out by using universal primers FD1 and rP1and products were sequenced commercially. These gene sequences were compared with other gene sequences in the GenBank databases to find the closely related sequences. The alignment of these sequences with sequences available from GenBank database was carried out to construct a phylogenetic tree for these bacteria. These genes were deposited to GenBank and accession numbers were obtained. Most of the isolates belonged to different species of genus Bacillus, sharing 92-99% 16S rDNA identity with the respective type strain. Other isolates had close similarities with Escherichia coli, Staphylococcus arlettae and Staphylococcus gallinarum with 97%, 98% and 99% 16S rDNA similarity respectively. The abilities of isolates to produce industrial enzymes (amylase, carboxymethylcellulase, xylanase, cellulase and protease) were checked. All isolates were tested against starch, carboxymethylcellulose (CMC), xylane, cellulose, and casein degradation in plate assays. BPT-5, 11,18,19 and 25 indicated the production of copious amounts of carbohydrates and protein degrading enzymes. Based on this study it can be concluded that Khewra salt mine is populated with diverse bacterial groups, which are potential source of industrial enzymes for commercial applications.