Genomic characterization of a new endophytic Streptomyces kebangsaanensis identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production
Background Streptomyces are well known for their capability to produce many bioactive secondary metabolites with medical and industrial importance. Here we report a novel bioactive phenazine compound, 6-((2-hydroxy-4-methoxyphenoxy) carbonyl) phenazine-1-carboxylic acid (HCPCA) extracted from Streptomyces kebangsaanensis, an endophyte isolated from the ethnomedicinal Portulaca oleracea. Methods The HCPCA chemical structure was determined using nuclear magnetic resonance spectroscopy. We conducted whole genome sequencing for the identification of the gene cluster(s) believed to be responsible for phenazine biosynthesis in order to map its corresponding pathway, in addition to bioinformatics analysis to assess the potential of S. kebangsaanensis in producing other useful secondary metabolites. Results The S. kebangsaanensis genome comprises an 8,328,719 bp linear chromosome with high GC content (71.35%) consisting of 12 rRNA operons, 81 tRNA, and 7,558 protein coding genes. We identified 24 gene clusters involved in polyketide, nonribosomal peptide, terpene, bacteriocin, and siderophore biosynthesis, as well as a gene cluster predicted to be responsible for phenazine biosynthesis. Discussion The HCPCA phenazine structure was hypothesized to derive from the combination of two biosynthetic pathways, phenazine-1,6-dicarboxylic acid and 4-methoxybenzene-1,2-diol, originated from the shikimic acid pathway. The identification of a biosynthesis pathway gene cluster for phenazine antibiotics might facilitate future genetic engineering design of new synthetic phenazine antibiotics. Additionally, these findings confirm the potential of S. kebangsaanensis for producing various antibiotics and secondary metabolites.
Remali et al. (2017) Genomic characterization of a new endophytic Streptomyces kebangsaanensis identifies biosynthetic pathway gene clusters for novel phenazine antibiotic production. PeerJ 5:e3738 https://doi.org/10.7717/peerj.3738
Geological duration of ammonoids controlled their geographical range of fossil distribution
The latitudinal distributions in Devonian–Cretaceous ammonoids were analyzed at the genus level, and were compared with the hatchling sizes (i.e., ammonitella diameters) and the geological durations. The results show that (1) length of temporal ranges of ammonoids effected broader ranges of fossil distribution and paleobiogeography of ammonoids, and (2) the hatchling size was not related to the geographical range of fossil distribution of ammonoids. Reducing the influence of geological duration in this analysis implies that hatchling size was one of the controlling factors that determined the distribution of ammonoid habitats at any given period in time: ammonoids with smaller hatchling sizes tended to have broader ammonoid habitat ranges. These relationships were somewhat blurred in the Devonian, Carboniferous, Triassic, and Jurassic, which is possibly due to (1) the course of development of a reproductive strategy with smaller hatchling sizes in the Devonian and (2) the high origination rates after the mass extinction events.
Wani (2017) Geological duration of ammonoids controlled their geographical range of fossil distribution. PeerJ 5:e4108 https://doi.org/10.7717/peerj.4108
Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1
Background Methylophaga nitratireducenticrescens strain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate ( ${\mathrm{NO}}_{3}^{-}$ NO 3 − ) to nitrite ( ${\mathrm{NO}}_{2}^{-}$ NO 2 − ). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. Here we characterized further the denitrifying activities of M. nitratireducenticrescens JAM1. Methods Series of oxic and anoxic cultures of strain JAM1 were performed with N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − or sodium nitroprusside, and growth and N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − , ${\mathrm{NO}}_{2}^{-}$ NO 2 − and N2 concentrations were measured. Ammonium ( ${\mathrm{NH}}_{4}^{+}$ NH 4 + )-free cultures were also tested to assess the dynamics of N2O, ${\mathrm{NO}}_{3}^{-}$ NO 3 − and ${\mathrm{NO}}_{2}^{-}$ NO 2 − . Isotopic labeling of N2O was performed in 15NH4+-amended cultures. Cultures with the JAM1ΔnarG1narG2 double mutant were performed to assess the involvement of the Nar systems on N2O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochrome bc-type nitric oxide reductase (cnorB1 and cnorB2) and nitrous oxide reductase (nosZ), and also nnrS and norR that encode NO-sensitive regulators. Results Strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory ${\mathrm{NO}}_{2}^{-}$ NO 2 − reductase, ${\mathrm{NO}}_{3}^{-}$ NO 3 − -amended cultures produce N2O, representing up to 6% of the N-input. ${\mathrm{NO}}_{2}^{-}$ NO 2 − was shown to be the key intermediate of this production process. Upregulation in the expression of cnorB1, cnorB2, nnrS and norR during the growth and the N2O accumulation phases suggests NO production in strain JAM1 cultures. Discussion By showing that all the three denitrification reductases are active, this demonstrates that M. nitratireducenticrescens JAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such an incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of the denitrification reactor from which it originates. The production of N2O in strain JAM1 did not involve Nar, contrary to what was demonstrated in Escherichia coli. M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using ${\mathrm{NO}}_{3}^{-}$ NO 3 − and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.
Mauffrey et al. (2017) Denitrifying metabolism of the methylotrophic marine bacterium Methylophaga nitratireducenticrescens strain JAM1. PeerJ 5:e4098 https://doi.org/10.7717/peerj.4098
The presence of genes encoding enzymes that digest carbohydrates in coral genomes and analysis of their activities
Numerous enzymes that digest carbohydrates, such as cellulases and chitinases, are present in various organisms (e.g., termites, nematodes, and so on). Recently, the presence of cellulases and chitinases has been reported in marine organisms such as urchin and bivalves, and their several roles in marine ecosystems have been proposed. In this study, we reported the presence of genes predicted to encode proteins similar to cellulases and chitinases in the genome of the coral Acropora digitifera, their gene expression patterns at various life stages, and cellulose- and chitin-degrading enzyme activities in several coral species (A. digitifera, Galaxea fascicularis, Goniastrea aspera, Montipora digitata, Pavona divaricata, Pocillopora damicornis, and Porites australiensis). Our gene expression analysis demonstrated the expressions of these cellulase- and chitinase-like genes during various life stages, including unfertilized eggs, fertilized eggs, zygotes, planula larvae, primary polyps and adults of A. digitifera. Agar plate assays confirmed cellulase and chitinase activities in the tissues extracted from adult branches of several coral species. These results suggested that corals are able to utilize cellulases and chitinases in their life histories.
Yoshioka et al. (2017) The presence of genes encoding enzymes that digest carbohydrates in coral genomes and analysis of their activities. PeerJ 5:e4087 https://doi.org/10.7717/peerj.4087
Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae)
Hormones play an important role in the regulation of physiological, developmental and behavioural processes. Many of these mechanisms in insects, however, are still not well understood. One way to investigate hormonal regulation is to analyse gene expression patterns of hormones and their receptors by real-time quantitative polymerase chain reaction (RT-qPCR). This method, however, requires stably expressed reference genes for normalisation. In the present study, we evaluated 11 candidate housekeeping genes as reference genes in samples of Lethrus apterus, an earth-boring beetle with biparental care, collected from a natural population. For identifying the most stable genes we used the following computational methods: geNorm, NormFinder, BestKeeper, comparative delta Ct method and RefFinder. Based on our results, the two body regions sampled (head and thorax) differ in which genes are most stably expressed. We identified two candidate reference genes for each region investigated: ribosomal protein L7A and RP18 in samples extracted from the head, and ribosomal protein L7A and RP4 extracted from the muscles of the thorax. Additionally, L7A and RP18 appear to be the best reference genes for normalisation in all samples irrespective of body region. These reference genes can be used to study the hormonal regulation of reproduction and parental care in Lethrus apterus in the future.
Nagy et al. (2017) Evaluation of potential reference genes for real-time qPCR analysis in a biparental beetle, Lethrus apterus (Coleoptera: Geotrupidae). PeerJ 5:e4047 https://doi.org/10.7717/peerj.4047
Phylogeographic and population insights of the Asian common toad (Bufo gargarizans) in Korea and China: population isolation and expansions as response to the ice ages
The effects of ice ages on speciation have been well documented for many European and North American taxa. In contrast, very few studies have addressed the consequences of such environmental and topographical changes in North East Asian species. More precisely, the Korean Peninsula offers a unique model to assess patterns and processes of speciation as it hosts the northern- and eastern-most distribution limit of some widespread Asian taxa. Despite this, studies addressing phylogeographic patterns and population genetics in the peninsula and surrounding countries are few and studies for most families are lacking. Here we inferred the phylogenetic relationships of the common toad (Bufo gargarizans) from South Korea and their North East Asian counterpart populations, based on mitochondrial data. Korean B. gargarizans GenBank BLASTs matched few individuals from nearby China, but the presence of a Korean clade suggests isolation on the Korean Peninsula, previous to the last glacial maximum, linked to sea level resurgence. Molecular clock calibrations within this group were used to date the divergence between clades and their relationship to paleo-climatic events in the area. Lack of genetic structure among South Korean populations and strong homogeneity between the Korean and some Chinese localities suggest weak isolation and recent expansion. Geographical projection of continuous coalescent maximum-clade-credibility trees shows an original Chinese expansion towards the Korean Peninsula through the Yellow Sea circa two million years ago with colonisation events dating circa 800 thousand years ago (K. y. a.). Following this colonisation, the data point to outgoing Korean Peninsula dispersal events throughout different periods, towards the North through land, and West through land bridge formations over the Yellow Sea during sea level falls. In accordance, demographic analyses revealed a population expansion in the Koran Peninsula circa 300 K. y. a., likely attributed to glacial cycle fluctuations.
Borzée et al. (2017) Phylogeographic and population insights of the Asian common toad (Bufo gargarizans) in Korea and China: population isolation and expansions as response to the ice ages. PeerJ 5:e4044 https://doi.org/10.7717/peerj.4044
Impacts of diarrhea on the immune system, intestinal environment, and expression of PGRPs in New Zealand rabbits
Diarrhea is a syndrome of digestive disorders in young rabbits and may lead to secondary infections resulting in reduced immunity and higher mortality in baby rabbits, with serious impacts on rabbit farming. In this study, we investigated the effects of diarrhea on the health of baby rabbits in terms of intestinal mucosal development, immune function, and intestinal microbial diversity. We found that the duodenal villus length and the villus/crypt ratio in rabbits with diarrhea were significantly reduced compared with those in healthy rabbits (P < 0.01). Rabbits with diarrhea had significantly lower concentrations of acetic acid (P < 0.05), higher pH levels (P < 0.05), and higher levels of ammonia nitrogen (P < 0.01) in the cecum. Moreover, diarrhea in baby rabbits led to significantly reduced levels of total serum protein (P < 0.05) and markedly increased levels of alkaline phosphatase, urea nitrogen, TNF-α, and IL-6 (P < 0.05). Transcriptional analysis of peptidoglycan recognition proteins (PGRPs, including PGLYRP-1, PGLYRP-2, and PGLYRP-3) using real-time PCR revealed that diarrhea induced the upregulation of PGRPs in the cecum and duodenum. Furthermore, through pyrosequencing of the 16S rRNA V4 region in cecum samples, we found that the total number and diversity of microbes were not significantly different between healthy rabbits and those with diarrhea, though there were noticeable differences in the prevalences of Clostridium, Roseburia, and Alistipes. Our results will contribute to a better understanding of the pathological mechanisms of diarrhea in young rabbits.
Chen et al. (2017) Impacts of diarrhea on the immune system, intestinal environment, and expression of PGRPs in New Zealand rabbits. PeerJ 5:e4100 https://doi.org/10.7717/peerj.4100
Diagnostic value of serum procalcitonin, lactate, and high-sensitivity C-reactive protein for predicting bacteremia in adult patients in the emergency department
Background Few studies compared the diagnostic value of procalcitonin with a combination of other tests including lactate and high-sensitivity C-reactive protein in the prediction of pathogenic bacteremia in emergency department adult patients. Methods We performed a retrospective study assessing the differences in performances of procalcitonin at a cutoff of 0.5 ng/mL, lactate at a cutoff of 19.8 mg/dL, high-sensitivity C-reactive protein at a cutoff of 0.8 mg/dL and their combinations for predicting bacteremia in emergency department adult patients. Sensitivity, specificity, overall accuracy, positive-test and negative-test likelihood, and diagnostic odds ratio with 95% confidence interval for each test combination were calculated for comparison. The receiver operating characteristic curve for every single test were compared using DeLong’s method. We also performed a sensitivity analysis in two expanded patient cohorts to assess the discriminative ability of procalcitonin or test combination. Results A total of 886 patients formed the initial patient cohort. The area under the receiver operating characteristic curve for discriminating positive blood culture was: procalcitonin = 0.72 (95% CI [0.69–0.75]) with a derived optimal cutoff at 3.9 ng/mL; lactate 0.69 (0.66–0.72) with an optimal cutoff at 17.9 mg/dL; high-sensitivity C-reactive protein 0.56 (0.53–0.59) with an optimal cutoff of 13 mg/dL; with pairwise comparisons showing statistically significant better performance of either procalcitonin or lactate outperforming high-sensitivity C-reactive protein. To predict positive blood cultures, the diagnostic odds ratio for procalcitonin was 3.64 (95% CI [2.46–5.51]), lactate 2.93 (2.09–4.14), and high-sensitivity C-reactive protein 0.91 (0.55–1.55; P = 0.79). About combined tests, the diagnostic odds ratio for procalcitonin and lactate increases were 3.98 (95% CI [2.81–5.63]) for positive blood culture prediction. Elevated procalcitonin level rendered a six-fold increased risk of positive gram-negative bacteremia with a diagnostic odds ratio of 6.44 (95% CI [3.65–12.15]), which showed no further improvement in any test combinations. In the sensitivity analysis, as a single test to predict unspecified, gram-negative and gram-positive bacteremia, procalcitonin performed even better in an expanded cohort of 2,234 adult patients in terms of the diagnostic odds ratio. Discussions For adult emergency patients, procalcitonin has an acceptable discriminative ability for bacterial blood culture and a better discriminative ability for gram-negative bacteremia when compared with lactate and high-sensitivity C-reactive protein. High-sensitivity C-reactive protein at a cutoff of 0.8 mg/dL performed poorly for the prediction of positive bacterial culture.
Lin et al. (2017) Diagnostic value of serum procalcitonin, lactate, and high-sensitivity C-reactive protein for predicting bacteremia in adult patients in the emergency department. PeerJ 5:e4094 https://doi.org/10.7717/peerj.4094
The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function
Previous studies have used the zebrafish to investigate the biology of lens crystallin proteins and their roles in development and disease. However, little is known about zebrafish α-crystallin promoter function, how it compares to that of mammals, or whether mammalian α-crystallin promoter activity can be assessed using zebrafish embryos. We injected a variety of α-crystallin promoter fragments from each species combined with the coding sequence for green fluorescent protein (GFP) into zebrafish zygotes to determine the resulting spatiotemporal expression patterns in the developing embryo. We also measured mRNA levels and protein abundance for all three zebrafish α-crystallins. Our data showed that mouse and zebrafish αA-crystallin promoters generated similar GFP expression in the lens, but with earlier onset when using mouse promoters. Expression was also found in notochord and skeletal muscle in a smaller percentage of embryos. Mouse αB-crystallin promoter fragments drove GFP expression primarily in zebrafish skeletal muscle, with less common expression in notochord, lens, heart and in extraocular regions of the eye. A short fragment containing only a lens-specific enhancer region increased lens and notochord GFP expression while decreasing muscle expression, suggesting that the influence of mouse promoter control regions carries over into zebrafish embryos. The two paralogous zebrafish αB-crystallin promoters produced subtly different expression profiles, with the aBa promoter driving expression equally in notochord and skeletal muscle while the αBb promoter resulted primarily in skeletal muscle expression. Messenger RNA for zebrafish αA increased between 1 and 2 days post fertilization (dpf), αBa increased between 4 and 5 dpf, but αBb remained at baseline levels through 5 dpf. Parallel reaction monitoring (PRM) mass spectrometry was used to detect αA, aBa, and αBb peptides in digests of zebrafish embryos. In whole embryos, αA-crystallin was first detected by 2 dpf, peaked in abundance by 4–5 dpf, and was localized to the eye. αBa was detected in whole embryo at nearly constant levels from 1–6 dpf, was also localized primarily to the eye, and its abundance in extraocular tissues decreased from 4–7 dpf. In contrast, due to its low abundance, no αBb protein could be detected in whole embryo, or dissected eye and extraocular tissues. Our results show that mammalian α-crystallin promoters can be efficiently screened in zebrafish embryos and that their controlling regions are well conserved. An ontogenetic shift in zebrafish aBa-crystallin promoter activity provides an interesting system for examining the evolution and control of tissue specificity. Future studies that combine these promoter based approaches with the expanding ability to engineer the zebrafish genome via techniques such as CRISPR/Cas9 will allow the manipulation of protein expression to test hypotheses about lens crystallin function and its relation to lens biology and disease.
Posner et al. (2017) The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function. PeerJ 5:e4093 https://doi.org/10.7717/peerj.4093
The relationship between egg size and helper number in cooperative breeders: a meta-analysis across species
Background Life history theory predicts that mothers should adjust reproductive investment depending on benefits of current reproduction and costs of reduced future reproductive success. These costs and benefits may in turn depend on the breeding female’s social environment. Cooperative breeders provide an ideal system to test whether changes in maternal investment are associated with the social conditions mothers experience. As alloparental helpers assist in offspring care, larger groups might reduce reproductive costs for mothers or alternatively indicate attractive conditions for reproduction. Thus, mothers may show reduced (load-lightening) or increased (differential allocation) reproductive investment in relation to group size. A growing number of studies have investigated how cooperatively breeding mothers adjust pre-natal investment depending on group size. Our aim was to survey these studies to assess, first, whether mothers consistently reduce or increase pre-natal investment when in larger groups and, second, whether these changes relate to variation in post-natal investment. Methods We extracted data on the relationship between helper number and maternal pre-natal investment (egg size) from 12 studies on 10 species of cooperatively breeding vertebrates. We performed meta-analyses to calculate the overall estimated relationship between egg size and helper number, and to quantify variation among species. We also tested whether these relationships are stronger in species in which the addition of helpers is associated with significant changes in maternal and helper post-natal investment. Results Across studies, there is a significant negative relationship between helper number and egg size, suggesting that in most instances mothers show reduced reproductive investment in larger groups, in particular in species in which mothers also show a significant reduction in post-natal investment. However, even in this limited sample, substantial variation exists in the relationship between helper number and egg size, and the overall effect appears to be driven by a few well-studied species. Discussion Our results, albeit based on a small sample of studies and species, indicate that cooperatively breeding females tend to produce smaller eggs in larger groups. These findings on prenatal investment accord with previous studies showing similar load-lightening reductions in postnatal parental effort (leading to concealed helper effects), but do not provide empirical support for differential allocation. However, the considerable variation in effect size across studies suggests that maternal investment is mitigated by additional factors. Our findings indicate that variation in the social environment may influence life-history strategies and suggest that future studies investigating within-individual changes in maternal investment in cooperative breeders offer a fruitful avenue to study the role of adaptive plasticity.
Dixit et al. (2017) The relationship between egg size and helper number in cooperative breeders: a meta-analysis across species. PeerJ 5:e4028 https://doi.org/10.7717/peerj.4028