Mirror-image T7 transcription of chirally inverted ribosomal and functional RNAs.

To synthesize a chirally inverted ribosome with the goal of building mirror-image biology systems requires the preparation of kilobase-long mirror-image ribosomal RNAs that make up the structural and catalytic core and about two-thirds of the molecular mass of the mirror-image ribosome. Here, we chemically synthesized a 100-kilodalton mirror-image T7 RNA polymerase, which enabled efficient and faithful transcription of the full-length mirror-image 5S, 16S, and 23S ribosomal RNAs from enzymatically assembled long mirror-image genes. We further exploited the versatile mirror-image T7 transcription system for practical applications such as biostable mirror-image riboswitch sensor, long-term storage of unprotected kilobase-long l-RNA in water, and l-ribozyme-catalyzed l-RNA polymerization to serve as a model system for basic RNA research.

Changes induced by chronic exposure to high arsenic concentrations in the intestine and its microenvironment.

The perturbation of intestinal microbes may serve as a mechanism by which arsenic exposure causes or exacerbates diseases in humans. However, the changes in the intestinal microbiome and metabolome induced by long-term exposure to high concentrations of arsenic have not been extensively studied. In this study, C57BL/6 mice were exposed to sodium arsenite (As) (50 ppm) for 6 months. Our results show that long-term exposure to high As concentrations changed the structure of intestinal tissues and the expression of As resistance related genes in intestinal microbes. In addition, 16S rRNA gene sequencing revealed that As exposure significantly affected the Beta diversity of intestinal flora but had no significant effect on the Alpha diversity (except ACE index). Moreover, As exposure altered the composition of the intestinal microbiota from phylum to species. Non-targeted metabolomics profiling revealed that As exposure significantly changed the composition of metabolites, specifically those related to phenylalanine metabolism. Correlation analysis demonstrated that the changes in microbial communities and metabolites were highly correlated under As exposure. Overall, this study demonstrates that long-term exposure to high As concentrations disrupted the intestinal microbiome and metabolome, which may indicate the role of As exposure at inducing human diseases under similar conditions.

Perfluorooctane sulfonate alters gut microbiota-host metabolic homeostasis in mice.

Perfluorooctane sulfonate (PFOS) is a persistent environmental chemical whose biological effects are mediated by multiple mechanisms. Recent evidence suggests that the gut microbiome may be directly impacted by and/or alter the fate and effects of environmental chemicals in the host. Thus, the aim of this study was to determine whether PFOS influences the gut microbiome and its metabolism, and the host metabolome. Four groups of male C57BL/6 J mice were fed a diet with or without 0.003 %, 0.006 %, or 0.012 % PFOS, respectively. 16S rRNA gene sequencing, metabolomic, and molecular analyses were used to examine the gut microbiota of mice after dietary PFOS exposure. Dietary PFOS exposure caused a marked change in the gut microbiome compared to controls. Dietary PFOS also caused dose-dependent changes in hepatic metabolic pathways including those involved in lipid metabolism, oxidative stress, inflammation, TCA cycle, glucose, and amino acid metabolism. Changes in the metabolome correlated with changes in genes that regulate these pathways. Integrative analyses also demonstrated a strong correlation between the alterations in microbiota composition and host metabolic profiles induced by PFOS. Further, using isolated mouse cecal contents, PFOS exposure directly affected the gut microbiota metabolism. Results from these studies demonstrate that the molecular and biochemical changes induced by PFOS are mediated in part by the gut microbiome, which alters gene expression and the host metabolome in mice.

The organophosphate malathion disturbs gut microbiome development and the quorum-Sensing system.

The gut microbiome has tremendous potential to impact health and disease. Various environmental toxicants, including insecticides, have been shown to alter gut microbiome community structures. However, the mechanism that compositionally and functionally regulates gut microbiota remains unclear. Quorum sensing is known to modulate intra- and interspecies gene expression and coordinate population responses. It is unknown whether quorum sensing is disrupted when environmental toxicants cause perturbations in the gut microbiome community structure. To reveal the response of the quorum-sensing system to environmental exposure, we use a combination of Illumina-based 16S rRNA gene amplicon and shotgun metagenome sequencing to examine the impacts of a widely used organophosphate insecticide, malathion, on the gut microbiome trajectory, quorum sensing system and behaviors related to quorum sensing, such as motility and pathogenicity. Our results demonstrated that malathion perturbed the gut microbiome development, quorum sensing and quorum sensing related behaviors. These findings may provide a novel mechanistic understanding of the role of quorum-sensing in the gut microbiome toxicity of malathion.

Bioprospecting Red Sea Coastal Ecosystems for Culturable Microorganisms and Their Antimicrobial Potential.

Microorganisms that inhabit unchartered unique soil such as in the highly saline and hot Red Sea lagoons on the Saudi Arabian coastline, represent untapped sources of potentially new bioactive compounds. In this study, a culture-dependent approach was applied to three types of sediments: mangrove mud (MN), microbial mat (MM), and barren soil (BS), collected from Rabigh harbor lagoon (RHL) and Al-Kharrar lagoon (AKL). The isolated bacteria were evaluated for their potential to produce bioactive compounds. The phylogenetic characterization of 251 bacterial isolates based on the 16S rRNA gene sequencing, supported their assignment to five different phyla: Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Planctomycetes. Fifteen putative novel species were identified based on a 16S rRNA gene sequence similarity to other strain sequences in the NCBI database, being ≤98%. We demonstrate that 49 of the 251 isolates exhibit the potential to produce antimicrobial compounds. Additionally, at least one type of biosynthetic gene sequence, responsible for the synthesis of secondary metabolites, was recovered from 25 of the 49 isolates. Moreover, 10 of the isolates had a growth inhibition effect towards Staphylococcus aureus, Salmonella typhimurium and Pseudomonas syringae. We report the previously unknown antimicrobial activity of B. borstelensis, P. dendritiformis and M. salipaludis against all three indicator pathogens. Our study demonstrates the evidence of diverse cultured microbes associated with the Red Sea harbor/lagoon environments and their potential to produce antimicrobial compounds.

Molecular characterization of the intestinal microbiota in patients with and without abdominal bloating.

Recent studies have demonstrated differences in the intestinal microbiota between patients with irritable bowel syndrome (IBS) and healthy controls (HC), suggesting a role for the intestinal microbiota in the pathogenesis of IBS. Alterations in the microbiota have also been implicated in the pathogenesis of abdominal bloating, a commonly reported symptom in IBS. We investigated the relationship between the intestinal microbiota, abdominal bloating, and altered bowel patterns in a cohort of patients with IBS and HC. The 16S rRNA gene from fresh fecal samples was amplified and pyrosequenced by using Roche-454 Titanium chemistry. A Core Measurable Microbiome (CMM) was generated for Operational Taxonomic Unit (OTU) detected in >75% of all samples and compositional features of CMM were compared between groups by Linear Discriminant Analysis (LDA). IBS differentiated from HC by LDA using continuous variation in the species/OTUs or the CMM genera. When subcategorized based on bloating symptoms and bowel characteristics, the same subjects were also well differentiated from one another and from HC. ANOVA analysis showed quantitative species/OTU differences between the subgroups including IBS with and without bloating, and subtypes based on bowel characteristics. The clear LDA differentiation and the significant microbial taxa differences between the groups imply a significant association of the microbiota with bloating symptoms and bowel characteristics in IBS. These changes in the microbiota may serve as a biomarker for IBS and its clinical subtypes and suggest a role for the intestinal microbiota in the pathogenesis of the main symptoms of the disorder.

The human milk oligosaccharide 2'-fucosyllactose augments the adaptive response to extensive intestinal.

Intestinal resection resulting in short bowel syndrome (SBS) carries a heavy burden of long-term morbidity, mortality, and cost of care, which can be attenuated with strategies that improve intestinal adaptation. SBS infants fed human milk, compared with formula, have more rapid intestinal adaptation. We tested the hypothesis that the major noncaloric human milk oligosaccharide 2'-fucosyllactose (2'-FL) contributes to the adaptive response after intestinal resection. Using a previously described murine model of intestinal adaptation, we demonstrated increased weight gain from 21 to 56 days (P < 0.001) and crypt depth at 56 days (P < 0.0095) with 2'-FL supplementation after ileocecal resection. Furthermore, 2'-FL increased small bowel luminal content microbial alpha diversity following resection (P < 0.005) and stimulated a bloom in organisms of the genus Parabacteroides (log2-fold = 4.1, P = 0.035). Finally, transcriptional analysis of the intestine revealed enriched ontologies and pathways related to antimicrobial peptides, metabolism, and energy processing. We conclude that 2'-FL supplementation following ileocecal resection increases weight gain, energy availability through microbial community modulation, and histological changes consistent with improved adaptation.

Skin Microbiome in Patients With Psoriasis Before and After Balneotherapy at the Thermal Care Center of La Roche-Posay.

BACKGROUND: Changes in the composition of microbial communities that colonize skin have been linked to several diseases including psoriasis. Nevertheless, the intra-individual dynamics and how these communities respond to balneotherapy remain poorly understood. METHODS: This open label study was conducted between July and September 2012. Microbial communities of patients with psoriasis vulgaris were characterized prior and post a 3-week selenium-rich water balneotherapy treatment at the thermal care center La Roche-Posay (La Roche-Posay, France). Balneotherapy consisted of high-pressure filiform showers, baths, facial, and body spray treatments as well as La Roche-Posay thermal spring water (LRP-TSW) consumption. Swabs were taken from affected and proximal unaffected skin and the 16S rRNA bacterial gene was used to analyze the composition of bacterial communities. Using the same 16S rRNA gene tool, we tried to describe the LRP-TSW bacterial landscape. RESULTS: This study included 54 patients diagnosed with moderate to severe forms of psoriasis vulgaris. After eliminating individuals lacking paired samples from both visits, 29 individuals were analyzed for their microbiome profile. Shannon Diversity Index and global bacterial landscape indicate similar microbial communities on both unaffected and adjacent affected skin. PASI values decreased post-balneotherapy implying improvement of disease severity. No significant change in the Shannon Diversity Index was noticed at the end of the third week. The average taxonomic composition of skin microbial communities associated with unaffected and affected skin of psoriatic patients post-balneotherapy shows that treatment with LRP-TSW significantly increased the level of Xanthomonas genus and, to a lesser extent, Corynebacterium genus. The Xanthomonas genus belongs to the main Xanthomonadaceae family found in LRP-TSW and also on healthy skin. CONCLUSIONS: In psoriatic patients, a poor bacterial biodiversity was noticed and the bacterial communities were similar on unaffected and affected adjacent skin. Family analysis identified, for the first time, Xanthomonadaceae belonging to Proteobacteria phylum and known to be keratolytic, associated with the clinical improvement observed after a 3-week balneotherapy treatment. This data supports the interest of selenium-rich thermal spring water in the treatment of psoriasis vulgaris.

Daily expression pattern of protein-encoding genes and small noncoding RNAs in synechocystis sp. strain PCC 6803.

Many organisms harbor circadian clocks with periods close to 24 h. These cellular clocks allow organisms to anticipate the environmental cycles of day and night by synchronizing circadian rhythms with the rising and setting of the sun. These rhythms originate from the oscillator components of circadian clocks and control global gene expression and various cellular processes. The oscillator of photosynthetic cyanobacteria is composed of three proteins, KaiA, KaiB, and KaiC, linked to a complex regulatory network. Synechocystis sp. strain PCC 6803 possesses the standard cyanobacterial kaiABC gene cluster plus multiple kaiB and kaiC gene copies and antisense RNAs for almost every kai transcript. However, there is no clear evidence of circadian rhythms in Synechocystis sp. PCC 6803 under various experimental conditions. It is also still unknown if and to what extent the multiple kai gene copies and kai antisense RNAs affect circadian timing. Moreover, a large number of small noncoding RNAs whose accumulation dynamics over time have not yet been monitored are known for Synechocystis sp. PCC 6803. Here we performed a 48-h time series transcriptome analysis of Synechocystis sp. PCC 6803, taking into account periodic light-dark phases, continuous light, and continuous darkness. We found that expression of functionally related genes occurred in different phases of day and night. Moreover, we found day-peaking and night-peaking transcripts among the small RNAs; in particular, the amounts of kai antisense RNAs correlated or anticorrelated with those of their respective kai target mRNAs, pointing toward the regulatory relevance of these antisense RNAs. Surprisingly, we observed that the amounts of 16S and 23S rRNAs in this cyanobacterium fluctuated in light-dark periods, showing maximum accumulation in the dark phase. Importantly, the amounts of all transcripts, including small noncoding RNAs, did not show any rhythm under continuous light or darkness, indicating the absence of circadian rhythms in Synechocystis.

Phylogenetic diversity and biological activity of actinobacteria isolated from the Chukchi Shelf marine sediments in the Arctic Ocean.

Marine environments are a rich source of Actinobacteria and have the potential to produce a wide variety of biologically active secondary metabolites. In this study, we used four selective isolation media to culture Actinobacteria from the sediments collected from the Chukchi Shelf in the Arctic Ocean. A total of 73 actinobacterial strains were isolated. Based on repetitive DNA fingerprinting analysis, we selected 30 representatives for partial characterization according to their phylogenetic diversity, antimicrobial activities and secondary-metabolite biosynthesis genes. Results from the 16S rRNA gene sequence analysis indicated that the 30 strains could be sorted into 18 phylotypes belonging to 14 different genera: Agrococcus, Arsenicicoccus, Arthrobacter, Brevibacterium, Citricoccus, Janibacter, Kocuria, Microbacterium, Microlunatus, Nocardioides, Nocardiopsis, Saccharopolyspora, Salinibacterium and Streptomyces. To our knowledge, this paper is the first report on the isolation of Microlunatus genus members from marine habitats. Of the 30 isolates, 11 strains exhibited antibacterial and/or antifungal activity, seven of which have activities against Bacillus subtilis and Candida albicans. All 30 strains have at least two biosynthetic genes, one-third of which possess more than four biosynthetic genes. This study demonstrates the significant diversity of Actinobacteria in the Chukchi Shelf sediment and their potential for producing biologically active compounds and novel material for genetic manipulation or combinatorial biosynthesis.

Anti-Chikungunya viral activities of aplysiatoxin-related compounds from the marine cyanobacterium Trichodesmium erythraeum.

Tropical filamentous marine cyanobacteria have emerged as a viable source of novel bioactive natural products for drug discovery and development. In the present study, aplysiatoxin (1), debromoaplysiatoxin (2) and anhydrodebromoaplysiatoxin (3), as well as two new analogues, 3-methoxyaplysiatoxin (4) and 3-methoxydebromoaplysiatoxin (5), are reported for the first time from the marine cyanobacterium Trichodesmium erythraeum. The identification of the bloom-forming cyanobacterial strain was confirmed based on phylogenetic analysis of its 16S rRNA sequences. Structural determination of the new analogues was achieved by extensive NMR spectroscopic analysis and comparison with NMR spectral data of known compounds. In addition, the antiviral activities of these marine toxins were assessed using Chikungunya virus (CHIKV)-infected cells. Post-treatment experiments using the debrominated analogues, namely compounds 2, 3 and 5, displayed dose-dependent inhibition of CHIKV when tested at concentrations ranging from 0.1 µM to 10.0 µM. Furthermore, debromoaplysiatoxin (2) and 3-methoxydebromoaplysiatoxin (5) exhibited significant anti-CHIKV activities with EC50 values of 1.3 µM and 2.7 µM, respectively, and selectivity indices of 10.9 and 9.2, respectively.

Establishing a relationship between bacteria in the human gut and complex regional pain syndrome.

Complex Regional Pain Syndrome (CRPS) is a serious and painful condition involving the peripheral and central nervous systems. Full comprehension of the disorder's pathophysiology remains incomplete, but research implicates the immune system as a contributor to chronic pain. Because of the impact gastrointestinal bacteria have in the development and behavior of the immune system, this study compares the GI microbial communities of 16 participants with CRPS (5 of whom have intestinal discomforts) and 16 healthy controls using 454 sequencing technology. CRPS subjects were found to have significantly less diversity than their healthy counterparts. Statistical analysis of the phylogenetic classifications revealed significantly increased levels of Proteobacteria and decreased levels of Firmicutes in CRPS subjects. Clustering analysis showed significant separation between healthy controls and CRPS subjects. These results support the hypothesis that the GI microbial communities of CRPS participants differ from those of their healthy counterparts. These variations may hold the key to understanding how CRPS develops and provide information that could yield a potential treatment.

Nus transcription elongation factors and RNase III modulate small ribosome subunit biogenesis in Escherichia coli.

Escherichia coli NusA and NusB proteins bind specific sites, such as those in the leader and spacer sequences that flank the 16S region of the ribosomal RNA transcript, forming a complex with RNA polymerase that suppresses Rho-dependent transcription termination. Although antitermination has long been the accepted role for Nus factors in rRNA synthesis, we propose that another major role for the Nus-modified transcription complex in rrn operons is as an RNA chaperone insuring co-ordination of 16S rRNA folding and RNase III processing that results in production of proper 30S ribosome subunits. This contrarian proposal is based on our studies of nusA and nusB cold-sensitive mutations that have altered translation and at low temperature accumulate 30S subunit precursors. Both phenotypes are suppressed by deletion of RNase III. We argue that these results are consistent with the idea that the nus mutations cause altered rRNA folding that leads to abnormal 30S subunits and slow translation. According to this idea, functional Nus proteins stabilize an RNA loop between their binding sites in the 5' RNA leader and on the transcribing RNA polymerase, providing a topological constraint on the RNA that aids normal rRNA folding and processing.

Cyprinid herpesvirus 3 infection disrupts the skin barrier of common carp (Cyprinus carpio L.).

Cyprinid herpesvirus-3 (CyHV-3) is recognised as a pathogen which causes mass mortality in populations of carp, Cyprinus carpio. One of the characteristic symptoms of the disease associated with CyHV-3 infection is the occurrence of skin lesions, sloughing off the epithelium and a lack of mucus. Furthermore, fish then seem to be more susceptible to secondary infections by bacterial, parasitic or fungal pathogens which may cause further mortality within the population. The observed pathological alterations lead to the assumption that the carp skin barrier is strongly challenged during CyHV-3 associated disease. Therefore we examined mRNA expression of genes encoding inflammatory mediators, type I interferons, and the following skin defence molecules: antimicrobial peptides, claudins, and mucin. In addition, we monitored changes in the bacterial flora of the skin during disease conditions. Our results show that CyHV-3 associated disease in the skin of common carp leads to a reduction in mRNA expression of genes encoding several important components of the mucosal barrier, in particular mucin 5B, beta defensin 1 and 2, and the tight junction proteins claudin 23 and 30. This caused changes in the bacterial flora and the development of secondary bacterial infection among some individual fish. To our knowledge this is the first report showing that under disease conditions associated with virus infection, the mucosal barrier of fish skin is disrupted resulting in a higher susceptibility to secondary infections. The reported clinical signs of CyHV-3 skin infection can now be explained by our results at the molecular level, although the mechanism of a probable virus induced immunomodulation has to be investigated further.

Differential in vivo gene expression of major Leptospira proteins in resistant or susceptible animal models.

Transcripts of Leptospira 16S rRNA, FlaB, LigB, LipL21, LipL32, LipL36, LipL41, and OmpL37 were quantified in the blood of susceptible (hamsters) and resistant (mice) animal models of leptospirosis. We first validated adequate reference genes and then evaluated expression patterns in vivo compared to in vitro cultures. LipL32 expression was downregulated in vivo and differentially regulated in resistant and susceptible animals. FlaB expression was also repressed in mice but not in hamsters. In contrast, LigB and OmpL37 were upregulated in vivo. Thus, we demonstrated that a virulent strain of Leptospira differentially adapts its gene expression in the blood of infected animals.

Isolation of a potent antibiotic producer bacterium, especially against MRSA, from northern region of the Persian Gulf.

Nowadays, emergence and prevalence of MRSA (Methicillin Resistant Staphylococcus aureus) strain have become a great global concern in 21st century, so, it is necessary to discover new antibiotics against this pathogen. The aim of this study was isolation and evaluation marine bacteria from the Persian Gulf in order to finding antibiotic compounds against some pathogenic bacteria. For this purpose, water and sediment samples were collected from the Persian Gulf during March to October 2009. The antibacterial activity of the isolated bacteria was assessed using disc diffusion method. The Growth Curve Interference (GCI) parameter against MRSA was determined for the high potential antibiotic producing strain. The most important factors affecting fermentation conditions in antibiotic production were also optimized. Definite identification of intended isolate was confirmed by 16S rRNA sequencing. Altogether, 51 bacterial colony was isolated and among them only 3 bacterium showed antibacterial activity. Pseudoalteromonas piscicida PG-01 isolated from a sediment sample was chosen as the best antibiotic producing strain. This strain was effective against all tested Gram-positive bacteria, had good anti-MRSA activity and also GCI value against MRSA was two times lower than MIC value. Among the optimized fermentation parameters, carbon and nitrogen sources play major role in efficacy of optimized antibiotic production. Ultrastructural study on the effect of intended antibiotic compounds on MRSA using TEM revealed that the target site for this compound is cell wall. Considering the antibacterial effect of PG-01 strain especially against MRSA, intended antibiotic compounds can gives hope for treatment of diseases caused by multi-drug resistant bacteria.

[Prolonged cultivation of an anaerobic bacterial community producing hydrogen].

This paper studies various methods of long-term maintenance of the process of hydrogen evolution during the growth of an aerobic bacterial community on a starch-containing environment. When cultured in separable trip fermentation mode for 72 days, from 0.10 to 0.23 H2/l of medium/day was formed. The regime of regular reseeding lasted more than 100 days, forming an average of 0.81 1 H2/l of medium/day. The advantages and disadvantages of different methods of microbial hydrogen production during a dark starch fermentation process are presented. From the obtained H2 forming microbial communities, we isolated an anaerobic spore-forming bacterium (strain BF). Phylogenetic analysis of the 16S RNA gene sequence of the new strain showed that according to its genotype it belongs to the Clostridium butyricum species.

Investigation of unusual growth and phenotypic characteristics of plasmid-containing and plasmid-free strains of oligotrophic bacterium Ancylobacter vacuolatus.

The oligotrophic bacterium Ancylobacter vacuolatus contains two large plasmids pREV1 and pREV2 (about 150 and 250 kb, respectively). Plasmid pREV1 carries the genes responsible for resistance to chloramphenicol, trimethoprim and y-irradiation. Plasmid pREV2 carries the genes responsible for resistance to beta-lactam antibiotics and formation of gas vacuoles. The ability to grow under oligotrophic conditions did not depend directly on either plasmid and was probably chromosome-encoded. Nevertheless, strains lacking the pREV2 plasmid had an improved capacity for growth in enriched media, as is evident from the following findings: 1) the growth rate of the strains lacking pREV2 was about 60% higher with an induction time of about two times less than those for strains carrying the plasmid; 2) the overall cell yield in rich media and colony size on non-oligotrophic agarized media increased with removal of pREV2; 3) the characteristic change in cell morphology occurring in the wild type ofA. vacuolatus when switched from oligotrophic to eutrophic growth conditions was not observed in the strains lacking pREV2; 4) bacterial strains lacking pREV2 exhibited significantly higher rRNA content than the parent strain. As a possible explanation for these phenomena, we suggest that the pREV2 plasmid carries gene(s) for protein(s) acting as repressor(s) of expression of some enzymes involved in eutrophic metabolism. Such protein(s) probably participate in switching between the oligotrophic and eutrophic types of metabolism in response to changing nutrient supply in the environment.