Finding candidate genes under positive selection in Non‐model species: examples of genes involved in host specialization in pathogens

Numerous genes in diverse organisms have been shown to be under positive selection, especially genes involved in reproduction, adaptation to contrasting environments, hybrid inviability, and host‐pathogen interactions. Looking for genes under positive selection in pathogens has been a priority in efforts to investigate coevolution dynamics and to develop vaccines or drugs. To elucidate the functions involved in host specialization, here we aimed at identifying candidate sequences that could have evolved under positive selection among closely related pathogens specialized on different hosts. For this goal, we sequenced c. 17 000–32 000 ESTs from each of four Microbotryum species, which are fungal pathogens responsible for anther smut disease on host plants in the Caryophyllaceae. Forty‐two of the 372 predicted orthologous genes showed significant signal of positive selection, which represents a good number of candidate genes for further investigation. Sequencing 16 of these genes in 9 additional Microbotryum species confirmed that they have indeed been rapidly evolving in the pathogen species specialized on different hosts. The genes showing significant signals of positive selection were putatively involved in nutrient uptake from the host, secondary metabolite synthesis and secretion, respiration under stressful conditions and stress response, hyphal growth and differentiation, and regulation of expression by other genes. Many of these genes had transmembrane domains and may therefore also be involved in pathogen recognition by the host. Our approach thus revealed fruitful and should be feasible for many non‐model organisms for which candidate genes for diversifying selection are needed.     

Genomic signatures of obligate host dependence in the luminous bacterial symbiont of a vertebrate

The majority of bacteria engaged in bioluminescent symbiosis are environmentally acquired and facultatively symbiotic. A few enigmatic bioluminescent symbionts have not been successfully cultured, which has led to speculation that they may be obligately dependent on their hosts. Here, we report the draft genome of the uncultured luminous symbiont of an anomalopid flashlight fish, ‘Candidatus Photodesmus katoptron’. The genome of the anomalopid symbiont is reduced by 80% compared with close relatives and lacks almost all genes necessary for amino acid synthesis and for metabolism of energy sources other than glucose, supporting obligate dependence on the host for growth. ‘Candidatus Photodesmus katoptron’ is the first described obligate mutualistic symbiont of a vertebrate. Unlike most other obligate mutualists, the anomalopid symbiont genome has retained complete pathways for chemotaxis and motility as well as most genes involved in cell wall production, consistent with the hypothesis that these bacteria may be transmitted environmentally during an extra‐host phase.     

Burkholderia BcpA mediates biofilm formation independently of interbacterial contact‐dependent growth inhibition

Contact‐dependent growth inhibition (CDI) is a phenomenon in which Gram‐negative bacteria use the toxic C‐terminus of a large surface‐exposed exoprotein to inhibit the growth of susceptible bacteria upon cell–cell contact. Little is known about when and where bacteria express the genes encoding CDI system proteins and how these systems contribute to the survival of bacteria in their natural niche. Here we establish that, in addition to mediating interbacterial competition, the Burkholderia thailandensis CDI system exoprotein BcpA is required for biofilm development. We also provide evidence that the catalytic activity of BcpA and extracellular DNA are required for the characteristic biofilm pillars to form. We show using a bcpA–gfp fusion that within the biofilm, expression of the CDI system‐encoding genes is below the limit of detection for the majority of bacteria and only a subset of cells express the genes strongly at any given time. Analysis of a strain constitutively expressing the genes indicates that native expression is critical for biofilm architecture. Although CDI systems have so far only been demonstrated to be involved in interbacterial competition, constitutive production of the system's immunity protein in the entire bacterial population did not alter biofilm formation, indicating a CDI‐independent role for BcpA in this process. We propose, therefore, that bacteria may use CDI proteins in cooperative behaviours, like building biofilm communities, and in competitive behaviours that prevent non‐self bacteria from entering the community.     

Organic and conventional fruits and vegetables contain equivalent counts of Gram‐negative bacteria expressing resistance to antibacterial agents

Resistance to antibiotics is a major public health problem which might culminate in outbreaks caused by pathogenic bacteria untreatable by known antibiotics. Most of the genes conferring resistance are acquired horizontally from already resistant commensal or environmental bacteria. Food contamination by resistant bacteria might be a significant source of resistance genes for human bacteria but has never been precisely assessed, nor is it known whether organic products differ in this respect from conventionally produced products. We showed here, on a large year‐long constructed sample set containing 399 products that, irrespective of their mode of production, raw fruits and vegetables are heavily contaminated by Gram‐negative bacteria (GNB) resistant to multiple antibiotics. Most of these bacteria originate in the soil and environment. We focused on non‐oxidative GNB resistant to third‐generation cephalosporins, because of their potential impact on human health. Among them, species potentially pathogenic for immunocompetent hosts were rare. Of the products tested, 13% carried bacteria producing extended – spectrum beta‐lactamases, all identified as Rahnella sp. which grouped into two phylotypes and all carrying the bla_RAHN gene. Thus, both organic and conventional fruits and vegetables may constitute significant sources of resistant bacteria and of resistance genes.     

Proteins from the FLOWERING LOCUS T‐like subclade of the PEBP family act antagonistically to regulate floral initiation in tobacco

Flowering is an important agronomic trait that often depends on the integration of photoperiod, vernalization, gibberellin and/or autonomous signaling pathways by regulatory proteins such as FLOWERING LOCUS T (FT), a member of the phosphatidylethanolamine‐binding protein (PEBP) family. Six PEBP family proteins control flowering in the model plant Arabidopsis thaliana, and their regulatory functions are well established, but variation in the number and structural diversity of PEBPs in different species means their precise functions must be determined on a case‐by‐case basis. We isolated four novel FT‐like genes from Nicotiana tabacum (tobacco), and determined their expression profiles in wild‐type plants and their overexpression phenotypes in transgenic plants. We found that all four genes were expressed in leaves under short‐day conditions, and at least NtFT3 expression was restricted to phloem companion cells. We also found that the NtFT1, NtFT2 and NtFT3 proteins are floral inhibitors (atypical for FT‐like proteins), whereas only NtFT4 is a floral inducer. We were unable to detect the expression of these genes under long‐day conditions, suggesting that all four tobacco FT‐like proteins may control flowering in response to short days. Phylogenetic analysis of PEBP family proteins and their functions in different solanaceous species confirmed that gene duplication and divergence within the FT‐like clade has led to the evolution of antagonistic regulators that may help to fine‐tune floral initiation in response to environmental cues.     

Bacterial endosymbiont infections in ‘living fossils’: a case study of North American vaejovid scorpions

Bacterial endosymbionts are common among arthropods, and maternally inherited forms can affect the reproductive and behavioural traits of their arthropod hosts. The prevalence of bacterial endosymbionts and their role in scorpion evolution have rarely been investigated. In this study, 61 samples from 40 species of scorpion in the family Vaejovidae were screened for the presence of the bacterial endosymbionts Cardinium, Rickettsia, Spiroplasma and Wolbachia. No samples were infected by these bacteria. However, one primer pair specifically designed to amplify Rickettsia amplified nontarget genes of other taxa. Similar off‐target amplification using another endosymbiont‐specific primer was also found during preliminary screenings. Results caution against the overreliance on previously published screening primers to detect bacterial endosymbionts in host taxa and suggest that primer specificity may be higher in primers targeting nuclear rather than mitochondrial genes.     

The Japanese ladybirds,Coccinula crotchi and Coccinula sinensis,are infected with very closely related strains of male-killing Flavobacterium

Male-killing is 1 of 4 known strategies that inherited parasitic endosymbionts have evolved to manipulate their host＇s reproduction. In early male-killing, infected male offspring are killed early in embryogenesis. Within the Insecta, male-killing bacteria have been found in a wide range of hosts. The Coccinellidae families of beetles, better known as ladybirds, are particularly prone to male-killer invasion. In samples of the coccinellid, Coc- cinula crotchi, from Japan, a new male-killing bacterium was revealed by phenotypic assay. Molecular genetic analysis revealed the identity to be a tetracycline-sensitive Flavobac- terium that causes female-biased offspring sex ratio. Furthermore, that Flavobacterium strain was found to be closely related to the Flavobacterium causing male-killing in the congeneric Japanese coccinellid, Coccinula sinensis, which was collected from the same region. However, we found slightly different Flavobacterium strains infecting C. sinen- sis from regions with different environmental conditions. This may be an indication of horizontal transmission of male-killing Flavobacterium between these 2 ladybird spices. Finally, environmental conditions may affect the spread of male-killing bacteria among their hosts.     

蚜虫与其胞内共生细菌的相互作用

蚜虫—巴克纳氏菌之间是一种典型的互利共生关系,两者相互依存,缺少一方,另一方便不能生存。研究表明,共生细菌能为寄主蚜虫提供必需氨基酸和维生索,并对寄主具有一些非营养功能,如促进蚜虫传播循环性病毒等。寄主蚜虫则是为共生菌提供一个合适的生存场所,并对共生菌的生长和繁殖进行调控。现代分子生物学技术从基因水平证明了蚜虫与共生菌的相互依赖性。     

A putative antimicrobial peptide from Hymenoptera in the megaplasmid pSCL4 of Streptomyces clavuligerus ATCC 27064 reveals a singular case of horizontal gene transfer with potential applications

Streptomyces clavuligerus is a Gram‐positive bacterium that is a high producer of secondary metabolites with industrial applications. The production of antibiotics such as clavulanic acid or cephamycin has been extensively studied in this species; nevertheless, other aspects, such as evolution or ecology, have received less attention. Furthermore, genes that arise from ancient events of lateral transfer have been demonstrated to be implicated in important functions of host species. This approximation discovered relevant genes that genomic analyses overlooked. Thus, we studied the impact of horizontal gene transfer in the S. clavuligerus genome. To perform this task, we applied whole‐genome analysis to identify a laterally transferred sequence from different domains. The most relevant result was a putative antimicrobial peptide (AMP) with a clear origin in the Hymenoptera order of insects. Next, we determined that two copies of these genes were present in the megaplasmid pSCL4 but absent in the S. clavuligerus ATCC 27064 chromosome. Additionally, we found that these sequences were exclusive to the ATCC 27064 strain (and so were not present in any other bacteria) and we also verified the expression of the genes using RNAseq data. Next, we used several AMP predictors to validate the original annotation extracted from Hymenoptera sequences and explored the possibility that these proteins had post‐translational modifications using peptidase cleavage prediction. We suggest that Hymenoptera AMP‐like proteins of S. clavuligerus ATCC 27064 may be useful for both species adaptation and as an antimicrobial molecule with industrial applications.     

Varying degree of physiological integration among host instars and their endoparasitoid affects stress‐induced mortality

In natural populations of insect herbivores, genetic differentiation is likely to occur due to variation in host plant utilization and selection by the local community of organisms with which they interact. In parasitoids, engaging in intimate associations with their host during immature development, local variation may exist in host quality for parasitoid development. We compared the development of a gregarious endoparasitoid, Cotesia glomerata L. (Hymenoptera: Braconidae), collected in The Netherlands, in three strains and three caterpillar instars (L1–L3) of its main host, Pieris brassicae L. (Lepidoptera: Pieridae). Hosts had been collected in The Netherlands and France, and were reared in the laboratory for one generation. We also used an established Dutch laboratory strain that had not been exposed to parasitoids for at least 24 generations. Parasitoid survival to adulthood was inversely correlated with host instar at parasitism. Adult parasitoid body mass was largest when hosts were parasitized as L1 and smallest when hosts were parasitized as L3, whereas egg‐to‐adult development time was quickest on L3 hosts and slowest on L1 hosts. Higher survival and faster development of C. glomerata on French L2 hosts also showed that there is variation in host‐instar‐related suitability. Many L2 and most L3 caterpillars that were parasitized exhibited signs of pathogen infection and perished within a few days of parasitism, whereas this never happened when hosts were parasitized as L1 or in non‐parasitized control caterpillars. Our results reveal that, irrespective of the host strain, L1 hosts are optimally synchronized with C. glomerata development. By contrast, the high precocious mortality of L3 larvae may be due to stress‐induced regulation by the parasitoid in order to ‘force’ its developmental program into synchrony with the developing parasitoid larvae. Our results underscore a potentially important role played by pathogens in mediating herbivore–parasitoid interactions that are host‐instar‐dependent in their expression.     

Aquatic animals promote antibiotic resistance gene dissemination in water via conjugation: Role of different regions within the zebra fish intestinal tract,and impact on fish intestinal microbiota

The aqueous environment is one of many reservoirs of antibiotic resistance genes (ARGs). Fish, as important aquatic animals which possess ideal intestinal niches for bacteria to grow and multiply, may ingest antibiotic resistance bacteria from aqueous environment. The fish gut would be a suitable environment for conjugal gene transfer including those encoding antibiotic resistance. However, little is known in relation to the impact of ingested ARGs or antibiotic resistance bacteria (ARB) on gut microbiota. Here, we applied the cultivation method, qPCR, nuclear molecular genetic marker and 16S rDNA amplicon sequencing technologies to develop a plasmid‐mediated ARG transfer model of zebrafish. Furthermore, we aimed to investigate the dissemination of ARGs in microbial communities of zebrafish guts after donors carrying self‐transferring plasmids that encode ARGs were introduced in aquaria. On average, 15% of faecal bacteria obtained ARGs through RP4‐mediated conjugal transfer. The hindgut was the most important intestinal region supporting ARG dissemination, with concentrations of donor and transconjugant cells almost 25 times higher than those of other intestinal segments. Furthermore, in the hindgut where conjugal transfer occurred most actively, there was remarkable upregulation of the mRNA expression of the RP4 plasmid regulatory genes, trbBp and trfAp. Exogenous bacteria seem to alter bacterial communities by increasing Escherichia and Bacteroides species, while decreasing Aeromonas compared with control groups. We identified the composition of transconjugants and abundance of both cultivable and uncultivable bacteria (the latter accounted for 90.4%–97.2% of total transconjugants). Our study suggests that aquatic animal guts contribute to the spread of ARGs in water environments.     

Molecular phylogenetic and divergence time estimation analyses of the sawfly subfamily Selandriinae (Hymenoptera: Tenthredinidae)

Selandriinae, a subfamily of family Tenthredinidae (Hymenoptera: Symphyta), comprises multiple tribes, each of which has a relationship with specific plant group. The host specificity of the Selandriinae taxa provides a good model to examine the coevolution between hosts and insects. However, few phylogenetic studies for the Selandriinae obscure the evolutionary scenario with their host‐plants. The present study is a molecular phylogenetic analysis of 19 selandriine species based on mitochondrial genes (12S: 461 sites, 16S: 262sites and COI: 495 sites) and nuclear genes (18S: 773 sites and 28S: 495 sites). The results suggested three of six studied tribes are genetically isolated. Moreover, estimation of the time of molecular divergence showed that the Selandriinae clearly diverged at the same time as their host‐plants (monocots and ferns). These results suggested that the Selandriinae species might have codiversified with their hosts.     

Genetic characterization of caffeine degradation by bacteria and its potential applications

The ability of bacteria to grow on caffeine as sole carbon and nitrogen source has been known for over 40 years. Extensive research into this subject has revealed two distinct pathways, N‐demethylation and C‐8 oxidation, for bacterial caffeine degradation. However, the enzymological and genetic basis for bacterial caffeine degradation has only recently been discovered. This review article discusses the recent discoveries of the genes responsible for both N‐demethylation and C‐8 oxidation. All of the genes for the N‐demethylation pathway, encoding enzymes in the Rieske oxygenase family, reside on 13.2‐kb genomic DNA fragment found in Pseudomonas putida CBB5. A nearly identical DNA fragment, with homologous genes in similar orientation, is found in Pseudomonas sp. CES. Similarly, genes for C‐8 oxidation of caffeine have been located on a 25.2‐kb genomic DNA fragment of Pseudomonas sp. CBB1. The C‐8 oxidation genes encode enzymes similar to those found in the uric acid metabolic pathway of Klebsiella pneumoniae. Various biotechnological applications of these genes responsible for bacterial caffeine degradation, including bio‐decaffeination, remediation of caffeine‐contaminated environments, production of chemical and fuels and development of diagnostic tests have also been demonstrated.