Achieving robust synthetic tolerance in industrial E. coli through negative auto-regulation of a DsrA-Hfq module.

In industrial fermentation processes, microorganisms often encounter acid stress, which significantly impact their productivity. This study focused on the acid-resistant module composed of small RNA (sRNA) DsrA and the sRNA chaperone Hfq. Our previous study had shown that this module improved the cell growth of Escherichia coli MG1655 at low pH, but failed to obtain this desired phenotype in industrial strains. Here, we performed a quantitative analysis of DsrA-Hfq module to determine the optimal expression mode. We then assessed the potential of the CymR-based negative auto-regulation (NAR) circuit for industrial application, under different media, strains and pH levels. Growth assay at pH 4.5 revealed that NAR-05D04H circuit was the best acid-resistant circuit to improve the cell growth of E. coli MG1655. This circuit was robust and worked well in the industrial lysine-producing strain E. coli SCEcL3 at a starting pH of 6.8 and without pH control, resulting in a 250 % increase in lysine titer and comparable biomass in shaking flask fermentation compared to the parent strain. This study showed the practical application of NAR circuit in regulating DsrA-Hfq module, effectively and robustly improving the acid tolerance of industrial strains, which provides a new approach for breeding industrial strains with tolerance phenotype.

Differential evaluation of sulfur oxides in the natural lake water samples by carbazole-furan fluorescent probe.

Water bodies are frequently polluted, with sulfur oxides being the most common form of water pollution. Therefore, developing a detection mechanism for sulfur oxides in water bodies is particularly urgent. A new fluorescent probe YX-KZBD was designed and developed. This probe releases fluorescent signals with its own sulfurous acid recognition site, detects sulfurous acid based on the Michael addition reaction, and evaluates the pollution degree of sulfur oxides in the water environment through the transformation mode of the sulfur cycle. This probe has high energy transfer efficiency in aqueous solutions. In addition, the fluorescence data obtained by analyzing the water samples were linearly fitted with the gene abundance values of the functional genes of sulfur-producing bacteria, and a significant correlation was obtained. The Kriging interpolation model was used to evaluate the sulfate content distribution at each sampling point to understand the distribution of sulfur oxides in natural water intuitively. The fluorescence signal excited by the probe was also combined with a real-time quantitative polymerase chain reaction (qPCR), and sulfate-reducing and sulfur-oxidizing bacteria were introduced in the sulfur cycle, providing a new method to assess the extent of water pollution effectively.

Circular and Linear Dichroism for the Analysis of Small Noncoding RNA Properties.

Useful structural information about the conformation of nucleic acids can be quickly acquired by circular and linear dichroism (CD/LD) spectroscopy. These techniques, rely on the differential absorption of polarised light and are indeed extremely sensitive to subtle changes in the structure of chiral biomolecules. Many CD analyses of DNA or DNA:protein complexes have been conducted with substantial data acquisitions. Conversely, CD RNA analysis are still scarce, despite the fact that RNA plays a wide cellular function. This chapter seeks to introduce the reader to the use of circular, linear dichroism and in particular the use of Synchrotron Radiation for such samples. The use of these techniques on small noncoding RNA (sRNA) will be exemplified by analyzing changes in base stacking and/or helical parameters for the understanding of sRNA structure and function, especially by translating the dynamics of RNA:RNA annealing but also to access RNA stability or RNA:RNA alignment. The effect of RNA remodeling proteins will also be addressed. These analyses are especially useful to decipher the mechanisms by which sRNA will adopt the proper conformation thanks to the action of proteins such as Hfq or ProQ in the regulation of the expression of their target mRNAs.

Involvement of OxyR and Dps in the repression of replication initiation by DsrA small RNA in Escherichia coli.

Regulation of the cell cycle process is an effective measure to ensure the stability and fidelity of genetic material during the reproduction of bacteria under different stresses. The small RNA DsrA helps bacteria adapt to environments by binding to multiple targets, but its association with the cell cycle remains unclear. Detection by flow cytometry, we first found that the knockout of dsrA promoted replication initiation, and corresponding overexpression of DsrA inhibited replication initiation in Escherichia coli. The absence of the chaperone protein Hfq, the DNA replication negative regulator protein Dps, or the transcription factor OxyR, was found to cause DsrA to no longer inhibit replication initiation. Excess DsrA promotes expression of the oxyR and dps gene, whereas ß-galactosidase activity assay showed that deleting oxyR limited the enhancement of dps promoter transcriptional activity by DsrA. OxyR is a known positive regulator of Dps. Our data suggests that the effect of DsrA on replication initiation requires Hfq and that the upregulation of Dps expression by OxyR in response to DsrA levels may be a potential regulatory pathway for the negative regulation of DNA replication initiation.

[Arsenic Methylation Efficiency Changes During Paddy Soil Drying and Its Key Influencing Factors Analysis].

The straight head disease of rice is one of the main problems limiting rice production. Arsenic (As) methylation in paddy soils is considered to be highly related to the occurrence of the straight head disease. As a typical field practice, rice fields are usually drained during the late tillering stage and the mid-late grain filling stage. Nevertheless, the key influencing factors on the As methylation efficiency during paddy soil drying remain unclear. In this study, an indoor cultivation experiment was set up to simulate the drying process of paddy soil. Two As-contaminated soils collected from Xingren (XR) in Guizhou province and Nandan (ND) in Guangxi province were used as test soils. Each soil was treated with the addition of rice straw (RS) and without rice straw (CK). With the drying of paddy soil (0, 24, 36, 48, and 60 h), the changes in soil Eh, pH, total organic carbon (TOC), and As chemical species in the porewater were determined. The abundance of the As methylation functional gene (arsM), sulfate-reducing bacteria (harboring dsrA, As methylation-related microorganism), and methanogens (harboring mcrA, As demethylation-related microorganism), as well as the diversity of arsM-harboring microorganisms, were also observed. The results showed that during the process of drying paddy soil, soil Eh changed from -300--200 mV under complete flooding to -150--50 mV after drying; however, the change in soil pH was not obvious. The concentrations of inorganic As (iAs) and dimethylarsenic (DMAs) in porewater significantly increased (P<0.05) with the drying process. Additionally, the concentration of DMAs in the RS treatment was prominently higher than that in CK. Compared with XR soil, the concentration of DMAs in ND soil was higher. As a function of soil drying time, the As methylation efficiency of XR soil (XR-CK and XR-RS) slightly increased but was not significant (P>0.05), whereas the As methylation efficiency of ND soil (ND-CK and ND-RS) increased significantly (P<0.05). After the drying time reached 60 h, the As methylation efficiency of ND-CK and ND-RS increased by 61.8% and 23.2%, respectively, compared with those at the early stage of drying (0 hours). The copy numbers of the arsM and dsrA genes greatly increased with the extension of drying time, whereas an opposite trend was observed for the copy number of the mcrA gene. Furthermore, the addition of straw obviously increased the gene abundance of whole bacteria and arsM-, dsrA-, and mcrA-harboring bacteria. Based on the multi-factor analysis of variance and the redundancy analysis, it was found that the test soil type, straw addition, drying time, and their interaction had a critical influence on the changes in As species, As methylation efficiency, and the gene abundance in soils. TOC, Eh, and the functional genes associated with As methylation were positively linked with the methylated As content in soil porewater but negatively correlated with that of iAs. According to the sequence of the arsM-harboring microbe, it was clearly demonstrated that a community shift of As-methylating microbe occurred with the soil drying. Here, the following conclusions were derived:① the drying process did not lower the As methylation efficiency in paddy soil. On the contrary, in this study, the As methylation efficiency, especially that for ND soil, remarkably improved. The addition of straw notably promoted the As methylation efficiency and the content of DMAs in porewater. ② An increasing tendency was observed for the abundance of microbes related to As methylation, whereas a reverse trend was indicated for microbes related to As demethylation. The community shift of arsM-harboring microbes might be the crucial reason for the improved As methylation efficiency during the soil drying. These observations contribute to a better understanding of the As methylation process during paddy soil drying and will shed light on the future mitigation of rice straight head disease in paddy soils.

Microbial Activities and Selection from Surface Ocean to Subseafloor on the Namibian Continental Shelf.

Oxygen minimum zones (OMZs) are hot spots for redox-sensitive nitrogen transformations fueled by sinking organic matter. In comparison, the regulating role of sulfur-cycling microbes in marine OMZs, their impact on carbon cycling in pelagic and benthic habitats, and activities below the seafloor remain poorly understood. Using 13C DNA stable isotope probing (SIP) and metatranscriptomics, we explored microbial guilds involved in sulfur and carbon cycling from the ocean surface to the subseafloor on the Namibian shelf. There was a clear separation in microbial community structure across the seawater-seafloor boundary, which coincided with a 100-fold-increased concentration of microbial biomass and unique gene expression profiles of the benthic communities. 13C-labeled 16S rRNA genes in SIP experiments revealed carbon-assimilating taxa and their distribution across the sediment-water interface. Most of the transcriptionally active taxa among water column communities that assimilated 13C from diatom exopolysaccharides (mostly Bacteroidetes, Actinobacteria, Alphaproteobacteria, and Planctomycetes) also assimilated 13C-bicarbonate under anoxic conditions in sediment incubations. Moreover, many transcriptionally active taxa from the seafloor community (mostly sulfate-reducing Deltaproteobacteria and sulfide-oxidizing Gammaproteobacteria) that assimilated 13C-bicarbonate under sediment anoxic conditions also assimilated 13C from diatom exopolysaccharides in the surface ocean and OMZ waters. Despite strong selection at the sediment-water interface, many taxa related to either planktonic or benthic communities were found to be present at low abundance and actively assimilating carbon under both sediment and water column conditions. In austral winter, mixing of shelf waters reduces stratification and suspends sediments from the seafloor into the water column, potentially spreading metabolically versatile microbes across niches. IMPORTANCE Microbial activities in oxygen minimum zones (OMZs) transform inorganic fixed nitrogen into greenhouse gases, impacting the Earth's climate and nutrient equilibrium. Coastal OMZs are predicted to expand with global change and increase carbon sedimentation to the seafloor. However, the role of sulfur-cycling microbes in assimilating carbon in marine OMZs and related seabed habitats remain poorly understood. Using 13C DNA stable isotope probing and metatranscriptomics, we explore microbial guilds involved in sulfur and carbon cycling from ocean surface to subseafloor on the Namibian shelf. Despite strong selection and differential activities across the sediment-water interface, many active taxa were identified in both planktonic and benthic communities, either fixing inorganic carbon or assimilating organic carbon from algal biomass. Our data show that many planktonic and benthic microbes linked to the sulfur cycle can cross redox boundaries when mixing of the shelf waters reduces stratification and suspends seafloor sediment particles into the water column.

DsrA Modulates Central Carbon Metabolism and Redox Balance by Directly Repressing pflB Expression in Salmonella Typhimurium.

Bacterial small RNAs (sRNAs) function as vital regulators in response to various environmental stresses by base pairing with target mRNAs. The sRNA DsrA, an important posttranscriptional regulator, has been reported to play a crucial role in defense against oxidative stress in Salmonella enterica serovar Typhimurium, but its regulatory mechanism remains unclear. The transcriptome sequencing (RNA-seq) results in this study showed that the genes involved in glycolysis, pyruvate metabolism, the tricarboxylic acid (TCA) cycle, and NADH-dependent respiration exhibited significantly different expression patterns between S. Typhimurium wild type (WT) and the dsrA deletion mutant (ΔdsrA strain) before and after H2O2 treatment. This indicated the importance of DsrA in regulating central carbon metabolism (CCM) and NAD(H) homeostasis of S. Typhimurium. To reveal the direct target of DsrA action, fusion proteins of six candidate genes (acnA, srlE, tdcB, nuoH, katG, and pflB) with green fluorescent protein (GFP) were constructed, and the fluorescence analysis showed that the expression of pflB encoding pyruvate-formate lyase was repressed by DsrA. Furthermore, site-directed mutagenesis and RNase E-dependent experiments showed that the direct base pairing of DsrA with pflB mRNA could recruit RNase E to degrade pflB mRNA and reduce the stability of pflB mRNA. In addition, the NAD+/NADH ratio in WT-ppflB-pdsrA was significantly lower than that in WT-ppflB, suggesting that the repression of pflB by DsrA could contribute greatly to the redox balance in S. Typhimurium. Taken together, a novel target of DsrA was identified, and its regulatory role was clarified, which demonstrated that DsrA could modulate CCM and redox balance by directly repressing pflB expression in S. Typhimurium. IMPORTANCE Small RNA DsrA plays an important role in defending against oxidative stress in bacteria. In this study, we identified a novel target (pflB, encoding pyruvate-formate lyase) of DsrA and demonstrated its potential regulatory mechanism in S. Typhimurium by transcriptome analysis. In silico prediction revealed a direct base pairing between DsrA and pflB mRNA, which was confirmed in site-directed mutagenesis experiments. The interaction of DsrA-pflB mRNA could greatly contribute to the regulation of central carbon metabolism and intracellular redox balance in S. Typhimurium. These findings provided a better understanding of the critical roles of small RNA in central metabolism and stress responses in foodborne pathogens.

The Amyloid Region of Hfq Riboregulator Promotes DsrA:rpoS RNAs Annealing.

Hfq is a bacterial RNA chaperone which promotes the pairing of small noncoding RNAs to target mRNAs, allowing post-transcriptional regulation. This RNA annealing activity has been attributed for years to the N-terminal region of the protein that forms a toroidal structure with a typical Sm-fold. Nevertheless, many Hfqs, including that of Escherichia coli, have a C-terminal region with unclear functions. Here we use a biophysical approach, Synchrotron Radiation Circular Dichroism (SRCD), to probe the interaction of the E. coli Hfq C-terminal amyloid region with RNA and its effect on RNA annealing. This C-terminal region of Hfq, which has been described to be dispensable for sRNA:mRNA annealing, has an unexpected and significant effect on this activity. The functional consequences of this novel property of the amyloid region of Hfq in relation to physiological stress are discussed.

Effectiveness of Metformin for Weight Reduction in Children and Adolescents Treated with Mixed Dopamine and Serotonin Receptor Antagonists: A Naturalistic Cohort Study.

Objectives: Mixed dopamine and serotonin receptor antagonists (DSRAs) are associated with significant weight gain and its complications. Our aim was to evaluate the effectiveness of metformin in reducing body mass index (BMI) and metabolic parameters in children treated with DSRAs. Methods: We report a naturalistic study of 49 children and adolescents (mean age 14.9 ± 3.7 years), with BMI >85 percentile for age, treated with DSRAs during 2018-2020 in a child psychiatry clinic. Clinical data, anthropometric measurements, and laboratory tests were compared between those who were (study group, n = 31) and were not (control group, n = 18) treated with metformin. Results: The mean study duration was 9.7 ± 5.9 months. The BMI standard deviation scores (BMI-SDS) of the study group declined significantly (from 2.08 ± 0.40 to 1.81 ± 0.54, p < 0.001), while the BMI-SDS of the control group did not change (from 2.03 ± 0.45 to 2.04 ± 0.47, p = 0.838). In the study group, the decline in the delta BMI-SDS/month was more robust among those with good than poor adherence to metformin (-0.047 ± 0.039 vs. -0.004 ± 0.017, p = 0.003). The decrease in BMI-SDS was greater for patients treated with risperidone and clothiapine than with other DSRAs. Fasting insulin and insulin resistance index (homeostasis model assessment of insulin resistance [HOMA-IR]) declined in the study group (from 25.4 ± 13.8 to 19.9 ± 10.7, p = 0.033 and from 5.4 ± 2.7 to 4.2 ± 2.1, p = 0.028, respectively). Conclusions: Metformin treatment was associated with significant decreases in BMI, fasting insulin, and HOMA-IR. The effect of metformin seems to be dependent on adherence and type of DSRAs.

Engineering of the Small Noncoding RNA (sRNA) DsrA Together with the sRNA Chaperone Hfq Enhances the Acid Tolerance of Escherichia coli.

Acid tolerance of microorganisms is a desirable phenotype for many industrial fermentation applications. In Escherichia coli, the stress response sigma factor RpoS is a promising target for engineering acid-tolerant phenotypes. However, the simple overexpression of RpoS alone is insufficient to confer these phenotypes. In this study, we show that the simultaneous overexpression of the noncoding small RNA (sRNA) DsrA and the sRNA chaperone Hfq, which act as RpoS activators, significantly increased acid tolerance in terms of cell growth under modest acidic pH, as well as cell survival upon extreme acid shock. Directed evolution of the DsrA-Hfq module further improved the acid tolerance, with the best mutants showing a 51 to 72% increase in growth performance at pH 4.5 compared with the starting strain, MG1655. Further analyses found that the improved acid tolerance of these DsrA-Hfq strains coincided with activation of genes associated with proton-consuming acid resistance system 2 (AR2), protein chaperone HdeB, and reactive oxygen species (ROS) removal in the exponential phase. This study illustrated that the fine-tuning of sRNAs and their chaperones can be a novel strategy for improving the acid tolerance of E. coliIMPORTANCE Many of the traditional studies on bacterial acid tolerance generally focused on improving cell survival under extreme-pH conditions, but cell growth under less harsh acidic conditions is more relevant to industrial applications. Under normal conditions, the general stress response sigma factor RpoS is maintained at low levels in the growth phase through a number of mechanisms. This study showed that RpoS can be activated prior to the stationary phase via engineering its activators, the sRNA DsrA and the sRNA chaperone Hfq, resulting in significantly improved cell growth at modest acidic pH. This work suggests that the sigma factors and likely other transcription factors can be retuned or retimed by manipulating the respective regulatory sRNAs along with the sufficient supply of the respective sRNA chaperones (i.e., Hfq). This provides a novel avenue for strain engineering of microbes.

Application of Synchrotron Radiation Circular Dichroism for RNA Structural Analysis.

Circular dichroism (CD) spectroscopy is a fast and simple technique providing important information about the conformation of nucleic acids, proteins, sugars, lipids, and their interactions between each other. This electronic absorption spectroscopy method is extremely sensitive to any change in molecular structure containing asymmetric molecules. While numerous reviews describe how to analyze deoxyribonucleic acid (DNA) structures using CD, analyses of ribonucleic acids (RNAs) are scarce. Nevertheless, RNAs are important molecules involved in a multitude of roles in the cell. In this chapter, we present applications of synchrotron radiation circular dichroism (SRCD) extending the spectral range down to 170 nm, improving structural analysis of RNA, including the analysis of helical parameters and alternative structures found in RNA. The effects of temperature to measure thermodynamic parameters and analyze ribonucleoprotein complexes will also be presented.

Mechanisms for Hfq-Independent Activation of rpoS by DsrA, a Small RNA, in Escherichia coli.

Many small RNAs (sRNAs) regulate gene expression by base pairing to their target messenger RNAs (mRNAs) with the help of Hfq in Escherichia coli. The sRNA DsrA activates translation of the rpoS mRNA in an Hfq-dependent manner, but this activation ability was found to partially bypass Hfq when DsrA is overproduced. The precise mechanism by which DsrA bypasses Hfq is unknown. In this study, we constructed strains lacking all three rpoS-activating sRNAs (i.e., ArcZ, DsrA, and RprA) in hfq+ and Hfq- backgrounds, and then artificially regulated the cellular DsrA concentration in these strains by controlling its ectopic expression. We then examined how the expression level of rpoS was altered by a change in the concentration of DsrA. We found that the translation and stability of the rpoS mRNA are both enhanced by physiological concentrations of DsrA regardless of Hfq, but that depletion of Hfq causes a rapid degradation of DsrA and thereby decreases rpoS mRNA stability. These results suggest that the observed Hfq dependency of DsrA-mediated rpoS activation mainly results from the destabilization of DsrA in the absence of Hfq, and that DsrA itself contributes to the translational activation and stability of the rpoS mRNA in an Hfq-independent manner.

Comparison of multi-criteria analysis methodologies for the prioritization of arsenic-contaminated sites in the southwest of China.

The issue of contaminated sites has been highlighted as an immediate priority in the 13th Five-Year Plan of China. Identification and prioritization of contaminated sites are of key importance for proposing effective strategies for the regional management of contaminated sites. In this study, three advanced multi-attribute methodologies, the risk-based priority methodology, the regional risk assessment methodology, and the dominance-based rough set approach (DRSA), were comparatively employed to screen contaminated sites in, Guangxi, Southwest of China. The results of the three prioritizations show that the highest ranking site identified by the three methods had great agreement. In regard to the screening attributers, while the risk-based prioritization methodology and regional risk assessment methodology allowed a high discrimination in the screening of contaminated sites associated with different attributes, such as farmland, residential areas, contaminant level, number of people, area, storage quality, site service life, and surrounding communities, the DRSA allowed the identification of contamination strength (CS) and contamination potential (CP).

A Modular Genetic System for High-Throughput Profiling and Engineering of Multi-Target Small RNAs.

RNA biology and RNA engineering are subjects of growing interest due to recent advances in our understanding of the diverse cellular functions of RNAs, including their roles as genetic regulators. The noncoding small RNAs (sRNAs) of bacteria are a fundamental basis of regulatory control that can regulate gene expression via antisense base-pairing to one or more target mRNAs. The sRNAs can be customized to generate a range of mRNA translation rates and stabilities. The sRNAs can be applied as a platform for metabolic engineering, to control expression of genes of interest by following relatively straightforward design rules (Kushwaha et al., ACS Synth Biol 5:795-809, 2016). However, the ab initio design of functional sRNAs to precise specifications of gene control is not yet possible. Consequently, there is a need for tools to rapidly profile uncharacterized sRNAs in vivo, to screen sRNAs against "new/novel" targets, and (in the case of metabolic engineering) to develop engineered sRNAs for regulatory function against multiple desired mRNA targets. To address this unmet need, we previously constructed a modular genetic system for assaying sRNA activity in vivo against specifiable mRNA sequences, using microtiter plate assays for high-throughput productivity. This sRNA design platform consists of three modular plasmids: one plasmid contains an inducible sRNA and the RNA chaperone Hfq; the second contains an inducible fluorescent reporter protein and a LacY mutant transporter protein for inducer molecules; and the third plasmid contains a second inducible fluorescent reporter protein. The second reporter gene makes it possible to screen for sRNA regulators that have activity against multiple mRNAs. We describe the protocol for engineering sRNAs with novel regulatory activity using this system. This sRNA prototyping regimen could also be employed for validating predicted mRNA targets of uncharacterized, naturally occurring sRNAs or for testing hypotheses about the predicted roles of genes, including essential genes, in cellular metabolism and other processes, by using customized antisense sRNAs to knock down or tune down gene expression.

Survey of sulfur-oxidizing bacterial community in the Pearl River water using soxB, sqr, and dsrA as molecular biomarkers.

In this study, we surveyed the abundance and diversity of three sulfur oxidation genes (sqr, soxB, and dsrA) using quantitative assays and Miseq high-throughput sequencing. The quantitative assays revealed that soxB is more abundant than sqr and dsrA and is the main contributor to sulfur oxidation. In the diversity analysis, the SOB community mainly comprised the classes Nitrospira, Alphaproteobacteria, Betaproteobacteria, and Gammaproteobacteria. The genera Gallionella, Hydrogenophaga, Limnohabitans, Methylomonas, Nitrospira, Rhodoferax, and Sulfuritalea were abundant in the communities for sqr; Dechloromonas, Limnohabitans, Paracoccus, Sulfuritalea, Sulfitobacter, and Thiobacillus were abundant in communities for soxB; Sulfuritalea, Sulfurisoma, and Thiobacillus were abundant in communities for dsrA. This study presented a high diversity of SOB species and functional sulfur-oxidizing genes in Pearl River via high-throughput sequencing, suggesting that the aquatic ecosystem has great potential to scavenge the sulfur pollutants by itself.

Diversity and Composition of Sulfate-Reducing Microbial Communities Based on Genomic DNA and RNA Transcription in Production Water of High Temperature and Corrosive Oil Reservoir.

Deep subsurface petroleum reservoir ecosystems harbor a high diversity of microorganisms, and microbial influenced corrosion is a major problem for the petroleum industry. Here, we used high-throughput sequencing to explore the microbial communities based on genomic 16S rDNA and metabolically active 16S rRNA analyses of production water samples with different extents of corrosion from a high-temperature oil reservoir. Results showed that Desulfotignum and Roseovarius were the most abundant genera in both genomic and active bacterial communities of all the samples. Both genomic and active archaeal communities were mainly composed of Archaeoglobus and Methanolobus. Within both bacteria and archaea, the active and genomic communities were compositionally distinct from one another across the different oil wells (bacteria p = 0.002; archaea p = 0.01). In addition, the sulfate-reducing microorganisms (SRMs) were specifically assessed by Sanger sequencing of functional genes aprA and dsrA encoding the enzymes adenosine-5'-phosphosulfate reductase and dissimilatory sulfite reductase, respectively. Functional gene analysis indicated that potentially active Archaeoglobus, Desulfotignum, Desulfovibrio, and Thermodesulforhabdus were frequently detected, with Archaeoglobus as the most abundant and active sulfate-reducing group. Canonical correspondence analysis revealed that the SRM communities in petroleum reservoir system were closely related to pH of the production water and sulfate concentration. This study highlights the importance of distinguishing the metabolically active microorganisms from the genomic community and extends our knowledge on the active SRM communities in corrosive petroleum reservoirs.

Corrigendum: A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

[This corrects the article on p. 731 in vol. 6, PMID: 26236305.].

Immunization with the Haemophilus ducreyi trimeric autotransporter adhesin DsrA with alum, CpG or imiquimod generates a persistent humoral immune response that recognizes the bacterial surface.

The Ducreyi serum resistance A (DsrA) protein of Haemophilus ducreyi belongs to a large family of multifunctional outer membrane proteins termed trimeric autotransporter adhesins responsible for resistance to the bactericidal activity of human complement (serum resistance), agglutination and adhesion. The ability of DsrA to confer serum resistance and bind extracellular matrix proteins lies in its N-terminal passenger domain. We have previously reported that immunization with a recombinant form of the passenger domain of DsrA, rNT-DsrA, in complete/incomplete Freund's adjuvant, protects against a homologous challenge in swine. We present herein the results of an immunogenicity study in mice aimed at investigating the persistence, type of immune response, and the effect of immunization route and adjuvants on surrogates of protection. Our results indicate that a 20 µg dose of rNT-DsrA administered with alum elicited antisera with comparable bacterial surface reactivity to that obtained with complete/incomplete Freund's adjuvant. At that dose, high titers and bacterial surface reactivity persisted for 211 days after the first immunization. Administration of rNT-DsrA with CpG or imiquimod as adjuvants elicited a humoral response with similar quantity and quality of antibodies (Abs) as seen with Freund's adjuvant. Furthermore, intramuscular administration of rNT-DsrA elicited high-titer Abs with significantly higher reactivity to the bacterial surface than those obtained with subcutaneous immunization. All rNT-DsrA/adjuvant combinations tested, save CpG, elicited a Th2-type response. Taken together, these findings show that a 20 µg dose of rNT-DsrA administered with the adjuvants alum, CpG or imiquimod elicits high-quality Abs with reactivity to the bacterial surface that could protect against an H. ducreyi infection.

The spectrum of activity of the small RNA DsrA: not so narrow after all.

For a long time, the small regulatory RNA DsrA has been considered as a regulator with a narrow spectrum of action due to its restricted targetome. Since the first reports on DsrA characterization, only two targets of DsrA have been described: rpoS and hns mRNAs, encoding the sigma factor σS and the nucleoid-associated protein H-NS, respectively. Recently, the scope of DsrA targetome has been expanded by the characterization of two negatively regulated mRNAs, mreB and rbsD, involved in cell wall biosynthesis and ribose metabolism, respectively. In this review, we summarize new insights in DsrA-mediated regulation and emphasize the versatility of DsrA modes of action.

A flexible and economical barcoding approach for highly multiplexed amplicon sequencing of diverse target genes.

High throughput sequencing of phylogenetic and functional gene amplicons provides tremendous insight into the structure and functional potential of complex microbial communities. Here, we introduce a highly adaptable and economical PCR approach to barcoding and pooling libraries of numerous target genes. In this approach, we replace gene- and sequencing platform-specific fusion primers with general, interchangeable barcoding primers, enabling nearly limitless customized barcode-primer combinations. Compared to barcoding with long fusion primers, our multiple-target gene approach is more economical because it overall requires lower number of primers and is based on short primers with generally lower synthesis and purification costs. To highlight our approach, we pooled over 900 different small-subunit rRNA and functional gene amplicon libraries obtained from various environmental or host-associated microbial community samples into a single, paired-end Illumina MiSeq run. Although the amplicon regions ranged in size from approximately 290 to 720 bp, we found no significant systematic sequencing bias related to amplicon length or gene target. Our results indicate that this flexible multiplexing approach produces large, diverse, and high quality sets of amplicon sequence data for modern studies in microbial ecology.