Preparation of hyaluronan oligosaccharides by a prokaryotic beta-glucuronidase: Characterization of free and immobilized forms of the enzyme.

Popularity of hyaluronan (HA) in the cosmetics and pharmaceutical industries, led to the investigation and development of new HA-based materials, with enzymes playing a key role. Beta-D-glucuronidases catalyze the hydrolysis of a beta-D-glucuronic acid residue from the non-reducing end of various substrates. However, lack of specificity towards HA for most beta-D-glucuronidases, in addition to the high cost and low purity of those active on HA, have prevented their widespread application. In this study, we investigated a recombinant beta-glucuronidase from Bacteroides fragilis (rBfGUS). We demonstrated the rBfGUS's activity on native, modified, and derivatized HA oligosaccharides (oHAs). Using chromogenic beta-glucuronidase substrate and oHAs, we characterized the enzyme's optimal conditions and kinetic parameters. Additionally, we evaluated rBfGUS's activity towards oHAs of various sizes and types. To increase reusability and ensure the preparation of enzyme-free oHA products, rBfGUS was immobilized on two types of magnetic macroporous bead cellulose particles. Both immobilized forms of rBfGUS demonstrated suitable operational and storage stabilities, and their activity parameters were comparable to the free form. Our findings suggest that native and derivatized oHAs can be prepared using this bacterial beta-glucuronidase, and a novel biocatalyst with enhanced operational parameters has been developed with a potential for industrial use.

Polyenic Antibiotics and Other Antifungal Compounds Produced by Hemolytic Streptomyces Species.

Streptomyces are of great interest in the pharmaceutical industry as they produce a plethora of secondary metabolites that act as antibacterial and antifungal agents. They may thrive on their own in the soil, or associate with other organisms, such as plants or invertebrates. Some soil-derived strains exhibit hemolytic properties when cultivated on blood agar, raising the question of whether hemolysis could be a virulence factor of the bacteria. In this work we examined hemolytic compound production in 23 ß-hemolytic Streptomyces isolates; of these 12 were soil-derived, 10 were arthropod-associated, and 1 was plant-associated. An additional human-associated S. sp. TR1341 served as a control. Mass spectrometry analysis suggested synthesis of polyene molecules responsible for the hemolysis: candicidins, filipins, strevertene A, tetrafungin, and tetrin A, as well as four novel polyene compounds (denoted here as polyene A, B, C, and D) in individual liquid cultures or paired co-cultures. The non-polyene antifungal compounds actiphenol and surugamide A were also identified. The findings indicate that the ability of Streptomyces to produce cytolytic compounds (here manifested by hemolysis on blood agar) is an intrinsic feature of the bacteria in the soil environment and could even serve as a virulence factor when colonizing available host organisms. Additionally, a literature review of polyenes and non-polyene hemolytic metabolites produced by Streptomyces is presented.

6S-Like scr3559 RNA Affects Development and Antibiotic Production in Streptomyces coelicolor.

Regulatory RNAs control a number of physiological processes in bacterial cells. Here we report on a 6S-like RNA transcript (scr3559) that affects both development and antibiotic production in Streptomyces coelicolor. Its expression is enhanced during the transition to stationary phase. Strains that over-expressed the scr3559 gene region exhibited a shortened exponential growth phase in comparison with a control strain; accelerated aerial mycelium formation and spore maturation; alongside an elevated production of actinorhodin and undecylprodigiosin. These observations were supported by LC-MS analyses of other produced metabolites, including: germicidins, desferrioxamines, and coelimycin. A subsequent microarray differential analysis revealed increased expression of genes associated with the described morphological and physiological changes. Structural and functional similarities between the scr3559 transcript and 6S RNA, and its possible employment in regulating secondary metabolite production are discussed.

Educational Program Improved Senior Preparedness to Call 911 as a Response to Stroke.

OBJECTIVES: Stroke predominantly affects the elderly. Universities of the Third Age (U3A) are presented with an opportunity to target them. The goal of our study was to improve older adults' preparedness to call 911 as a response to symptoms of stroke. MATERIALS AND METHODS: Participants were recruited from U3A in Brno, Czech Republic in year 2018. The program included an educational movie about stroke and testing with pretest posttest design. Stroke awareness was measured by Stroke Action Test and video-clips portraying stroke and stroke mimicking symptoms. Respondents had to answer close-ended questions. Composite scores were compared using paired t-test. RESULTS: Data were obtained from 206 attendees of the program, that is 2% of all students, from 4 of 5 U3A in Brno. The mean test score improved from 80% to 87% (paired p < 0.001). Participants with a lower baseline knowledge improved by 12% (95% CI 9% to 16%) and with a higher baseline knowledge by 0% (95% CI 3% to 4%). The score for calling 911 for stroke mimicking symptoms improved from 29% to 20% (paired p < 0.001). CONCLUSIONS: Video-based educational program improved senior preparedness to call 911 as a response to stroke. The improvement was mild, which is at least partly due to a high baseline level of preparedness of seniors active in U3A. A lower baseline knowledge was however associated with a bigger improvement, which might be important for use in a less active/educated population. Educational intervention also decreased intention to call 911 for stroke mimicking symptoms, which could have important implications for decreasing unnecessary activation of pre-hospital services.

Onset of calciotropic receptors during the initiation of mandibular/alveolar bone formation.

Mandibular/alveolar (m/a) bone, as a component of the periodontal apparatus, allows for the proper tooth anchorage and function of dentition. Bone formation around the tooth germs starts prenatally and, in the mouse model, the mesenchymal condensation turns into a complex vascularized bone (containing osteo-blasts, -cytes, -clasts) within only two days. This very short but critical period is characterized by synchronized cellular and molecular events. The m/a bone, as others, is subjected to endocrine regulations. This not only requires vasculature to allow the circulation of active molecules (ligands), but also the expression of corresponding cell receptors to define target tissues. This contribution aimed at following the dynamics of calciotropic receptors´ expression during morphological transformation of a mesenchymal condensation into the initial m/a bone structure. Receptors for all three calciotropic systemic regulators: parathormone, calcitonin and activated vitamin D (calcitriol), were localized on serial histological sections using immunochemistry and their relative expression was quantified by q-PCR. The onset of calciotropic receptors was followed along with bone cell differentiation (as checked using osteocalcin, sclerostin, RANK and TRAP) and vascularization (CD31) during mouse prenatal/embryonic (E) days 13-15 and 18. Additionally, the timing of calciotropic receptor appearance was compared with that of estrogen receptors (ESR1, ESR2). PTH receptor (PTH1r) appeared in the bone already at E13, when the first osteocalcin-positive cells were detected within the mesenchymal condensation forming the bone anlage. At this stage, blood vessels were only lining the condensation. At E14, the osteoblasts started to express the receptor for activated vitamin D (VDR). At this stage, the vasculature just penetrated the forming bone. On the same day, the first TRAP-positive (but not yet multinucleated) osteoclastic cells were identified. However, calcitonin receptor was detected only one day later. The first Sost-positive osteocytes, present at E15, were PTH1r and VDR positive. ESR1 almost copied the expression pattern of PTH1r, and ESR2 appearance was similar with VDR with a significant increase between E15 and E18. This report focuses on the in vivo situation and links morphological transformation of the mesenchymal cell condensation into a bone structure with dynamics of cell differentiation/maturation, vascularization and onset of receptors for calciotropic endocrine signalling in developing m/a bone.

Tuftelin and HIFs expression in osteogenesis.

Tuftelin was originally discovered and mostly studied in the tooth, but later found also in other organs. Despite its wide distribution among tissues, tuftelin's function has so far been specified only in the formation of enamel crystals. Nevertheless, in many cases, tuftelin was suggested to be associated with cellular adaptation to hypoxia and recently even with cell differentiation. Therefore, we aimed to investigate tuftelin expression along with hypoxia-inducible factors (HIFs) during the early development of the mandibular/alveolar (m/a) bone, when osteoblasts started to differentiate in vivo and to compare their expression levels in undifferentiated versus differentiated osteoblastic cells in vitro. Immunohistochemistry demonstrated the presence of tuftelin already in osteoblastic precursors which were also HIF1-positive, but HIF2-negative. Nevertheless, HIF2 protein appeared when osteoblasts differentiated, one day later. This is in agreement with observations made with MC3T3-E1 cells, where there was no significant difference in tuftelin and Hif1 expression in undifferentiated vs. differentiated cells, although Hif2 increased upon differentiation induction. In differentiated osteoblasts of the m/a bone, all three proteins accumulated, first, prenatally, in the cytoplasm and later, particularly at postnatal stages, they displayed also peri/nuclear localization. Such a dynamic time-space pattern of tuftelin expression has recently been reported in neurons, which, as the m/a bone, differentiate under less hypoxic conditions as indicated also by a prevalent cytoplasmic expression of HIF1 in osteoblasts. However, unlike what was shown in cultured neurons, tuftelin does not seem to participate in final osteoblastic differentiation and its functions, thus, appears to be tissue specific.

Osteogenic and Angiogenic Profiles of Mandibular Bone-Forming Cells.

The mandible is a tooth-bearing structure involving one of the most prominent bones of the facial region. Mesenchymal cell condensation is the first morphological sign of osteogenesis, and several studies have focused on this stage also in the mandible. Little information is available about the early post-condensation period, during which avascular soft condensation turns into vascularized bone, and all three major bone cell types, osteoblasts, osteocytes, and osteoclasts, differentiate. In the mouse first lower molar region, the post-condensation period corresponds to the prenatal days 13-15. If during this critical period, when osteogenesis reaches the point of major bone cell differentiation, vascularization already has to play a critical role, one should be able to show molecular changes which support both types of cellular events. The aim of the present report was to follow in organ context the expression of major osteogenic and angiogenic markers and identify those that are up- or downregulated during this period. To this end, PCR Array was applied covering molecules involved in osteoblastic cell proliferation, commitment or differentiation, extracellular matrix (ECM) deposition, mineralisation, osteocyte maturation, angiogenesis, osteoclastic differentiation, and initial bone remodeling. From 161 analyzed osteogenic and angiogenic factors, the expression of 37 was altered when comparing the condensation stage with the bone stage. The results presented here provide a molecular survey of the early post-condensation stage of mandibular/alveolar bone development which has not yet been investigated in vivo.

A Human Lung-Associated Streptomyces sp. TR1341 Produces Various Secondary Metabolites Responsible for Virulence, Cytotoxicity and Modulation of Immune Response.

Streptomycetes, typical soil dwellers, can be detected as common colonizers of human bodies, especially the skin, the respiratory tract, the guts and the genital tract using molecular techniques. However, their clinical manifestations and isolations are rare. Recently they were discussed as possible "coaches" of the human immune system in connection with certain immune disorders and cancer. This work aimed for the characterization and evaluation of genetic adaptations of a human-associated strain Streptomyces sp. TR1341. The strain was isolated from sputum of a senior male patient with a history of lung and kidney TB, recurrent respiratory infections and COPD. It manifested remarkably broad biological activities (antibacterial, antifungal, beta-hemolytic, etc.). We found that, by producing specific secondary metabolites, it is able to modulate host immune responses and the niche itself, which increase its chances for long-term survival in the human tissue. The work shows possible adaptations or predispositions of formerly soil microorganism to survive in human tissue successfully. The strain produces two structural groups of cytotoxic compounds: 28-carbon cytolytic polyenes of the filipin type and actinomycin X2. Additionally, we summarize and present data about streptomycete-related human infections known so far.

DNA mapping and kinetic modeling of the HrdB regulon in Streptomyces coelicolor.

HrdB in streptomycetes is a principal sigma factor whose deletion is lethal. This is also the reason why its regulon has not been investigated so far. To overcome experimental obstacles, for investigating the HrdB regulon, we constructed a strain whose HrdB protein was tagged by an HA epitope. ChIP-seq experiment, done in 3 repeats, identified 2137 protein-coding genes organized in 337 operons, 75 small RNAs, 62 tRNAs, 6 rRNAs and 3 miscellaneous RNAs. Subsequent kinetic modeling of regulation of protein-coding genes with HrdB alone and with a complex of HrdB and a transcriptional cofactor RbpA, using gene expression time series, identified 1694 genes that were under their direct control. When using the HrdB-RbpA complex in the model, an increase of the model fidelity was found for 322 genes. Functional analysis revealed that HrdB controls the majority of gene groups essential for the primary metabolism and the vegetative growth. Particularly, almost all ribosomal protein-coding genes were found in the HrdB regulon. Analysis of promoter binding sites revealed binding motif at the -10 region and suggested the possible role of mono- or di-nucleotides upstream of the -10 element.

A Waking Review: Old and Novel Insights into the Spore Germination in Streptomyces.

The complex development undergone by Streptomyces encompasses transitions from vegetative mycelial forms to reproductive aerial hyphae that differentiate into chains of single-celled spores. Whereas their mycelial life - connected with spore formation and antibiotic production - is deeply investigated, spore germination as the counterpoint in their life cycle has received much less attention. Still, germination represents a system of transformation from metabolic zero point to a new living lap. There are several aspects of germination that may attract our attention: (1) Dormant spores are strikingly well-prepared for the future metabolic restart; they possess stable transcriptome, hydrolytic enzymes, chaperones, and other required macromolecules stabilized in a trehalose milieu; (2) Germination itself is a specific sequence of events leading to a complete morphological remodeling that include spore swelling, cell wall reconstruction, and eventually germ tube emergences; (3) Still not fully unveiled are the strategies that enable the process, including a single cell's signal transduction and gene expression control, as well as intercellular communication and the probability of germination across the whole population. This review summarizes our current knowledge about the germination process in Streptomyces, while focusing on the aforementioned points.

RNase III-Binding-mRNAs Revealed Novel Complementary Transcripts in Streptomyces.

cis-Antisense RNAs (asRNAs) provide very simple and effective gene expression control due to the perfect complementarity between regulated and regulatory transcripts. In Streptomyces, the antibiotic-producing clade, the antisense control system is not yet understood, although it might direct the organism's complex development. Initial studies in Streptomyces have found a number of asRNAs. Apart from this, hundreds of mRNAs have been shown to bind RNase III, the double strand-specific endoribonuclease. In this study, we tested 17 mRNAs that have been previously co-precipitated with RNase III for antisense expression. Our RACE mapping showed that all of these mRNAs possess cognate asRNA. Additional tests for antisense expression uncovered as-adpA, as-rnc, as3983, as-sigB, as-sigH, and as-sigR RNAs. Northern blots detected the expression profiles of 18 novel transcripts. Noteworthy, we also found that only a minority of asRNAs respond to the absence of RNase III enzyme by increasing their cellular levels. Our findings suggest that antisense expression is widespread in Streptomyces, including genes of such important developmental regulators, as AdpA, RNase III, and sigma factors.

Secondary Metabolites Produced during the Germination of Streptomyces coelicolor.

Spore awakening is a series of actions that starts with purely physical processes and continues via the launching of gene expression and metabolic activities, eventually achieving a vegetative phase of growth. In spore-forming microorganisms, the germination process is controlled by intra- and inter-species communication. However, in the Streptomyces clade, which is capable of developing a plethora of valuable compounds, the chemical signals produced during germination have not been systematically studied before. Our previously published data revealed that several secondary metabolite biosynthetic genes are expressed during germination. Therefore, we focus here on the secondary metabolite production during this developmental stage. Using high-performance liquid chromatography-mass spectrometry, we found that the sesquiterpenoid antibiotic albaflavenone, the polyketide germicidin A, and chalcone are produced during germination of the model streptomycete, S. coelicolor. Interestingly, the last two compounds revealed an inhibitory effect on the germination process. The secondary metabolites originating from the early stage of microbial growth may coordinate the development of the producer (quorum sensing) and/or play a role in competitive microflora repression (quorum quenching) in their nature environments.

Changes in activity of metabolic and regulatory pathways during germination of S. coelicolor.

BACKGROUND: Bacterial spore germination is a developmental process during which all required metabolic pathways are restored to transfer cells from their dormant state into vegetative growth. Streptomyces are soil dwelling filamentous bacteria with complex life cycle, studied mostly for they ability to synthesize secondary metabolites including antibiotics. RESULTS: Here, we present a systematic approach that analyzes gene expression data obtained from 13 time points taken over 5.5 h of Streptomyces germination. Genes whose expression was significantly enhanced/diminished during the time-course were identified, and classified to metabolic and regulatory pathways. The classification into metabolic pathways revealed timing of the activation of specific pathways during the course of germination. The analysis also identified remarkable changes in the expression of specific sigma factors over the course of germination. Based on our knowledge of the targets of these factors, we speculate on their possible roles during germination. Among the factors whose expression was enhanced during the initial part of germination, SigE is though to manage cell wall reconstruction, SigR controls protein re-aggregation, and others (SigH, SigB, SigI, SigJ) control osmotic and oxidative stress responses. CONCLUSIONS: From the results, we conclude that most of the metabolic pathway mRNAs required for the initial phases of germination were synthesized during the sporulation process and stably conserved in the spore. After rehydration in growth medium, the stored mRNAs are being degraded and resynthesized during first hour. From the analysis of sigma factors we conclude that conditions favoring germination evoke stress-like cell responses.

6S RNA modulates growth and antibiotic production in Streptomyces coelicolor.

The aim of this study was to contribute to clarifying the role of 6S RNA in the development and control of antibiotic biosynthesis in Streptomyces coelicolor. Due to the low energetic cost of gene silencing via 6S RNA, it is an easy and rapid means of down-regulating the expression of specific genes in response to signals from changes in the environment. The expression of 6S RNA in S. coelicolor is not constitutive, and its accumulation is adapted to changes in nutritional conditions. The 6S RNA of S. coelicolor is capable of interacting with RNA polymerase ß ß' subunits and is a template for the transcription of short pRNAs. Deletion of the ssrS gene from S. coelicolor affects the growth rate and causes changes in the expression of several pathway-specific genes involved in actinorhodin biosynthesis. The complementation of the ΔssrS strain with ssrS gene restored the wild-type levels of growth and actinorhodin production. We conclude that 6S RNA contributes to the optimization of cellular adaptation and is an important factor involved in the regulation of growth and expression of key genes for the biosynthesis of actinorhodin.

Global features of gene expression on the proteome and transcriptome levels in S. coelicolor during germination.

Streptomycetes have been studied mostly as producers of secondary metabolites, while the transition from dormant spores to an exponentially growing culture has largely been ignored. Here, we focus on a comparative analysis of fluorescently and radioactively labeled proteome and microarray acquired transcriptome expressed during the germination of Streptomyces coelicolor. The time-dynamics is considered, starting from dormant spores through 5.5 hours of growth with 13 time points. Time series of the gene expressions were analyzed using correlation, principal components analysis and an analysis of coding genes utilization. Principal component analysis was used to identify principal kinetic trends in gene expression and the corresponding genes driving S. coelicolor germination. In contrast with the correlation analysis, global trends in the gene/protein expression reflected by the first principal components showed that the prominent patterns in both the protein and the mRNA domains are surprisingly well correlated. Analysis of the number of expressed genes identified functional groups activated during different time intervals of the germination.

Systems insight into the spore germination of Streptomyces coelicolor.

An example of bacterium, which undergoes a complex development, is the genus of Streptomyces whose importance lies in their wide capacity to produce secondary metabolites, including antibiotics. In this work, a proteomic approach was applied to the systems study of germination as a transition from dormancy to the metabolically active stage. The protein expression levels were examined throughout the germination time course, the kinetics of the accumulated and newly synthesized proteins were clustered, and proteins detected in each group were identified. Altogether, 104 2DE gel images at 13 time points, from dormant state until 5.5 h of growth, were analyzed. The mass spectrometry identified proteins were separated into functional groups and their potential roles during germination were further assessed. The results showed that the full competence of spores to effectively undergo active metabolism is derived from the sporulation step, which facilitates the rapid initiation of global protein expression during the first 10 min of cultivation. Within the first hour, the majority of proteins were synthesized. From this stage, the full capability of regulatory mechanisms to respond to environmental cues is presumed. The obtained results might also provide a data source for further investigations of the process of germination.

The suboptimal structures find the optimal RNAs: homology search for bacterial non-coding RNAs using suboptimal RNA structures.

Non-coding RNAs (ncRNAs) are regulatory molecules encoded in the intergenic or intragenic regions of the genome. In prokaryotes, biocomputational identification of homologs of known ncRNAs in other species often fails due to weakly evolutionarily conserved sequences, structures, synteny and genome localization, except in the case of evolutionarily closely related species. To eliminate results from weak conservation, we focused on RNA structure, which is the most conserved ncRNA property. Analysis of the structure of one of the few well-studied bacterial ncRNAs, 6S RNA, demonstrated that unlike optimal and consensus structures, suboptimal structures are capable of capturing RNA homology even in divergent bacterial species. A computational procedure for the identification of homologous ncRNAs using suboptimal structures was created. The suggested procedure was applied to strongly divergent bacterial species and was capable of identifying homologous ncRNAs.

Phosphorylation of ribosomal proteins influences subunit association and translation of poly (U) in Streptomyces coelicolor.

The occurrence of phosphorylated proteins in ribosomes of Streptomyces coelicolor was investigated. Little is known about which biological functions these posttranslational modifications might fulfil. A protein kinase associated with ribosomes phosphorylated six ribosomal proteins of the small subunit (S3, S4, S12, S13, S14 and S18) and seven ribosomal proteins of the large subunit (L2, L3, L7/L12, L16, L17, L23 and L27). The ribosomal proteins were phosphorylated mainly on the Ser/Thr residues. Phosphorylation of the ribosomal proteins influences ribosomal subunits association. Ribosomes with phosphorylated proteins were used to examine poly (U) translation activity. Phosphorylation induced about 50% decrease in polyphenylalanine synthesis. After preincubation of ribosomes with alkaline phosphatase the activity of ribosomes was greatly restored. Small differences were observed between phosphorylated and unphosphorylated ribosomes in the kinetic parameters of the binding of Phe-tRNA to the A-site of poly (U) programmed ribosomes, suggesting that the initial binding of Phe-tRNA is not significantly affected by phosphorylation. On contrary, the rate of peptidyl transferase was about two-fold lower than that in unphosphorylated ribosomes. The data presented demonstrate that phosphorylation of ribosomal proteins affects critical steps of protein synthesis.

tmRNA of Streptomyces collinus and Streptomyces griseus during the growth and in the presence of antibiotics.

Streptomycetes are soil microorganisms with the potential to produce a broad spectrum of secondary metabolities. The production of antibiotics is accompanied by a decrease in protein synthesis, which raises the question of how these bacteria survived the transition from the primary to the secondary metabolism. Translating ribosomes incapable to properly elongate or terminate polypeptide chain activate bacterial trans-translation system. Abundance and stability of the tmRNA during growth of Streptomyces collinus and Streptomyces griseus producing kirromycin and streptomycin, respectively, was analysed. The level of tmRNA is mostly proportional to the activity of the translational system. We demonstrate that the addition of sub-inhibitory concentrations of produced antibiotics to the cultures from the beginning of the exponential phase of growth leads to an increase in tmRNA levels and to an incorporation of amino acids into the tag-peptides at trans-translation of stalled ribosomes. These findings suggest that produced antibiotics induce tmRNA that facilitate reactivation of stalled complex of ribosomes and maintain viability. The effect of antibiotics that inhibit the cell-wall turnover, DNA, RNA or protein synthesis on the level of tmRNA was examined. Antibiotics interfering with ribosomal target sites are more effective at stimulation of the tmRNA level in streptomycetes examined than those affecting the synthesis of DNA, RNA or the cell wall.

Small non-coding RNAs in Streptomyces coelicolor.

In bacteria, small RNAs (sRNAs) make important regulatory contributions to an ever increasing number of cellular processes. To expand the repertoire of known sRNAs, we sought to identify novel sRNAs in the differentiating, multicellular bacterium Streptomyces coelicolor. We describe a combined bioinformatic and experimental approach that enabled the identification and characterization of nine novel sRNAs in S. coelicolor, including a cis-encoded antisense sRNA. We examined sRNA expression throughout the S. coelicolor developmental cycle, which progresses from vegetative mycelium formation, to aerial mycelium formation and finally sporulation. We further determined the effects of growth medium composition (rich versus minimal medium) on sRNA gene expression, and compared wild-type sRNA expression profiles with those of four developmental mutants. All but two of the sRNAs exhibited some degree of medium dependence, with three sRNAs being expressed exclusively during growth on one medium type. Unlike most sRNAs characterized thus far, several sRNA genes in S. coelicolor were expressed constitutively (apart from during late sporulation), suggesting a possible housekeeping role for these transcripts. Others were expressed at specific developmental stages, and their expression profiles were altered in response to developmental mutations. Expression of one sRNA in particular was dependent upon the sporulation-specific sigma factor sigma(WhiG).