The Serine Biosynthesis of Paenibacillus polymyxa WLY78 Is Regulated by the T-Box Riboswitch.

Serine is important for nearly all microorganisms in protein and downstream amino acids synthesis, however, the effect of serine on growth and nitrogen fixation was not completely clear in many bacteria, besides, the regulatory mode of serine remains to be fully established. In this study, we demonstrated that L-serine is essential for growth and nitrogen fixation of Paenibacillus polymyxa WLY78, but high concentrations of L-serine inhibit growth, nitrogenase activity, and nifH expression. Then, we revealed that expression of the serA whose gene product catalyzes the first reaction in the serine biosynthetic pathway is regulated by the T-box riboswitch regulatory system. The 508 bp mRNA leader region upstream of the serA coding region contains a 280 bp T-box riboswitch. The secondary structure of the T-box riboswitch with several conserved features: three stem-loop structures, a 14-bp T-box sequence, and an intrinsic transcriptional terminator, is predicted. Mutation and the transcriptional leader-lacZ fusions experiments revealed that the specifier codon of serine is AGC (complementary to the anticodon sequence of tRNAser). qRT-PCR showed that transcription of serA is induced by serine starvation, whereas deletion of the specifier codon resulted in nearly no expression of serA. Deletion of the terminator sequence or mutation of the continuous seven T following the terminator led to constitutive expression of serA. The data indicated that the T-box riboswitch, a noncoding RNA segment in the leader region, regulates expression of serA by a transcription antitermination mechanism.

Characterization of a transcriptional TPP riboswitch in the human pathogen Neisseria meningitidis.

Increasing evidence has demonstrated that regulatory RNA elements such as riboswitches (RS) play a pivotal role in the fine-tuning of bacterial gene expression. In this study, we investigated and characterized a novel transcriptional thiamine pyrophosphate (TPP) RS in the obligate human pathogen N. meningitidis MC58 (serogroup B). This RS is located in the 5´ untranslated region upstream of thiC gene, encoding a protein involved in TPP biosynthesis, an essential cofactor for all living beings. Primer extension revealed the transcriptional start site of thiC. Northern blot analysis of thiC mRNA and reporter gene studies confirmed the presence of an active TPP-sensing RS. Expression patterns of the wild-type RS and site-specific mutants showed that it is an OFF switch that controls transcription elongation of thiC mRNA. Interestingly, the regulatory mechanism of the meningococcal thiC RS resembles the Gram-positive Bacillus subtilis thiC RS rather than the Gram-negative Escherichia coli thiC RS. Therefore, the meningococcal thiC RS represents a rare example of transcriptional RS in a Gram-negative bacterium. We further observed that the RS is actively involved in modulating gene expression in response to different growth media and to supplemented bacterial and eukaryotic cell lysates as possible sources of nutrients in the nasopharynx. Our results suggest that RS-mediated gene regulation could influence meningococcal fitness, through the fine-tuning of biosynthesis and scavenging of nutrients and cofactors, such as thiamine.

Transcriptional analysis of RstA/RstB in avian pathogenic Escherichia coli identifies its role in the regulation of hdeD-mediated virulence and survival in chicken macrophages.

Avian pathogenic Escherichia coli (APEC) causes avian colibacillosis in poultry, which is characterized by systemic infections such as septicemia, air sacculitis, and pericarditis. APEC uses two-component regulatory systems (TCSs) to handle the stressful environments present in infected hosts. While many TCSs in E. coli have been well characterized, the RstA/RstB system in APEC has not been thoroughly investigated. The involvement of the RstA regulator in APEC pathogenesis was demonstrated during previous studies investigating its role in APEC persistence in chicken macrophages and respiratory infections. However, the mechanism underlying this phenomenon has not been clarified. Transcriptional analysis of the effect of rstAB deletion was therefore performed to improve the understanding of the RstA/RstB regulatory mechanism, and particularly its role in virulence. The transcriptomes of the rstAB mutant and the wild-type strain E058 were compared during their growth in the bloodstreams of challenged chickens. Overall, 198 differentially expressed (DE) genes were identified, and these indicated that RstA/RstB mainly regulates systems involved in nitrogen metabolism, iron acquisition, and acid resistance. Phenotypic assays indicated that the rstAB mutant responded more to an acidic pH than the wild-type strain did, possibly because of the repression of the acid-resistance operons hdeABD and gadABE by the deletion of rstAB. Based on the reported RstA box motif TACATNTNGTTACA, we identified four possible RstA target genes (hdeD, fadE, narG, and metE) among the DE genes. An electrophoretic mobility shift assay confirmed that RstA binds directly to the promoter of hdeD, and ß-galactosidase assays showed that hdeD expression was reduced by rstAB deletion, indicating that RstA directly regulates hdeD expression. The hdeD mutation resulted in virulence attenuation in both cultured chicken macrophages and experimentally infected chickens. In conclusion, our data suggest that RstA affects APEC E058 virulence partly by directly regulating the acidic resistance gene hdeD.

Effect of Vitamin D Supplementation on Faecal Microbiota: A Randomised Clinical Trial.

In animal studies, vitamin D supplementation has been shown to improve gut microbiota and intestinal inflammation. However, limited evidence exists on the effect of vitamin D supplementation on the human gut microbiota. We examined the effect of vitamin D supplementation on faecal microbiota in 26 vitamin D-deficient (25-hydroxyvitamin D (25(OH)D) ≤50 nmol/L), overweight or obese (BMI ≥25 kg/m2) otherwise healthy adults. Our study was ancillary to a community based double-blind randomised clinical trial, conducted between 2014 and 2016. The participants provided stool samples at baseline and after 100,000 international units (IU) loading dose of cholecalciferol followed by 4000 IU daily or matching placebo for 16 weeks. Faecal microbiota was analysed using 16S rRNA sequencing; V6-8 region. There was no significant difference in microbiome α-diversity between vitamin D and placebo groups at baseline and follow-up (all p > 0.05). In addition, no clustering was found based on vitamin D supplementation at follow-up (p = 0.3). However, there was a significant association between community composition and vitamin D supplementation at the genus level (p = 0.04). The vitamin D group had a higher abundance of genus Lachnospira, and lower abundance of genus Blautia (linear discriminate analysis >3.0). Moreover, individuals with 25(OH)D >75 nmol/L had a higher abundance of genus Coprococcus and lower abundance of genus Ruminococcus compared to those with 25(OH)D <50 nmol/L. Our findings suggest that vitamin D supplementation has some distinct effects on faecal microbiota. Future studies need to explore whether these effects would translate into improved clinical outcomes.

Escherichia coli tRNA 2-selenouridine synthase (SelU) converts S2U-RNA to Se2U-RNA via S-geranylated-intermediate.

To date the only tRNAs containing nucleosides modified with a selenium (5-carboxymethylaminomethyl-2-selenouridine and 5-methylaminomethyl-2-selenouridine) have been found in bacteria. By using tRNA anticodon-stem-loop fragments containing S2U, Se2U, or geS2U, we found that in vitro tRNA 2-selenouridine synthase (SelU) converts S2U-RNA to Se2U-RNA in a two-step process involving S2U-RNA geranylation (with ppGe) and subsequent selenation of the resulting geS2U-RNA (with SePO33- ). No 'direct' S2U-RNA→Se2U-RNA replacement is observed in the presence of SelU/SePO33- only (without ppGe). These results suggest that the in vivo S2U→Se2U and S2U→geS2U transformations in tRNA, so far claimed to be the elementary reactions occurring independently in the same domain of the SelU enzyme, should be considered a combination of two consecutive events - geranylation (S2U→geS2U) and selenation (geS2U→Se2U).

Isolation and Characterization of a microRNA-size Secretable Small RNA in Streptococcus sanguinis.

MicroRNAs in eukaryotic cells are thought to control highly complex signal transduction and other biological processes by regulating coding transcripts, accounting for their important role in cellular events in eukaryotes. Recently, a novel class of bacterial RNAs similar in size [18-22 nucleotides (nt)] to microRNAs has been reported. Herein, we describe microRNAs, small RNAs from the oral pathogen Streptococcus sanguinis. The bacteria are normally present in the oral cavities and cause endocarditis by contaminating bloodstreams. Small RNAs were analyzed by deep sequencing. Selected highly expressed small RNAs were further validated by real-time polymerase chain reaction and northern blot analyses. We found that skim milk supplement changed the expression of small RNAs S.S-1964 in tandem with the nearby SSA_0513 gene involved in vitamin B12 conversion. We furthermore observed small RNAs secreted via bacterial membrane vesicles. Although their precise function remains unclear, secretable small RNAs may represent an entirely new area of study in bacterial genetics.

Changes of cecal microflora in chickens following Eimeria tenella challenge and regulating effect of coated sodium butyrate.

Eimeria tenella, one of the most important parasitic protozoa in the genus Eimeria, is responsible for chicken caecal coccidiosis resulting in huge economic losses to poultry industry. The present study investigated the changes in caecal microflora of E. tenella-infected chickens and the regulating effect of coated sodium butyrate, a potential alternative to antibiotics. Using high-throughput sequencing of 16S rRNA V3-V4 region of bacteria we found significant changes in caecal microflora of E. tenella-infected chickens indicated by an increase of Firmicutes (mainly Ruminococcaceae, Lachnospiraceae and vadin BB60) and Proteobacteria (mainly Enterobacteriaceae) and a decrease of Bacteroidetes (predominantly Bacteroidaceae). Inclusion of coated sodium butyrate in the diet of chickens per se had no significant effect on caecal microflora of normal healthy chickens but significantly prevented the increase in Firmicute abundance and decrease of Bacteroidetes abundance in E. tenella-infected birds. No significant changes to caecal microflora were observed at the phylum level between control and E. tenella-infected birds given coated sodium butyrate. In conclusion, our results show that coated sodium butyrate can balance the disorders of cecal microflora caused by E. tenella; thus, it can be a useful supplement for the control of avian coccidiosis.

Transcriptomic and Proteomic Profiling of Anabaena sp. Strain 90 under Inorganic Phosphorus Stress.

Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyanobacterium Anabaena sp. strain 90 to phosphorus deprivation, aiming at the identification of candidate genes to monitor the Pi status in cyanobacteria. Furthermore, this study increased the basic understanding of how phosphorus affects diazotrophic and bloom-forming cyanobacteria as a major growth-limiting factor. Based on RNA sequencing data, we identified 246 differentially expressed genes after phosphorus starvation and 823 differentially expressed genes after prolonged Pi limitation, most of them related to central metabolism and cellular growth. The transcripts of the genes related to phosphorus transport and assimilation (pho regulon) were most upregulated during phosphorus depletion. One of the most increased transcripts encodes a giant protein of 1,869 amino acid residues, which contains, among others, a phytase-like domain. Our findings predict its crucial role in phosphorus starvation, but future studies are still needed. Using two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found 43 proteins that were differentially expressed after prolonged phosphorus stress. However, correlation analysis unraveled an association only to some extent between the transcriptomic and proteomic abundances. Based on the present results, we suggest that the method used for monitoring the Pi status in cyanobacterial bloom should contain wider combinations of pho regulon genes (e.g., PstABCS transport systems) in addition to the commonly used alkaline phosphatase gene alone.

GFAJ-1 is an arsenate-resistant, phosphate-dependent organism.

The bacterial isolate GFAJ-1 has been proposed to substitute arsenic for phosphorus to sustain growth. We have shown that GFAJ-1 is able to grow at low phosphate concentrations (1.7 µM), even in the presence of high concentrations of arsenate (40 mM), but lacks the ability to grow in phosphorus-depleted (<0.3 µM), arsenate-containing medium. High-resolution mass spectrometry analyses revealed that phosphorylated central metabolites and phosphorylated nucleic acids predominated. A few arsenylated compounds, including C6 sugar arsenates, were detected in extracts of GFAJ-1, when GFAJ-1 was incubated with arsenate, but further experiments showed they formed abiotically. Inductively coupled plasma mass spectrometry confirmed the presence of phosphorus in nucleic acid extracts, while arsenic could not be detected and was below 1 per mil relative to phosphorus. Taken together, we conclude that GFAJ-1 is an arsenate-resistant, but still a phosphate-dependent, bacterium.

Esre: a novel essential non-coding RNA in Escherichia coli.

YigP gene (GeneID: 948915) locates between ubiquinone biosynthetic genes ubiE and ubiB in Escherichia coli. GeneBank annotates yigP as a putative protein-coding gene. In this study, we found a new essential sRNA gene, esre, locates within the region of yigP. The E. coli strain with inactive esre must rely on a complementary plasmid to survive. Moreover, RACE experiments showed esre encodes an RNA molecule of 252 nt. Further experiments revealed esre gene is immune to frame shift mutations and the function of esre depends mostly on the RNA secondary structure, which are typical traits of sRNA. Since it is difficult to predict the target of an essential sRNA, more research is needed to reveal the function and mechanism of esre.

Quantifying the sequence-function relation in gene silencing by bacterial small RNAs.

Sequence-function relations for small RNA (sRNA)-mediated gene silencing were quantified for the sRNA RyhB and some of its mRNA targets in Escherichia coli. Numerous mutants of RyhB and its targets were generated and their in vivo functions characterized at various levels of target and RyhB expression. Although a core complementary region is required for repression by RyhB, variations in the complementary sequences of the core region gave rise to a continuum of repression strengths, correlated exponentially with the computed free energy of RyhB-target duplex formation. Moreover, sequence variations in the linker region known to interact with the RNA chaperone Hfq also gave rise to a continuum of repression strengths, correlated exponentially with the computed energy cost of keeping the linker region open. These results support the applicability of the thermodynamic model in predicting sRNA-mRNA interaction and suggest that sequences at these locations may be used to fine-tune the degree of repression. Surprisingly, a truncated RyhB without the Hfq-binding region is found to repress multiple targets of the wild-type RyhB effectively, both in the presence and absence of Hfq, even though the former is required for the activity of wild-type RyhB itself. These findings challenge the commonly accepted model concerning the function of Hfq in gene silencing-both in providing stability to the sRNAs and in catalyzing the target mRNAs to take on active conformations-and raise the intriguing question of why many endogenous sRNAs subject their functions to Hfq-dependences.

The SAM-responsive S(MK) box is a reversible riboswitch.

The S(MK) (SAM-III) box is an S-adenosylmethionine (SAM)-responsive riboswitch found in the 5' untranslated region of metK genes, encoding SAM synthetase, in many members of the Lactobacillales. SAM binding causes a structural rearrangement in the RNA that sequesters the Shine-Dalgarno (SD) sequence by pairing with a complementary anti-SD (ASD) sequence; sequestration of the SD sequence inhibits binding of the 30S ribosomal subunit and prevents translation initiation. We observed a slight increase in the half-life of the metK transcript in vivo when Enterococcus faecalis cells were depleted for SAM, but no significant change in overall transcript abundance, consistent with the model that this riboswitch regulates at the level of translation initiation. The half-life of the SAM-S(MK) box RNA complex in vitro is shorter than that of the metK transcript in vivo, raising the possibility of reversible binding of SAM. We used a fluorescence assay to directly visualize reversible switching between the SAM-free and SAM-bound conformations. We propose that the S(MK) box riboswitch can make multiple SAM-dependent regulatory decisions during the lifetime of the transcript in vivo, acting as a reversible switch that allows the cell to respond rapidly to fluctuations in SAM pools by modulating expression of the SAM synthetase gene.

Jellyfish as vectors of bacterial disease for farmed salmon (Salmo salar).

Swarms or blooms of jellyfish are increasingly problematic and can result in high mortality rates of farmed fish. Small species of jellyfish, such as Phialella quadrata (13 mm in diameter), are capable of passing through the mesh of sea cages and being sucked into the mouth of fish during respiration. Results of the current study show that the initial damage to gills of farmed Atlantic salmon, likely produced by nematocyst-derived toxins from the jellyfish, was compounded by secondary bacterial infection with Tenacibaculum maritimum. Results also demonstrate that these filamentous bacteria were present on the mouth of the jellyfish and that their DNA sequences were almost identical to those of bacteria present on the salmon gills. This suggests that the bacterial lesions were not the result of an opportunistic infection of damaged tissue, as previously thought. Instead, P. quadrata is probably acting as a vector for this particular bacterial pathogen, and it is the first time that evidence to support such a link has been presented. No prior literature describing the presence of bacteria associated with jellyfish, except studies about their decay, could be found. It is not known if all jellyfish of this and other species carry similar bacteria or the relationship to each other. Their source, the role they play under other circumstances, and indeed whether the jellyfish were themselves diseased are also not known. The high proteolytic capabilities of T. maritimum mean that partially digested gill tissues were readily available to the jellyfish, which rely heavily on intracellular digestion for their nutrition.

Enhancing the prebiotic relevance of a set of covalently self-assembling, autorecombining RNAs through in vitro selection.

An in vitro form of the self-splicing group-I intron interrupting the Azoarcus tRNA(Ile) was shortened by ~10% with the removal of helix P6a. This deletion reduced the reverse-splicing activity of the ribozyme about 10-fold. Through in vitro selection, this activity was restored in several low-error mutants. A number of mutations were found that improved reverse-splicing activity through both increased k (obs) and better folding. The deletion mutant could be fragmented into as many as three discrete pieces, which, when incubated together, were capable of covalent self-assembly through energy-neutral transesterification reactions, a process called autorecombination. A subset of the mutations identified through in vitro selection for reverse-splicing were exaptations in that they were also shown to augment the autorecombination reactions, leading to higher yields of covalently self-assembled products, making this the smallest such system yet discovered.

Fluorescence probing of T box antiterminator RNA: insights into riboswitch discernment of the tRNA discriminator base.

The T box transcription antitermination riboswitch is one of the main regulatory mechanisms utilized by Gram-positive bacteria to regulate genes that are involved in amino acid metabolism. The details of the antitermination event, including the role that Mg(2+) plays, in this riboswitch have not been completely elucidated. In these studies, details of the antitermination event were investigated utilizing 2-aminopurine to monitor structural changes of a model antiterminator RNA when it was bound to model tRNA. Based on the results of these fluorescence studies, the model tRNA binds the model antiterminator RNA via an induced-fit. This binding is enhanced by the presence of Mg(2+), facilitating the complete base pairing of the model tRNA acceptor end with the complementary bases in the model antiterminator bulge.

Structure of the thiostrepton resistance methyltransferase.S-adenosyl-L-methionine complex and its interaction with ribosomal RNA.

The x-ray crystal structure of the thiostrepton resistance RNA methyltransferase (Tsr).S-adenosyl-L-methionine (AdoMet) complex was determined at 2.45-A resolution. Tsr is definitively confirmed as a Class IV methyltransferase of the SpoU family with an N-terminal "L30-like" putative target recognition domain. The structure and our in vitro analysis of the interaction of Tsr with its target domain from 23 S ribosomal RNA (rRNA) demonstrate that the active biological unit is a Tsr homodimer. In vitro methylation assays show that Tsr activity is optimal against a 29-nucleotide hairpin rRNA though the full 58-nucleotide L11-binding domain and intact 23 S rRNA are also effective substrates. Molecular docking experiments predict that Tsr.rRNA binding is dictated entirely by the sequence and structure of the rRNA hairpin containing the A1067 target nucleotide and is most likely driven primarily by large complementary electrostatic surfaces. One L30-like domain is predicted to bind the target loop and the other is near an internal loop more distant from the target site where a nucleotide change (U1061 to A) also decreases methylation by Tsr. Furthermore, a predicted interaction with this internal loop by Tsr amino acid Phe-88 was confirmed by mutagenesis and RNA binding experiments. We therefore propose that Tsr achieves its absolute target specificity using the N-terminal domains of each monomer in combination to recognize the two distinct structural elements of the target rRNA hairpin such that both Tsr subunits contribute directly to the positioning of the target nucleotide on the enzyme.

DNA-based stable isotope probing enables the identification of active bacterial endophytes in potatoes.

A (13)CO2 (99 atom-%, 350 ppm) incubation experiment was performed to identify active bacterial endophytes in two cultivars of Solanum tuberosum, cultivars Desirée and Merkur. We showed that after the assimilation and photosynthetic transformation of (13)CO2 into (13)C-labeled metabolites by the plant, the most directly active, cultivar specific heterotrophic endophytic bacteria that consume these labeled metabolite scan be identified by DNA stable isotope probing (DNA-SIP).Density-resolved DNA fractions obtained from SIP were subjected to 16S rRNA gene-based community analysis using terminal restriction fragment length polymorphism analysis and sequencing of generated gene libraries.Community profiling revealed community compositions that were dominated by plant chloroplast and mitochondrial 16S rRNA genes for the 'light' fractions of (13)CO2-incubated potato cultivars and of potato cultivars not incubated with (13)CO2. In the 'heavy' fractions of the (13)CO2-incubated endophyte DNA, a bacterial 492-bp terminal restriction fragment became abundant, which could be clearly identified as Acinetobacter and Acidovorax spp. in cultivars Merkur and Desirée,respectively, indicating cultivar-dependent distinctions in (13)C-label flow. These two species represent two common potato endophytes with known plant-beneficial activities.The approach demonstrated the successful detection of active bacterial endophytes in potato. DNA-SIP therefore offers new opportunities for exploring the complex nature of plant-microbe interactions and plant-dependent microbial metabolisms within the endosphere.

Rapid determination of the bacterial composition of commercial probiotic products by terminal restriction fragment length polymorphism analysis.

Label claims on probiotic products often do not represent the true constituents. With the increased use of probiotics in clinical studies, it is necessary to know the true composition of probiotic products to better interpret study outcomes. We used terminal restriction fragment length polymorphism analysis to rapidly determine the overall bacterial composition of 14 commercial probiotic products and validated the results with species-specific polymerase chain reaction. The results show that many probiotic products contain unadvertised additional lactobacilli and bifidobacteria, whereas others are missing species listed on the product label. In summary, terminal restriction fragment length polymorphism is a rapid method for profiling the microbial contents of probiotic products used in clinical studies.