RADReef: A global Holocene Reef Rate of Accretion Dataset.

Reef cores are a powerful tool for investigating temporal changes in reef communities. Radiometric dating facilitates the determination of vertical accretion rates, which has allowed for examination of local-regional controlling factors, such as subsidence and sea level changes. Coral reefs must grow at sufficient rates to keep up with sea level rise, or risk 'drowning.' As sea level is expected to rise significantly in the next 100 years and beyond, it is important to understand whether reefs will be able to survive. Historical records of reef accretion rates extracted from cores provide valuable insights into extrinsic controlling factors of reef growth and are instrumental in helping predict if future reefs can accrete at rates needed to overcome predicted sea level changes. While extensive research exists at local and regional scales, limited attention has been given to identifying global patterns and drivers. To address this, we present "RADReef": A global dataset of dated Holocene reef cores. RADReef serves as a foundation for further research on past, present and future reef accretion.

Ensifer canadensis sp. nov. strain T173T isolated from Melilotus albus (sweet clover) in Canada possesses recombinant plasmid pT173b harbouring symbiosis and type IV secretion system genes apparently acquired from Ensifer medicae.

A bacterial strain, designated T173T, was previously isolated from a root-nodule of a Melilotus albus plant growing in Canada and identified as a novel Ensifer lineage that shared a clade with the non-symbiotic species, Ensifer adhaerens. Strain T173T was also previously found to harbour a symbiosis plasmid and to elicit root-nodules on Medicago and Melilotus species but not fix nitrogen. Here we present data for the genomic and taxonomic description of strain T173T. Phylogenetic analyses including the analysis of whole genome sequences and multiple locus sequence analysis (MLSA) of 53 concatenated ribosome protein subunit (rps) gene sequences confirmed placement of strain T173T in a highly supported lineage distinct from named Ensifer species with E. morelensis Lc04T as the closest relative. The highest digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values of genome sequences of strain T173T compared with closest relatives (35.7 and 87.9%, respectively) are well below the respective threshold values of 70% and 95-96% for bacterial species circumscription. The genome of strain T173T has a size of 8,094,229 bp with a DNA G + C content of 61.0 mol%. Six replicons were detected: a chromosome (4,051,102 bp) and five plasmids harbouring plasmid replication and segregation (repABC) genes. These plasmids were also found to possess five apparent conjugation systems based on analysis of TraA (relaxase), TrbE/VirB4 (part of the Type IV secretion system (T4SS)) and TraG/VirD4 (coupling protein). Ribosomal RNA operons encoding 16S, 23S, and 5S rRNAs that are usually restricted to bacterial chromosomes were detected on plasmids pT173d and pT173e (946,878 and 1,913,930 bp, respectively) as well as on the chromosome of strain T173T. Moreover, plasmid pT173b (204,278 bp) was found to harbour T4SS and symbiosis genes, including nodulation (nod, noe, nol) and nitrogen fixation (nif, fix) genes that were apparently acquired from E. medicae by horizontal transfer. Data for morphological, physiological and symbiotic characteristics complement the sequence-based characterization of strain T173T. The data presented support the description of a new species for which the name Ensifer canadensis sp. nov. is proposed with strain T173T (= LMG 32374T = HAMBI 3766T) as the species type strain.

Examination of the horizontal gene transfer dynamics of an integrative and conjugative element encoding multidrug resistance in Histophilus somni.

Integrative and conjugative elements (ICEs) are self-transferable mobile genetic elements that play a significant role in disseminating antimicrobial resistance between bacteria via horizontal gene transfer. A recently identified ICE in a clinical isolate of Histophilus somni (ICEHs02) is 72 914 base pairs in length and harbours seven predicted antimicrobial resistance genes conferring resistance to tetracycline (tetR-tet(H)), florfenicol (floR), sulfonamide (Sul2), aminoglycosides (APH(3″)-Ib, APH(6)-Id, APH(3')-Ia), and copper (mco). This study investigated ICEHs02 host range, assessed effects of antimicrobial stressors on transfer frequency, and examined effects of ICEHs02 acquisition on hosts. Conjugation assays examined transfer frequency of ICEHs02 to H. somni and Pasteurella multocida strains. Polymerase chain reaction assays confirmed the presence of a circular intermediate, ICE-associated core genes, and cargo genes in recipient strains. Susceptibility testing examined ICEHs02-associated resistance phenotypes in recipient strains. Tetracycline and ciprofloxacin induction significantly increased the transfer rates of ICEHs02 in vitro. The copy numbers of the circular intermediate of ICEHs02 per chromosome exhibited significant increases of ∼37-fold after tetracycline exposure and ∼4-fold after ciprofloxacin treatment. The acquisition of ICEHs02 reduced the relative fitness of H. somni transconjugants (TG) by 28% (w = 0.72 ± 0.04) and the relative fitness of P. multocida TG was decreased by 15% (w = 0.85 ± 0.01).

Why are rhizobial symbiosis genes mobile?

Rhizobia are one of the most important and best studied groups of bacterial symbionts. They are defined by their ability to establish nitrogen-fixing intracellular infections within plant hosts. One surprising feature of this symbiosis is that the bacterial genes required for this complex trait are not fixed within the chromosome, but are encoded on mobile genetic elements (MGEs), namely plasmids or integrative and conjugative elements. Evidence suggests that many of these elements are actively mobilizing within rhizobial populations, suggesting that regular symbiosis gene transfer is part of the ecology of rhizobial symbionts. At first glance, this is counterintuitive. The symbiosis trait is highly complex, multipartite and tightly coevolved with the legume hosts, while transfer of genes can be costly and disrupt coadaptation between the chromosome and the symbiosis genes. However, horizontal gene transfer is a process driven not only by the interests of the host bacterium, but also, and perhaps predominantly, by the interests of the MGEs that facilitate it. Thus understanding the role of horizontal gene transfer in the rhizobium-legume symbiosis requires a 'mobile genetic element's-eye view' on the ecology and evolution of this important symbiosis. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element.

Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer.

Influence of the Chelation Process on the Stability of Organic Trace Mineral Supplements Used in Animal Nutrition.

The effect of the chelation process on the pH-dependent stability of organic trace minerals (OTMs) used as mineral supplements in animal nutrition was assessed using analytical techniques such as potentiometry, Fourier Transform Infrared Spectroscopy (FTIRS) and amino acid profiling. The aim was to understand the influence and relative importance of the manufacturing conditions on mineral chelation and the subsequent pH stability of OTMs. A selection of OTMs were assessed over a wide pH range to account for the typical environmental changes encountered in the gastrointestinal (GI) tract. In the case of proteinate type products, the potentiometric assessment of free mineral concentration indicated that the hydrolysis procedure used to generate the chelating peptides was the major influencer of the pH stability of the products. Many products are available under the umbrella term "OTMs", including amino acid complexes, amino acid chelates, polysaccharide complexes and proteinates. Significant differences in the pH-dependent stability of a range of commercially available OTMs were observed.

Bacterial cyclic diguanylate signaling networks sense temperature.

Many bacteria use the second messenger cyclic diguanylate (c-di-GMP) to control motility, biofilm production and virulence. Here, we identify a thermosensory diguanylate cyclase (TdcA) that modulates temperature-dependent motility, biofilm development and virulence in the opportunistic pathogen Pseudomonas aeruginosa. TdcA synthesizes c-di-GMP with catalytic rates that increase more than a hundred-fold over a ten-degree Celsius change. Analyses using protein chimeras indicate that heat-sensing is mediated by a thermosensitive Per-Arnt-SIM (PAS) domain. TdcA homologs are widespread in sequence databases, and a distantly related, heterologously expressed homolog from the Betaproteobacteria order Gallionellales also displayed thermosensitive diguanylate cyclase activity. We propose, therefore, that thermotransduction is a conserved function of c-di-GMP signaling networks, and that thermosensitive catalysis of a second messenger constitutes a mechanism for thermal sensing in bacteria.

Defining the Rhizobium leguminosarum Species Complex.

Bacteria currently included in Rhizobium leguminosarum are too diverse to be considered a single species, so we can refer to this as a species complex (the Rlc). We have found 429 publicly available genome sequences that fall within the Rlc and these show that the Rlc is a distinct entity, well separated from other species in the genus. Its sister taxon is R. anhuiense. We constructed a phylogeny based on concatenated sequences of 120 universal (core) genes, and calculated pairwise average nucleotide identity (ANI) between all genomes. From these analyses, we concluded that the Rlc includes 18 distinct genospecies, plus 7 unique strains that are not placed in these genospecies. Each genospecies is separated by a distinct gap in ANI values, usually at approximately 96% ANI, implying that it is a 'natural' unit. Five of the genospecies include the type strains of named species: R. laguerreae, R. sophorae, R. ruizarguesonis, "R. indicum" and R. leguminosarum itself. The 16S ribosomal RNA sequence is remarkably diverse within the Rlc, but does not distinguish the genospecies. Partial sequences of housekeeping genes, which have frequently been used to characterize isolate collections, can mostly be assigned unambiguously to a genospecies, but alleles within a genospecies do not always form a clade, so single genes are not a reliable guide to the true phylogeny of the strains. We conclude that access to a large number of genome sequences is a powerful tool for characterizing the diversity of bacteria, and that taxonomic conclusions should be based on all available genome sequences, not just those of type strains.

Molecular Biology in the Improvement of Biological Nitrogen Fixation by Rhizobia and Extending the Scope to Cereals.

The contribution of biological nitrogen fixation to the total N requirement of food and feed crops diminished in importance with the advent of synthetic N fertilizers, which fueled the "green revolution". Despite being environmentally unfriendly, the synthetic versions gained prominence primarily due to their low cost, and the fact that most important staple crops never evolved symbiotic associations with bacteria. In the recent past, advances in our knowledge of symbiosis and nitrogen fixation and the development and application of recombinant DNA technology have created opportunities that could help increase the share of symbiotically-driven nitrogen in global consumption. With the availability of molecular biology tools, rapid improvements in symbiotic characteristics of rhizobial strains became possible. Further, the technology allowed probing the possibility of establishing a symbiotic dialogue between rhizobia and cereals. Because the evolutionary process did not forge a symbiotic relationship with the latter, the potential of molecular manipulations has been tested to incorporate a functional mechanism of nitrogen reduction independent of microbes. In this review, we discuss various strategies applied to improve rhizobial strains for higher nitrogen fixation efficiency, more competitiveness and enhanced fitness under unfavorable environments. The challenges and progress made towards nitrogen self-sufficiency of cereals are also reviewed. An approach to integrate the genetically modified elite rhizobia strains in crop production systems is highlighted.

A bacteriophage infecting Mesorhizobium species has a prolate capsid and shows similarities to a family of Caulobacter crescentus phages.

Mesorhizobium phage vB_MloS_Cp1R7A-A1 was isolated from soil planted with chickpea in Saskatchewan. It is dissimilar in sequence and morphology to previously described rhizobiophages. It is a B3 morphotype virus with a distinct prolate capsid and belongs to the tailed phage family Siphoviridae. Its genome has a GC content of 60.3% and 238 predicted genes. Putative functions were predicted for 57 genes, which include 27 tRNA genes with anticodons corresponding to 18 amino acids. This represents the highest number of tRNA genes reported yet in a rhizobiophage. The gene arrangement shows a partially modular organization. Most of the structural genes are found in one module, whereas tRNA genes are in another. Genes for replication, recombination, and nucleotide metabolism form the third module. The arrangement of the replication module resembles the replication module of Enterobacteria phage T5, raising the possibility that it uses a recombination-based replication mechanism, but there is also a suggestion that a T7-like replication mechanism could be used. Phage termini appear to be long direct repeats of just over 12 kb in length. Phylogenetic analysis revealed that Cp1R7A-A1 is more closely related to PhiCbK-like Caulobacter phages and other B3 morphotype phages than to other rhizobiophages sequenced thus far.

Carbon Catabolite Repression Governs Diverse Physiological Processes and Development in Aspergillus nidulans.

Carbon catabolite repression (CCR) is a common phenomenon of microorganisms that enable efficient utilization of carbon nutrients, critical for the fitness of microorganisms in the wild and for pathogenic species to cause infection. In most filamentous fungal species, the conserved transcription factor CreA/Cre1 mediates CCR. Previous studies demonstrated a primary function for CreA/Cre1 in carbon metabolism; however, the phenotype of creA/cre1 mutants indicated broader roles. The global function and regulatory mechanism of this wide-domain transcription factor has remained elusive. Here, we applied two powerful genomics methods (transcriptome sequencing and chromatin immunoprecipitation sequencing) to delineate the direct and indirect roles of Aspergillus nidulans CreA across diverse physiological processes, including secondary metabolism, iron homeostasis, oxidative stress response, development, N-glycan biosynthesis, unfolded protein response, and nutrient and ion transport. The results indicate intricate connections between the regulation of carbon metabolism and diverse cellular functions. Moreover, our work also provides key mechanistic insights into CreA regulation and identifies CreA as a master regulator controlling many transcription factors of different regulatory networks. The discoveries for this highly conserved transcriptional regulator in a model fungus have important implications for CCR in related pathogenic and industrial species. IMPORTANCE The ability to scavenge and use a wide range of nutrients for growth is crucial for microorganisms' survival in the wild. Carbon catabolite repression (CCR) is a transcriptional regulatory phenomenon of both bacteria and fungi to coordinate the expression of genes required for preferential utilization of carbon sources. Since carbon metabolism is essential for growth, CCR is central to the fitness of microorganisms. In filamentous fungi, CCR is mediated by the conserved transcription factor CreA/Cre1, whose function in carbon metabolism has been well established. However, the global roles and regulatory mechanism of CreA/Cre1 are poorly defined. This study uncovers the direct and indirect functions of CreA in the model organism Aspergillus nidulans over diverse physiological processes and development and provides mechanistic insights into how CreA controls different regulatory networks. The work also reveals an interesting functional divergence between filamentous fungal and yeast CreA/Cre1 orthologues.

Genome Sequences of vB_RleM_RL38JI and vB_RleM_RL2RES, Two Virulent Rhizobium leguminosarum Transducing Phages.

Phages vB_RleM_RL38JI and vB_RleM_RL2RES are known to mediate generalized transduction in Rhizobium leguminosarum The RL38JI genome consists of 158,577 nucleotides and 270 predicted genes, whereas RL2RES has a 156,878-bp genome with 262 predicted genes. The two genomes are similar, with 82.88% nucleotide identity to each other.

Defining the requirements for the conjugative transfer of Rhizobium leguminosarum plasmid pRleVF39b.

Rhizobium leguminosarum strain VF39 contains a plasmid, pRleVF39b, which encodes a distinctive type of conjugation system (rhizobial type IVa) that is relatively widespread among rhizobial genomes. The cluster of genes encoding the transfer functions lacks orthologs to genes such as traCD, traF and traB, but contains 15 conserved genes of unknown function. We determined the importance of these genes in conjugation by constructing marked and unmarked mutations in each gene, and established that six genes, now designated trcA-F, played a significant role in plasmid transfer. Like the relaxase gene, traA, and the genes encoding the MPF system (trb genes), five of these genes, located in two divergently transcribed operons, are regulated by the Xre family repressor TrbR. The other gene, trcF encodes a protein with similarity to histidinol phosphatases, and its role in conjugation is unclear, but mutations in trcF are severely impaired for conjugation. TrcF does not play a role in regulation of other conjugation genes.

Delineation of the integrase-attachment and origin-of-transfer regions of the symbiosis island ICEMlSymR7A.

Integrative and conjugative elements (ICEs) are chromosomally-integrated mobile genetic elements that excise from their host chromosome and transfer to other bacteria via conjugation. ICEMlSymR7A is the prototypical member of a large family of "symbiosis ICEs" which confer upon their hosts the ability to form a nitrogen-fixing symbiosis with a variety of legume species. Mesorhizobial symbiosis ICEs carry a common core of mobilisation genes required for integration, excision and conjugative transfer. IntS of ICEMlSymR7A enables recombination between the ICEMlSymR7A attachment site attP and the 3' end of the phe-tRNA gene. Here we identified putative IntS attP arm (P) sites within the attP region and demonstrated that the outermost P1 and P5 sites demarcated the minimal region for efficient IntS-mediated integration. We also identified the ICEMlSymR7A origin-of-transfer (oriT) site directly upstream of the relaxase-gene rlxS. The ICEMlSymR7A conjugation system mobilised a plasmid carrying the cloned oriT to Escherichia coli in an rlxS-dependent manner. Surprisingly, an in-frame, markerless deletion mutation in the ICEMlSymR7A recombination directionality factor (excisionase) gene rdfS, but not a mutation in intS, abolished mobilisation, suggesting the rdfS deletion tentatively has downstream effects on conjugation or its regulation. In summary, this work defines two critical cis-acting regions required for excision and transfer of ICEMlSymR7A and related ICEs.

Quantitative assessment of copper proteinates used as animal feed additives using ATR-FTIR spectroscopy and powder X-ray diffraction (PXRD) analysis.

The aim of the present work was to establish a reliable analytical method to determine the degree of complexation in commercial metal proteinates used as feed additives in the solid state. Two complementary techniques were developed. Firstly, a quantitative attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopic method investigated modifications in vibrational absorption bands of the ligand on complex formation. Secondly, a powder X-ray diffraction (PXRD) method to quantify the amount of crystalline material in the proteinate product was developed. These methods were developed in tandem and cross-validated with each other. Multivariate analysis (MVA) was used to develop validated calibration and prediction models. The FTIR and PXRD calibrations showed excellent linearity (R2 > 0.99). The diagnostic model parameters showed that the FTIR and PXRD methods were robust with a root mean square error of calibration RMSEC ≤3.39% and a root mean square error of prediction RMSEP ≤7.17% respectively. Comparative statistics show excellent agreement between the MVA packages assessed and between the FTIR and PXRD methods. The methods can be used to determine the degree of complexation in complexes of both protein hydrolysates and pure amino acids.

Combined frequency domain photoacoustic and ultrasound imaging for intravascular applications.

Intravascular photoacoustic (IVPA) imaging has the potential to characterize lipid-rich structures based on the optical absorption contrast of tissues. In this study, we explore frequency domain photoacoustics (FDPA) for intravascular applications. The system employed an intensity-modulated continuous wave (CW) laser diode, delivering 1W over an intensity modulated chirp frequency of 4-12MHz. We demonstrated the feasibility of this approach on an agar vessel phantom with graphite and lipid targets, imaged using a planar acoustic transducer co-aligned with an optical fibre, allowing for the co-registration of IVUS and FDPA images. A frequency domain correlation method was used for signal processing and image reconstruction. The graphite and lipid targets show an increase in FDPA signal as compared to the background of 21dB and 16dB, respectively. Use of compact CW laser diodes may provide a valuable alternative for the development of photoacoustic intravascular devices instead of pulsed laser systems.

Chemotaxis signaling systems in model beneficial plant-bacteria associations.

Beneficial plant-microbe associations play critical roles in plant health. Bacterial chemotaxis provides a competitive advantage to motile flagellated bacteria in colonization of plant root surfaces, which is a prerequisite for the establishment of beneficial associations. Chemotaxis signaling enables motile soil bacteria to sense and respond to gradients of chemical compounds released by plant roots. This process allows bacteria to actively swim towards plant roots and is thus critical for competitive root surface colonization. The complete genome sequences of several plant-associated bacterial species indicate the presence of multiple chemotaxis systems and a large number of chemoreceptors. Further, most soil bacteria are motile and capable of chemotaxis, and chemotaxis-encoding genes are enriched in the bacteria found in the rhizosphere compared to the bulk soil. This review compares the architecture and diversity of chemotaxis signaling systems in model beneficial plant-associated bacteria and discusses their relevance to the rhizosphere lifestyle. While it is unclear how controlling chemotaxis via multiple parallel chemotaxis systems provides a competitive advantage to certain bacterial species, the presence of a larger number of chemoreceptors is likely to contribute to the ability of motile bacteria to survive in the soil and to compete for root surface colonization.

Design of a Subtarsal Ultrasonic Transducer for Mild Hyperthermia Treatment of Dry Eye Disease.

Dry eye disease is a disorder of the ocular surface that causes pain and low vision in a significant portion of the adult population. A common cause is obstructive Meibomian gland dysfunction, whereby the Meibomian glands secrete abnormal meibum with a melting point elevated by 3°C-4°C; hence, hyperthermia is the typical treatment. A design is proposed for an ultrasound hyperthermia device made of a transducer contained inside a contact lens with an internal air gap. The transducer heats the posterior of the tarsus, and the air gap provides an air backing to the transducer, preventing direct heating of the cornea. A prototype device was built, and hyperthermia experiments were performed on a porcine subject in vivo. A therapeutic temperature rise of 5°C-7°C was achievable in 10-15 min. The temperature of the cornea did not rise more than 2°C during any of the experiments.

Characterization of the temperate phage vB_RleM_PPF1 and its site-specific integration into the Rhizobium leguminosarum F1 genome.

Bacteriophages may play an important role in regulating population size and diversity of the root nodule symbiont Rhizobium leguminosarum, as well as participating in horizontal gene transfer. Although phages that infect this species have been isolated in the past, our knowledge of their molecular biology, and especially of genome composition, is extremely limited, and this lack of information impacts on the ability to assess phage population dynamics and limits potential agricultural applications of rhizobiophages. To help address this deficit in available sequence and biological information, the complete genome sequence of the Myoviridae temperate phage PPF1 that infects R. leguminosarum biovar viciae strain F1 was determined. The genome is 54,506 bp in length with an average G+C content of 61.9 %. The genome contains 94 putative open reading frames (ORFs) and 74.5 % of these predicted ORFs share homology at the protein level with previously reported sequences in the database. However, putative functions could only be assigned to 25.5 % (24 ORFs) of the predicted genes. PPF1 was capable of efficiently lysogenizing its rhizobial host R. leguminosarum F1. The site-specific recombination system of the phage targets an integration site that lies within a putative tRNA-Pro (CGG) gene in R. leguminosarum F1. Upon integration, the phage is capable of restoring the disrupted tRNA gene, owing to the 50 bp homologous sequence (att core region) it shares with its rhizobial host genome. Phage PPF1 is the first temperate phage infecting members of the genus Rhizobium for which a complete genome sequence, as well as other biological data such as the integration site, is available.

Synthesis of New Benzocyclotrimer Analogues: New Receptors for Tetramethylammonium Ion Recognition.

Using a [2 + 2 + 2] cycloaddition/Mitsunobu reaction sequence, a convenient synthesis to access new benzocyclotrimer analogues has been developed. The new receptors have the geometry and functionality capable of recognizing the tetramethylammonium ion in the gas phase and in solution.