Recombination and selectional forces in cyanopeptolin NRPS operons from highly similar, but geographically remote Planktothrix strains

<p>Abstract</p> <p>Background</p> <p>Cyanopeptolins are nonribosomally produced heptapetides showing a highly variable composition. The cyanopeptolin synthetase operon has previously been investigated in three strains from the genera <it>Microcystis</it>, <it>Planktothrix </it>and <it>Anabaena</it>. Cyanopeptolins are displaying protease inhibitor activity, but the biological function(s) is (are) unknown. Cyanopeptolin gene cluster variability and biological functions of the peptide variants are likely to be interconnected.</p> <p>Results</p> <p>We have investigated two cyanopeptolin gene clusters from highly similar, but geographically remote strains of the same genus. Sequencing of a nonribosomal peptide synthetase (NRPS) cyanopeptolin gene cluster from the Japanese strain <it>Planktothrix </it>NIES 205 (205-<it>oci</it>), showed the 30 kb gene cluster to be highly similar to the <it>oci </it>gene cluster previously described in <it>Planktothrix </it>NIVA CYA 116, isolated in Norway. Both operons contained seven NRPS modules, a sulfotransferase (S) and a glyceric acid loading (GA)-domain. Sequence analyses showed a high degree of conservation, except for the presence of an epimerase domain in NIES 205 and the regions around the epimerase, showing high substitution rates and Ka/Ks values above 1. The two strains produce almost identical cyanopeptolins, cyanopeptolin-1138 and oscillapeptin E respectively, but with slight differences regarding the production of minor cyanopeptolin variants. These variants may be the result of relaxed adenylation (A)-domain specificity in the nonribosomal enzyme complex. Other genetic markers (16S rRNA, <it>ntc</it>A and the phycocyanin <it>cpc</it>BA spacer) were identical, supporting that these geographically separated <it>Planktothrix </it>strains are closely related.</p> <p>Conclusion</p> <p>A horizontal gene transfer event resulting in exchange of a whole module-encoding region was observed. Nucleotide statistics indicate that both purifying selection and positive selection forces are operating on the gene cluster. The positive selection forces are acting within and around the epimerase insertion while purifying selection conserves the remaining (major) part of the gene cluster. The presence of an epimerase in the gene cluster is in line with the D-configuration of Htyr, determined experimentally in oscillapeptin E in a previous study.</p

Long-term follow-up of colorectal cancer screening attendees identifies differences in Phascolarctobacterium spp. using 16S rRNA and metagenome sequencing

BackgroundThe microbiome has been implicated in the initiation and progression of colorectal cancer (CRC) in cross-sectional studies. However, there is a lack of studies using prospectively collected samples.MethodsFrom the Norwegian Colorectal Cancer Prevention (NORCCAP) trial, we analyzed 144 archived fecal samples from participants who were diagnosed with CRC or high-risk adenoma (HRA) at screening and from participants who remained cancer-free during 17 years of follow-up. We performed 16S rRNA sequencing of all the samples and metagenome sequencing on a subset of 47 samples. Differences in taxonomy and gene content between outcome groups were assessed for alpha and beta diversity and differential abundance.ResultsDiversity and composition analyses showed no significant differences between CRC, HRA, and healthy controls. Phascolarctobacterium succinatutens was more abundant in CRC compared with healthy controls in both the 16S and metagenome data. The abundance of Bifidobacterium and Lachnospiraceae spp. was associated with time to CRC diagnosis.ConclusionUsing a longitudinal study design, we identified three taxa as being potentially associated with CRC. These should be the focus of further studies of microbial changes occurring prior to CRC diagnosis

Heavy Screen Use on Weekends in Childhood Predicts Increased Body Mass Index in Adolescence : A Three-Year Follow-Up Study

Purpose We aimed to examine whether sedentary screen time changes when transitioning from childhood to adolescence and whether children's screen time, separately for school days and weekends, affects body mass index (BMI) in adolescents. Methods This prospective 3-year follow-up study included 5,084 children with a mean (standard deviation) age of 11 (1) years at baseline and 14 (1) years at follow-up. Children reported screen time, more specifically, time spent viewing TV programs and using a computer outside school while sitting. We categorized children into light, medium, and heavy TV viewers and computer users separately for school days and weekends. We also calculated the age- and gender-specific BMI z-score (BMIz). Results Time spent viewing TV changed from baseline to follow-up on school days and on weekends (p <.001 for both); the proportion of heavy TV viewers on school days (≥3 h/d) increased from 16% to 23% and on weekends (≥4 h/d) from 19% to 30%. Heavy TV viewers and computer users on both school days and on weekends had a higher BMIz 3 years later (p <.001 for all). After adjusting for age, gender, language, baseline BMIz, sleep duration, eating habits, exercise, and the other screen-time variables, heavy TV viewing on weekends remained significantly associated with an increased BMIz at follow-up (B = .078; p = .013). Conclusions The proportion of heavy screen users increases when transitioning from childhood to adolescence. Moreover, heavy screen use, especially on weekends in 11-year-old children, is associated with an increased BMI 3 years later.Peer reviewe

A genome-wide analysis of nonribosomal peptide synthetase gene clusters and their peptides in a Planktothrix rubescens strain

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria often produce several different oligopeptides, with unknown biological functions, by nonribosomal peptide synthetases (NRPS). Although some cyanobacterial NRPS gene cluster types are well described, the entire NRPS genomic content within a single cyanobacterial strain has never been investigated. Here we have combined a genome-wide analysis using massive parallel pyrosequencing ("454") and mass spectrometry screening of oligopeptides produced in the strain <it>Planktothrix rubescens </it>NIVA CYA 98 in order to identify all putative gene clusters for oligopeptides.</p> <p>Results</p> <p>Thirteen types of oligopeptides were uncovered by mass spectrometry (MS) analyses. Microcystin, cyanopeptolin and aeruginosin synthetases, highly similar to already characterized NRPS, were present in the genome. Two novel NRPS gene clusters were associated with production of anabaenopeptins and microginins, respectively. Sequence-depth of the genome and real-time PCR data revealed three copies of the microginin gene cluster. Since NRPS gene cluster candidates for microviridin and oscillatorin synthesis could not be found, putative (gene encoded) precursor peptide sequences to microviridin and oscillatorin were found in the genes <it>mdn</it>A and <it>osc</it>A, respectively. The genes flanking the microviridin and oscillatorin precursor genes encode putative modifying enzymes of the precursor oligopeptides. We therefore propose ribosomal pathways involving modifications and cyclisation for microviridin and oscillatorin. The microviridin, anabaenopeptin and cyanopeptolin gene clusters are situated in close proximity to each other, constituting an oligopeptide island.</p> <p>Conclusion</p> <p>Altogether seven nonribosomal peptide synthetase (NRPS) gene clusters and two gene clusters putatively encoding ribosomal oligopeptide biosynthetic pathways were revealed. Our results demonstrate that whole genome shotgun sequencing combined with MS-directed determination of oligopeptides successfully can identify NRPS gene clusters and the corresponding oligopeptides. The analyses suggest independent evolution of all NRPS gene clusters as functional units. Our data indicate that the <it>Planktothrix </it>genome displays evolution of dual pathways (NRPS and ribosomal) for production of oligopeptides in order to maximize the diversity of oligopeptides with similar but functional discrete bioactivities.</p

Saliva microbiota differs between children with low and high sedentary screen times

Publisher Copyright: © 2021 The Author(s)This study examined whether the diversity, composition and functional capacity of the saliva microbiota differed between children with low and high sedentary screen times. We analyzed the saliva microbiota using 16S rRNA (V3–V4) sequencing from 193 children with low and 183 children with high TV/screen viewing times while sitting. Microbiota diversity was higher among children with low screen times compared to children with high screen times. Furthermore, microbiota composition differed between the screen time groups. In addition, we identified ten differentially abundant taxonomic groups, including Veillonella, Prevotella and Streptococcus, and five differentially present metabolic pathways between the screen time groups. Children with high screen times exhibited a higher capacity to synthesize the fatigue- and activity-related amino acids ornithine and arginine. To conclude, children with high sedentary screen (sitting) times exhibited a lower diversity and a different composition and functionality of the microbiota compared to children with low screen times.Peer reviewe

Genome-wide DNA methylation in saliva and body size of adolescent girls

Aim: We performed an epigenome-wide association study within the Finnish Health in Teens cohort to identify differential DNA methylation and its association with BMI in adolescents. Materials & methods: Differential DNA methylation analyses of 3.1 million CpG sites were performed in saliva samples from 50 lean and 50 heavy adolescent girls by genome-wide targeted bisulfite-sequencing. Results: We identified 100 CpG sites with p-values <0.000524, seven regions by 'bumphunting' and five CpG islands that differed significantly between the two groups. The ten CpG sites and regions most strongly associated with BMI substantially overlapped with obesity-and insulin-related genes, including MC2R, IGFBPL1, IP6K1 and IGF2BP1. Conclusion: Our findings suggest an association between the saliva methylome and BMI in adolescence.Peer reviewe

A 10-year prediagnostic follow-up study shows that serum RNA signals are highly dynamic in lung carcinogenesis

The majority of lung cancer (LC) patients are diagnosed at a late stage, and survival is poor. Circulating RNA molecules are known to have a role in cancer; however, their involvement before diagnosis remains an open question. In this study, we investigated circulating RNA dynamics in prediagnostic LC samples, focusing on smokers, to identify if and when disease-related signals can be detected in serum. We sequenced small RNAs in 542 serum LC samples donated up to 10 years before diagnosis and 519 matched cancer-free controls coming from 905 individuals in the Janus Serum Bank. This sample size provided sufficient statistical power to independently analyze time to diagnosis, stage, and histology. The results showed dynamic changes in differentially expressed circulating RNAs specific to LC histology and stage. The greatest number of differentially expressed RNAs was identified around 7 years before diagnosis for early-stage LC and 1–4 years prior to diagnosis for locally advanced and advanced-stage LC, regardless of LC histology. Furthermore, NSCLC and SCLC histologies have distinct prediagnostic signals. The majority of differentially expressed RNAs were associated with cancer-related pathways. The dynamic RNA signals pinpointed different phases of tumor development over time. Stage-specific RNA profiles may be associated with tumor aggressiveness. Our results improve the molecular understanding of carcinogenesis. They indicate substantial opportunity for screening and improved treatment and will guide further research on early detection of LC. However, the dynamic nature of the RNA signals also suggests challenges for prediagnostic biomarker discovery

Meal Regularity Plays a Role in Shaping the Saliva Microbiota

Background Diet may influence health directly or indirectly via the human microbiota, emphasizing the need to unravel these complex relationships for future health benefits. Associations between eating habits and gut microbiota have been shown, but less is known about the association between eating habits and saliva microbiota. Objective The aim of this study was to investigate if eating habits and meal patterns are associated with the saliva microbiota. Methods In total, 842 adolescents, aged 11-14 years, from the Finnish Health in Teens (Fin-HIT) study cohort were included in this study. Eating habits and breakfast and dinner patterns were derived from a web-based questionnaire answered in school. Three major eating habit groups were identified: fruit and vegetable avoiders (FV avoiders), healthy and unhealthy. Microbiota profiles were produced from 16S rRNA gene (V3-V4) sequencing of DNA from the saliva samples. Statistical models were adjusted for gender, age, parental language, body mass index (BMI) categories, and sequencing depth. Results Regular breakfast eaters had a higher alpha diversity (Shannon index with mean (standard error of means) 2.27 (0.03) vs. 2.22 (0.03), p = 0.06, inverse Simpson's index with 6.27 (0.17) vs. 5.80 (0.02), p = 0.01), and slight differences in bacterial composition (PERMANOVA: p = 0.001) compared with irregular breakfast eaters. A similar trend in alpha diversity was observed between regular and irregular dinner eaters (Shannon index with 2.27 (0.03) vs. 2.22 (0.03), p = 0.054, inverse Simpson's index with 6.23 (0.17) vs. 6.04 (0.22), p = 0.28), while no difference was found in composition (PERMANOVA: p = 0.08). No differences were identified between eating habit groups and saliva microbiota diversity (Shannon index p = 0.77, inverse Simpson's index p = 0.94) or composition (PERMANOVA: p = 0.13). FV avoiders, irregular breakfast eaters and irregular dinner eaters had high abundances of Prevotella. Conclusion Regularity of eating, especially breakfast eating, was associated with more diverse saliva microbiota and different composition compared with irregular eaters. However, the dissimilarities in composition were small between regular and irregular breakfast eaters. Our results suggest that Prevotella abundances in saliva were common in FV avoiders and meal skippers. However, the clinical implications of these findings need to be evaluated in future studies.Peer reviewe

Gender-Specific Associations Between Saliva Microbiota and Body Size

ObjectiveThe human intestinal microbiota likely play an important role in the development of overweight and obesity. However, the associations between saliva microbiota and body mass index (BMI) have been sparsely studied. The aim of this study was to identify the associations between saliva microbiota and body size in Finnish children.MethodsThe saliva microbiota of 900 Finnish children, aged 11–14 years with measured height and weight, was characterized using 16S rRNA (V3–V4) sequencing.ResultsThe core saliva microbiota consisted of 14 genera that were present in more than 95% of the Finnish children. The saliva microbiota profiles were gender-specific with higher alpha-diversity in boys than girls and significant differences between the genders in community composition and abundances. Alpha-diversity differed between normal weight and overweight girls and between normal weight and obese boys. The composition was dissimilar between normal weight and obese girls, but not in boys. The relative abundance profiles differed according to body size. Decrease in commensal saliva bacteria were observed in all the body sizes when compared to normal weight children. Notably, the relative abundance of bacteria related to, Veillonella, Prevotella, Selenomonas, and Streptococcus was reduced in obese children.ConclusionSaliva microbiota diversity and composition were significantly associated with body size and gender in Finnish children. Body size–specific saliva microbiota profiles open new avenues for studying the potential roles of microbiota in weight development and management

Reproducibility and repeatability of six high-throughput 16S rDNA sequencing protocols for microbiota profiling

Culture-independent molecular techniques and advances in next generation sequencing (NGS) technologies make large-scale epidemiological studies on microbiota feasible. A challenge using NGS is to obtain high reproducibility and repeatability, which is mostly attained through robust amplification. We aimed to assess the reproducibility of saliva microbiota by comparing triplicate samples. The microbiota was produced with simplified in-house 16S amplicon assays taking advantage of large number of barcodes. The assays included primers with Truseq (TS-tailed) or Nextera (NX-tailed) adapters and either with dual index or dual index plus a 6-nt internal index. All amplification protocols produced consistent microbial profiles for the same samples. Although, in our study, reproducibility was highest for the TS-tailed method. Five replicates of a single sample, prepared with the TS-tailed 1-step protocol without internal index sequenced on the HiSeq platform provided high alpha-diversity and low standard deviation (mean Shannon and Inverse Simpson diversity was 3.19 +/- 0.097 and 13.56 +/- 1.634 respectively). Large-scale profiling of microbiota can consistently be produced by all 16S amplicon assays. The TS-tailed-1S dual index protocol is preferred since it provides repeatable profiles on the HiSeq platform and are less labour intensive.Peer reviewe