Characterization of Drosophila CMP-sialic acid synthetase activity reveals unusual enzymatic properties.

CMP-sialic acid synthetase (CSAS) is a key enzyme of the sialylation pathway. CSAS produces the activated sugar donor, CMP-sialic acid, which serves as a substrate for sialyltransferases to modify glycan termini with sialic acid. Unlike other animal CSASs that normally localize in the nucleus, Drosophila melanogaster CSAS (DmCSAS) localizes in the cell secretory compartment, predominantly in the Golgi, which suggests that this enzyme has properties distinct from those of its vertebrate counterparts. To test this hypothesis, we purified recombinant DmCSAS and characterized its activity in vitro Our experiments revealed several unique features of this enzyme. DmCSAS displays specificity for N-acetylneuraminic acid as a substrate, shows preference for lower pH and can function with a broad range of metal cofactors. When tested at a pH corresponding to the Golgi compartment, the enzyme showed significant activity with several metal cations, including Zn(2+), Fe(2+), Co(2+) and Mn(2+), whereas the activity with Mg(2+) was found to be low. Protein sequence analysis and site-specific mutagenesis identified an aspartic acid residue that is necessary for enzymatic activity and predicted to be involved in co-ordinating a metal cofactor. DmCSAS enzymatic activity was found to be essential in vivo for rescuing the phenotype of DmCSAS mutants. Finally, our experiments revealed a steep dependence of the enzymatic activity on temperature. Taken together, our results indicate that DmCSAS underwent evolutionary adaptation to pH and ionic environment different from that of counterpart synthetases in vertebrates. Our data also suggest that environmental temperatures can regulate Drosophila sialylation, thus modulating neural transmission.

Enhanced biofilm formation and multi-host transmission evolve from divergent genetic backgrounds in Campylobacter jejuni.

Multicellular biofilms are an ancient bacterial adaptation that offers a protective environment for survival in hostile habitats. In microaerophilic organisms such as Campylobacter, biofilms play a key role in transmission to humans as the bacteria are exposed to atmospheric oxygen concentrations when leaving the reservoir host gut. Genetic determinants of biofilm formation differ between species, but little is known about how strains of the same species achieve the biofilm phenotype with different genetic backgrounds. Our approach combines genome-wide association studies with traditional microbiology techniques to investigate the genetic basis of biofilm formation in 102 Campylobacter jejuni isolates. We quantified biofilm formation among the isolates and identified hotspots of genetic variation in homologous sequences that correspond to variation in biofilm phenotypes. Thirteen genes demonstrated a statistically robust association including those involved in adhesion, motility, glycosylation, capsule production and oxidative stress. The genes associated with biofilm formation were different in the host generalist ST-21 and ST-45 clonal complexes, which are frequently isolated from multiple host species and clinical samples. This suggests the evolution of enhanced biofilm from different genetic backgrounds and a possible role in colonization of multiple hosts and transmission to humans.

The role of Drosophila cytidine monophosphate-sialic acid synthetase in the nervous system.

While sialylation plays important functions in the nervous system, the complexity of glycosylation pathways and limitations of genetic approaches preclude the efficient analysis of these functions in mammalian organisms. Drosophila has recently emerged as a promising model for studying neural sialylation. Drosophila sialyltransferase, DSiaT, was shown to be involved in the regulation of neural transmission. However, the sialylation pathway was not investigated in Drosophila beyond the DSiaT-mediated step. Here we focused on the function of Drosophila cytidine monophosphate-sialic acid synthetase (CSAS), the enzyme providing a sugar donor for DSiaT. Our results revealed that the expression of CSAS is tightly regulated and restricted to the CNS throughout development and in adult flies. We generated CSAS mutants and analyzed their phenotypes using behavioral and physiological approaches. Our experiments demonstrated that mutant phenotypes of CSAS are similar to those of DSiaT, including decreased longevity, temperature-induced paralysis, locomotor abnormalities, and defects of neural transmission at neuromuscular junctions. Genetic interactions between CSAS, DSiaT, and voltage-gated channel genes paralytic and seizure were consistent with the hypothesis that CSAS and DSiaT function within the same pathway regulating neural excitability. Intriguingly, these interactions also suggested that CSAS and DSiaT have some additional, independent functions. Moreover, unlike its mammalian counterparts that work in the nucleus, Drosophila CSAS was found to be a glycoprotein-bearing N-glycans and predominantly localized in vivo to the Golgi compartment. Our work provides the first systematic analysis of in vivo functions of a eukaryotic CSAS gene and sheds light on evolutionary relationships among metazoan CSAS proteins.

The role of protein N-glycosylation in neural transmission.

Recent studies have explored the function of N-linked glycosylation in the nervous system, demonstrating essential roles of carbohydrate structures in neural development. The function of N-glycans in neural physiology remains less understood; however, increasing evidence indicates that N-glycans can play specific modulatory roles controlling neural transmission and excitability of neural circuits. These roles are mediated via effects on synaptic proteins involved in neurotransmitter release, transporters that regulate nerotransmitter concentrations, neurotransmitter receptors, as well as via regulation of proteins that control excitability and response to milieu stimuli, such as voltage-gated ion channels and transient receptor potential channels, respectively. Sialylated N-glycan structures are among the most potent modulators of cell excitability, exerting prominent effects on voltage gated Na(+) and K(+) channels. This modulation appears to be underlain by complex molecular mechanisms involving electrostatic effects, as well as interaction modes based on more specific steric effects and interactions with lectins and other molecules. Data also indicate that particular features of N-glycans, such as their location on a protein and structural characteristics, can be specifically associated with the effect of glycosylation. These features and their functional implications can vary between different cell types, which highlight the importance of in vivo analyses of glycan functions. Experimental challenges are associated with the overwhelming complexity of the nervous system and glycosylation pathways in vertebrates, and thus model organisms like Drosophila should help elucidate evolutionarily conserved mechanisms underlying glycan functions. Recent studies supported this notion and shed light on functions of several glycosylation genes involved in the regulation of the nervous system.

Microbial community structure of a pilot-scale thermophilic anaerobic digester treating poultry litter.

The microbial community structure of a stable pilot-scale thermophilic continuous stirred tank reactor digester stabilized on poultry litter was investigated. This 40-m(3) digester produced biogas with 57% methane, and chemical oxygen demand removal of 54%. Bacterial and archaeal diversity were examined using both cloning and pyrosequencing that targeted 16S rRNA genes. The bacterial community was dominated by phylum Firmicutes, constituting 93% of the clones and 76% of the pyrotags. Of the Firmicutes, class Clostridia (52% pyrotags) was most abundant followed by class Bacilli (13% pyrotags). The bacterial libraries identified 94 operational taxonomic units (OTUs) and pyrosequencing identified 577 OTUs at the 97% minimum similarity level. Fifteen OTUs were dominant (≥2% abundance), and nine of these were novel unclassified Firmicutes. Several of the dominant OTUs could not be classified more specifically than Clostridiales, but were most similar to plant biomass degraders, including Clostridium thermocellum. Of the rare pyrotag OTUs (<0.5% abundance), 75% were Firmicutes. The dominant methanogen was Methanothermobacter which has hydrogenotrophic metabolism, and accounted for >99% of the archaeal clones. Based on the primary methanogen, as well as digester chemistry (high VA and ammonia levels), we propose that bacterial acetate oxidation is the primary pathway in this digester for the control of acetate levels.

Glia-neuron coupling via a bipartite sialylation pathway promotes neural transmission and stress tolerance in Drosophila.

Modification by sialylated glycans can affect protein functions, underlying mechanisms that control animal development and physiology. Sialylation relies on a dedicated pathway involving evolutionarily conserved enzymes, including CMP-sialic acid synthetase (CSAS) and sialyltransferase (SiaT) that mediate the activation of sialic acid and its transfer onto glycan termini, respectively. In Drosophila, CSAS and DSiaT genes function in the nervous system, affecting neural transmission and excitability. We found that these genes function in different cells: the function of CSAS is restricted to glia, while DSiaT functions in neurons. This partition of the sialylation pathway allows for regulation of neural functions via a glia-mediated control of neural sialylation. The sialylation genes were shown to be required for tolerance to heat and oxidative stress and for maintenance of the normal level of voltage-gated sodium channels. Our results uncovered a unique bipartite sialylation pathway that mediates glia-neuron coupling and regulates neural excitability and stress tolerance.

A hidden structural variation in a known IRD gene: a cautionary tale of two new disease candidate genes.

Rod-cone dystrophy (RCD), also known as retinitis pigmentosa, is an inherited condition leading to vision loss, affecting 1 in 3500 people. More than 270 genes are known to be implicated in the inherited retinal degenerations (IRDs), yet genetic diagnosis for ∼30% of IRD of patients remains elusive despite advances in sequencing technologies. The goal of this study was to determine the genetic causality in a family with RCD. Family members were given a full ophthalmic exam at the Retinal Service at Massachusetts Eye and Ear and consented to genetic testing. Whole-exome sequencing (WES) was performed and variants of interest were Sanger-validated. Functional assays were conducted in zebrafish along with splicing assays in relevant cell lines to determine the impact on retinal function. WES identified variants in two potential candidate genes that segregated with disease: GNL3 (G Protein Nucleolar 3) c.1187 + 3A > C and c.1568-8C > A; and PDE4DIP (Phosphodiester 4D Interacting Protein) c.3868G > A (p.Glu1290Lys) and c.4603G > A (p.Ala1535Thr). Both genes were promising candidates based on their retinal involvement (development and interactions with IRD-associated proteins); however, the functional assays did not validate either gene. Subsequent WES reanalysis with an updated bioinformatics pipeline and widened search parameters led to the detection of a 94-bp duplication in PRPF31 (pre-mRNA Processing Factor 31) c.73_266dup (p.Asp56GlyfsTer33) as the causal variant. Our study demonstrates the importance of thorough functional characterization of new disease candidate genes and the value of reanalyzing next-generation sequencing sequence data, which in our case led to identification of a hidden pathogenic variant in a known IRD gene.

Expanding the phenotypic spectrum in RDH12-associated retinal disease.

Retinol dehydrogenase 12, RDH12, plays a pivotal role in the visual cycle to ensure the maintenance of normal vision. Alterations in activity of this protein result in photoreceptor death and decreased vision beginning at an early age and progressing to substantial vision loss later in life. Here we describe 11 patients with retinal degeneration that underwent next-generation sequencing (NGS) with a targeted panel of all currently known inherited retinal degeneration (IRD) genes and whole-exome sequencing to identify the genetic causality of their retinal disease. These patients display a range of phenotypic severity prompting clinical diagnoses of macular dystrophy, cone-rod dystrophy, retinitis pigmentosa, and early-onset severe retinal dystrophy all attributed to biallelic recessive mutations in RDH12 We report 15 causal alleles and expand the repertoire of known RDH12 mutations with four novel variants: c.215A > G (p.Asp72Gly); c.362T > C (p.Ile121Thr); c.440A > C (p.Asn147Thr); and c.697G > A (p.Val233Ille). The broad phenotypic spectrum observed with biallelic RDH12 mutations has been observed in other genetic forms of IRDs, but the diversity is particularly notable here given the prior association of RDH12 primarily with severe early-onset disease. This breadth emphasizes the importance of broad genetic testing for inherited retinal disorders and extends the pool of individuals who may benefit from imminent gene-targeted therapies.

The transcriptional programme of Salmonella enterica serovar Typhimurium reveals a key role for tryptophan metabolism in biofilms.

BACKGROUND: Biofilm formation enhances the capacity of pathogenic Salmonella bacteria to survive stresses that are commonly encountered within food processing and during host infection. The persistence of Salmonella within the food chain has become a major health concern, as biofilms can serve as a reservoir for the contamination of food products. While the molecular mechanisms required for the survival of bacteria on surfaces are not fully understood, transcriptional studies of other bacteria have demonstrated that biofilm growth triggers the expression of specific sets of genes, compared with planktonic cells. Until now, most gene expression studies of Salmonella have focused on the effect of infection-relevant stressors on virulence or the comparison of mutant and wild-type bacteria. However little is known about the physiological responses taking place inside a Salmonella biofilm. RESULTS: We have determined the transcriptomic and proteomic profiles of biofilms of Salmonella enterica serovar Typhimurium. We discovered that 124 detectable proteins were differentially expressed in the biofilm compared with planktonic cells, and that 10% of the S. Typhimurium genome (433 genes) showed a 2-fold or more change in the biofilm compared with planktonic cells. The genes that were significantly up-regulated implicated certain cellular processes in biofilm development including amino acid metabolism, cell motility, global regulation and tolerance to stress. We found that the most highly down-regulated genes in the biofilm were located on Salmonella Pathogenicity Island 2 (SPI2), and that a functional SPI2 secretion system regulator (ssrA) was required for S. Typhimurium biofilm formation. We identified STM0341 as a gene of unknown function that was needed for biofilm growth. Genes involved in tryptophan (trp) biosynthesis and transport were up-regulated in the biofilm. Deletion of trpE led to decreased bacterial attachment and this biofilm defect was restored by exogenous tryptophan or indole. CONCLUSIONS: Biofilm growth of S. Typhimurium causes distinct changes in gene and protein expression. Our results show that aromatic amino acids make an important contribution to biofilm formation and reveal a link between SPI2 expression and surface-associated growth in S. Typhimurium.

Enrichment and identification of polycyclic aromatic compound-degrading bacteria enriched from sediment samples.

The degradation of polycyclic aromatic compounds (PACs) has been widely studied. Knowledge of the degradation of PACs by microbial populations can be utilized in the remediation of contaminated sites. To isolate and identify PAC-degrading bacteria for potential use in future bioremediation programmes, we established a series of PAC enrichments under the same experimental conditions from a single sediment sample taken from a highly polluted estuarine site. Enrichment cultures were established using the pollutants: anthracene, phenanthrene and dibenzothiophene as a sole carbon source. The shift in microbial community structure on each of these carbon sources was monitored by analysis of a time series of samples from each culture using 16S rRNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Significantly, our findings demonstrate that shifts in the constituent species within each degradative community are directly attributable to enrichment with different PACs. Subsequently, we characterized the microorganisms comprising the degradative communities within each enrichment using 16S rRNA sequence data. Our findings demonstrate that the ability to degrade PACs is present in five divisions of the Proteobacteria and Actinobacteria. By determining the precise identity of the PAC-degrading bacterial species isolated from a single sediment sample, and by comparing our findings with previously published research, we demonstrate how bacteria with similar PAC degrading capabilities and 16S rRNA signatures are found in similarly polluted environments in geographically very distant locations, e.g., China, Italy, Japan and Hawaii. Such a finding suggests that geographical barriers do not limit the distribution of key PAC-degrading bacteria; this finding is in accordance with the Baas-Becking hypothesis "everything is everywhere; the environment selects" and may have significant consequences for the global distribution of PAC-degrading bacteria and their use in bioremediation.

An observational study of spectators' step counts and reasons for attending a professional golf tournament in Scotland.

BACKGROUND: Spectators at several hundred golf tournaments on six continents worldwide may gain health-enhancing physical activity (HEPA) during their time at the event. This study aims to investigate spectators' reasons for attending and assess spectator physical activity (PA) (measured by step count). METHODS: Spectators at the Paul Lawrie Matchplay event in Scotland (August 2016) were invited to take part in this study. They were asked to complete a brief questionnaire with items to assess (1) demographics, (2) reasons for attendance and (3) baseline PA. In addition, participants were requested to wear a pedometer from time of entry to the venue until exit. RESULTS: A total of 339 spectators were recruited to the study and out of which 329 (97.2%) returned step-count data. Spectators took a mean of 11 589 steps (SD 4531). 'Fresh air' (rated median 9 out of 10) then 'watching star players', 'exercise/physical activity', 'time with friends and family' and 'atmosphere' (all median 8 out of 10) were rated the most important reasons for attending. CONCLUSION: This study is the first to assess spectator physical activity while watching golf (measured by step count). Obtaining exercise/PA is rated as an important reason for attending a tournament by many golf spectators. Spectating at a golf tournament can provide HEPA. 82.9% of spectators achieved the recommended daily step count while spectating. Further research directly assessing whether spectating may constitute a 'teachable moment', for increasing physical activity beyond the tournament itself, is merited.

Rains, drains and active strains: towards online assessment of wastewater bacterial communities.

Wastewater treatment is one of the largest scale and arguably the most commercially important biotechnological process in the world. Bacterial breakdown of waste materials facilitates the safe disposal of effluents into receiving water bodies. Given this significance, research has focused on identifying the keystone species on which efficient treatment is based. However, unravelling the microbial diversity within such systems has proven difficult. This is highlighted by our lack of detailed knowledge of the microbial interactions within these complex populations, limiting our ability to fully exploit bacterial degradative abilities. Even with the incorporation of new emerging molecular techniques, there have been no investigations linking genetic sequence to microbial function and successful treatment operation. To reach this goal, researchers need the ability to identify, enumerate and monitor the metabolic functions of subpopulations within these complex bacterial communities. Flow cytometry (FCM) combined with fluorescence-based molecular identification techniques provides a method for such studies. Moreover, single-cell sorting provides a unique opportunity to identify and remove individual cells of interest. Laboratory culture of sorted cells is often possible and permits the use of more traditional microbiological techniques to backup molecular investigations. Utilising this approach will advance our understanding of wastewater treatment processes and help maintain and enhance plant operation to improve efficiency.

Filament formation by Salmonella spp. inoculated into liquid food matrices at refrigeration temperatures, and growth patterns when warmed.

In this study, the formation of multicellular filamentous Salmonella cells in response to low temperatures was investigated by using isolates of Salmonella enterica serovar Enteritidis PT4 and S. enterica serovar Typhimurium DT104 as the inocula. The formation of filamentous cells in two liquid food matrices at the recommended maximum temperature for refrigeration (8 degrees C) was monitored and compared with that in tryptone soya broth. Giemsa staining was performed to locate nuclear material within the filaments. Single filaments were warmed on agar at 37 degrees C, and the subsequent rate of septation was quantified. For all strains tested, > 70% of the Salmonella cells inoculated had become filamentous after 4 days in media at 8 degrees C, indicating that filamentation could occur during the shelf life of most refrigerated foods. Strains with impaired RpoS expression were able to form filaments at 8 degrees C, although these filaments tended to be shorter and less numerous. All strains also formed filamentous cells at 8 degrees C in retail milk or chicken meat extract. Filaments often exceeded 100 microm in length and appeared straight-sided under the microscope in media and in foods, and Giemsa staining demonstrated that regularly spaced nucleoids were present. This phenotype indicates that an early block in cell septation is probably responsible for filamentation. When filaments were warmed on agar at 37 degrees C, there was a rapid completion of septation, and for one filament, a >200-fold increase in cell number was observed within 4 h. There are clear public health implications associated with the filamentation of Salmonella in contaminated foods at refrigeration temperatures, especially when the possibility of rapid septation of filamentous cells upon warming is considered.

N-glycosylation in regulation of the nervous system.

Protein N-glycosylation can influence the nervous system in a variety of ways by affecting functions of glycoproteins involved in nervous system development and physiology. The importance of N-glycans for different aspects of neural development has been well documented. For example, some N-linked carbohydrate structures were found to play key roles in neural cell adhesion and axonal targeting during development. At the same time, the involvement of glycosylation in the regulation of neural physiology remains less understood. Recent studies have implicated N-glycosylation in the regulation of neural transmission, revealing novel roles of glycans in synaptic processes and the control of neural excitability. N-Glycans were found to markedly affect the function of several types of synaptic proteins involved in key steps of synaptic transmission, including neurotransmitter release, reception, and uptake. Glycosylation also regulates a number of channel proteins, such as TRP channels that control responses to environmental stimuli and voltage-gated ion channels, the principal determinants of neuronal excitability. Sialylated carbohydrate structures play a particularly prominent part in the modulation of voltage-gated ion channels. Sialic acids appear to affect channel functions via several mechanisms, including charge interactions, as well as other interactions that probably engage steric effects and interactions with other molecules. Experiments also indicated that some structural features of glycans can be particularly important for their function. Since glycan structures can vary significantly between different cell types and depend on the metabolic state of the cell, it is important to analyze glycan functions using in vivo approaches. While the complexity of the nervous system and intricacies of glycosylation pathways can create serious obstacles for in vivo experiments in vertebrates, recent studies have indicated that more simple and experimentally tractable model organisms like Drosophila should provide important advantages for elucidating evolutionarily conserved functions of N-glycosylation in the nervous system.

Revealing a world of biofilms--the pioneering research of Bill Costerton.

Bill Costerton is recognized as the founding father of the field of biofilms, which is the study of microorganisms attached to surfaces. He was a true pioneer and was passionate about directly observing living complex microbial communities to learn how they function in different ecosystems. His multidisciplinary approach to the study of biofilms forged a common way of thinking about the ways in which microorganisms survive and function in the environment as well as in medical, dental, industrial, agricultural, engineering and other contexts. In this Essay, we outline some of the achievements that Bill made during his scientific journey.