Exchange of Microbiomes in Plant-Insect Herbivore Interactions.

Prokaryotic and eukaryotic microbial symbiotic communities span through kingdoms. The vast microbial gene pool extends the host genome and supports adaptations to changing environmental conditions. Plants are versatile hosts for the symbionts, carrying microbes on the surface, inside tissues, and even within the cells. Insects are equally abundantly colonized by microbial symbionts on the exoskeleton, in the gut, in the hemocoel, and inside the cells. The insect gut is a prolific environment, but it is selective on the microbial species that enter with food. Plants and insects are often highly dependent on each other and frequently interact. Regardless of the accumulating evidence on the microbiomes of both organisms, it remains unclear how much they exchange and modify each other's microbiomes. In this review, we approach this question from the point of view of herbivores that feed on plants, with a special focus on the forest ecosystems. After a brief introduction to the subject, we concentrate on the plant microbiome, the overlap between plant and insect microbial communities, and how the exchange and modification of microbiomes affects the fitness of each host.

Child type 1 diabetes associated with mother vaginal bacteriome and mycobiome.

Mother vaginal microbes contribute to microbiome of vaginally delivered neonates. Child microbiome can be associated with autoimmune diseases, such as type 1 diabetes (T1D). We collected vaginal DNA samples from 25 mothers with a vaginally delivered child diagnosed with T1D and samples from 24 control mothers who had vaginally delivered a healthy child and analyzed bacteriome and mycobiome of the samples. The total DNA of the samples was extracted, and ribosomal DNA regions (16S for bacteria, ITS2 for fungi) were amplified, followed by next-generation sequencing and machine learning. We found that alpha-diversity of bacteriome was increased (P < 0.002), whereas alpha-diversity of mycobiome was decreased (P < 0.001) in mothers with a diabetic child compared to the control mothers. Beta-diversity analysis suggested differences in mycobiomes between the mother groups (P = 0.001). Random forest models were able to effectively predict diabetes and control status of unknown samples (bacteria: 0.86 AUC, fungi: 0.96 AUC). Our data indicate several fungal genera and bacterial metabolic pathways of mother vaginal microbiome to be associated with child T1D. We suggest that early onset of T1D in a child has a relationship with altered mother vaginal microbiome and that both bacteriome and mycobiome contribute to this shift.

Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

Tonsillar microbiota in children with PFAPA (periodic fever, aphthous stomatitis, pharyngitis, and adenitis) syndrome.

Periodic fever, aphthous stomatitis, pharyngitis, and adenitis (PFAPA) is a childhood febrile syndrome of unknown origin that is often cured with tonsillectomy. We aimed to compare the bacterial microbiota of the tonsils removed from PFAPA patients with those of controls. We used next-generation sequencing technology to investigate the bacterial microbiota of the tonsils of 30 PFAPA patients and 24 controls. We found significant differences in the presence and relative abundance of many bacteria between PFAPA cases and controls. For example, cyanobacteria, potential producers of microcystins and other toxins, were more common in the case samples (14/30, 47 %) than in the controls (4/24, 17 %, p = 0.02), and the mean relative abundance of cyanobacteria was higher in the case samples (0.2 %) than in the controls (0.01 %, p = 0.01). Streptococci were present in all samples in both groups, but their mean relative abundance was lower in the case samples (3.7 %) than in the controls (9.6 %, p = 0.01). Typical nasopharyngeal microbes such as fusobacteria, Prevotella, Tannerella, Porphyromonas, and Parvimonas dominated the microbiota of the tonsils in both groups. The microbiota of the tonsils removed from PFAPA patients differed significantly from those of the controls. Tonsillar microbiota may play a role in triggering the inflammatory processes that lead to symptoms of PFAPA.

Faecal microbiome in new-onset juvenile idiopathic arthritis.

Alterations in the intestinal microbial flora have been linked with autoimmune diseases. Our objective was to analyse the composition of the faecal microbiome of children with new-onset juvenile idiopathic arthritis (JIA) compared to healthy controls, and to identify specific gut bacteria associated with JIA. Stool samples from patients were taken at the time of diagnosis of JIA. The microbiome profiles of samples of 30 children with JIA (mean age 6.2 years, 22 girls) were analysed with 16S region-based sequencing profiling and compared to the stool samples of healthy controls (n = 27, mean age 5.4 years, 18 girls). The proportion of bacteria belonging to the phylum Firmicutes was significantly lower in children with JIA [21 % (95 % confident interval [CI]: 17-25 %)] compared to controls [33 % (95 % CI: 26-41 %), p = 0.009]. Bacteria belonging to Bacteroidetes were significantly more abundant in JIA [78 % (95 % CI: 74-82 %)] than in control samples [65 % (95 % CI: 57-73 %), p = 0.008]. Shared operational taxonomic units (OTUs) between the groups revealed that genera Actinobacteria and Fusobacteria were present only in JIA patients and Lentisphaerae only in controls. In summary, faecal flora in JIA is characterised by a low level of Firmicutes and an abundance of Bacteroidetes, resembling the aberration reported in type 1 diabetes. We suggest that alterations in the intestinal microbial flora may challenge the mucosal immune system of genetically susceptible subjects predisposing to local proinflammatory cascades, thus contributing to the development of JIA.

Biofilm formation and virulence of uropathogenic Escherichia coli in urine after consumption of cranberry-lingonberry juice.

Cranberry-lingonberry juice (CLJ) was effective in preventing urinary tract infections (UTIs) in our earlier randomized clinical trial. We aimed to test whether consumption of CLJ at a similar dose to earlier reduces the biofilm formation and virulence of uropathogenic Escherichia coli in urine. Twenty healthy women drank 100 ml of CLJ daily for two weeks. Urine samples were obtained 2-4 hours after the last dose. Control samples were taken after a one-week period without berry consumption. Biofilm formation of 20 E. coli strains was measured at 72 hours by the polystyrene microtitre plate method. Quantitative real-time PCR analyses were performed for selected genes. Four of the 20 clinical strains produced more biofilm in urine after CLJ consumption (P < 0.05) and one produced less. Expression levels of the pga, cpxA, fimA and papF genes did not differ between bacteria grown in control urine and urine obtained after CLJ consumption, except for pga gene expression, which was reduced in one strain after CLJ (P = 0.04). It appears that the effect of CLJ in preventing UTIs is not explained by mechanisms that reduce biofilm formation or the expression of selected virulence genes of Escherichia coli in urine.

Methylobacterium sp. resides in unculturable state in potato tissues in vitro and becomes culturable after induction by Pseudomonas fluorescens IMGB163.

AIMS: To induce growth of endophytic bacteria residing in an unculturable state in tissues of in vitro-grown potato plantlets. To isolate and identify the induced bacteria and to localize the strains in tissues of in vitro-grown potato plantlets. METHODS AND RESULTS: The inoculation of in vitro-grown potato plants with Pseudomonas fluorescens IMBG163 led to induction of another bacterium, a pink-pigmented facultative methylotroph that was identified as Methylobacterium sp. using phylogenetic 16S rDNA approach. Two molecular methods were used for localizing methylobacteria in potato plantlets: PCR and in situ hybridization (ISH/FISH). A PCR product specific for the Methylobacterium genus was found in DNA isolated from the surface-sterilized plantlet leaves. Presence of Methylobacterium rRNA was detected by ISH/FISH in leaves and stems of inoculated as well as axenic potato plantlets although the bacterium cannot be isolated from the axenic plants. CONCLUSION: Methylobacterium sp. resides in unculturable state within tissues of in vitro-grown potato plants and becomes culturable after inoculation with P. fluorescens IMBG163. SIGNIFICANCE AND IMPACT OF THE STUDY: In order to develop endophytic biofertilizers and biocontrol agents, a detailed knowledge of the life-style of endophytes is essential. To our knowledge, this is the first report on increase of the culturability of endophytes in response to inoculation by nonpathogenic bacteria.

Phylogenetic background of Orange lily (Lilium bulbiferum s.l.) cultivars from a genetically isolated environment.

Domesticated Orange lily ( LILIUM BULBIFERUM s.l.) cultivars do not typically produce seeds, and Orange lily is not native to Finland. Therefore, back crossing of the cultivars with wild species has not been possible. Genetic variability and genuineness of eight Finnish traditionally-grown Orange lily cultivars was studied. RAPD patterns were compared between the cultivars and genuine Orange lily ( LILIUM BULBIFERUM L.), and a related Dauricum group. The results showed partition of tested genotypes into four groups, L. CANADENSE as the outgroup. The cultivars were divided into two subgroups where the trait to form bulbils was characteristic to subgroup I. The cultivated strains differed from each other as much as from the seedling strains, but were genetically closer to genuine Orange lily than the Dauricum group. This indicates that the cultivars are genuine forms of Orange lily species. The special morphological features of the cultivars have likely been formed during centuries-long genetic isolation from natural populations.

Expression profile analysis of wild-type and fcc1 mutant strains of Fusarium verticillioides during fumonisin biosynthesis.

Fusarium verticillioides produces a group of mycotoxins known as fumonisins that are associated with a variety of mycotoxicoses in humans and animals. In this study, DNA microarrays were constructed with expressed sequence tags (ESTs) from F. verticillioides. To identify genes with patterns of expression similar to the fumonisin biosynthetic (FUM) genes, the microarray was probed with labeled cDNAs originating from a wild-type strain and a fcc1 mutant grown on maize and in a defined medium adjusted to either pH 3 or pH 8. The comparative analyses revealed differential expression of genes corresponding to 116 ESTs when the fungal strains were grown on maize. Under different pH conditions, 166 ESTs were differentially expressed, and 19 ESTs were identified that displayed expression patterns similar to the FUM ESTs. These results provide candidate genes with potential roles in fumonisin biosynthesis.

Two endophytic fungi in different tissues of scots pine buds (Pinus sylvestris L.).

Two fungal species were isolated with different frequencies from pine tissue cultures originating from buds. One species was detected in 33.1% of the cultures initiated in March, and another was present in 1.7% of cultures initiated in June. Based on analyses of phylogenetic and physiological characteristics these fungi were identified as Hormonema dematioides (isolated in March) and Rhodotorula minuta (isolated in June). Probes targeted towards the 18S rRNA of H. dematioides and R. minuta were made. When in situ hybridizations were performed on pine bud tissue, R. minuta was detected inside the cells of meristematic tissue in 40% of the samples, in contrast to H. dematioides, which was not found in this tissue. Using light microscopy, H. dematioides was found to be localized in the scale tissues of the buds. Fungal endophytes have previously been detected in scale tissues, but not in the meristematic tissues of buds. The habitats of these fungi may reflect their different roles in the plant.

Isolation of high quality RNA from bilberry (Vaccinium myrtillus L.) fruit.

A simple and efficient method is described for isolating high quality RNA from bilberry fruit. The procedure is based on the use of hexadecyltrimethyl ammonium bromide (CTAB), polyvinylpyrrolidone (PVP), and beta-mercaptoethanol in an extraction buffer in order to eliminate the polysaccharides and prevent the oxidation of phenolic compounds. This method is a modification of the one described for pine trees, and yields high-quality RNA suitable for cDNA based methodologies. This method is applicable for a variety of plant tissues.

Detection of intracellular bacteria in the buds of Scotch pine (Pinus sylvestris L.) by in situ hybridization.

Bacterial isolates were obtained from pine (Pinus sylvestris L.) tissue cultures and identified as Methylobacterium extorquens and Pseudomonas synxantha. The existence of bacteria in pine buds was investigated by 16S rRNA in situ hybridization. Bacteria inhabited the buds of every tree examined, primarily colonizing the cells of scale primordia and resin ducts.

Cloning and characterization of a novel human lysyl hydroxylase isoform highly expressed in pancreas and muscle.

We report the isolation and characterization of cDNA clones for a novel isoform of lysyl hydroxylase (lysyl hydroxylase 2), a posttranslational enzyme of collagen biosynthesis. The open reading frame predicted a protein of 737 amino acids, including an amino-terminal signal peptide. The amino acid sequence has overall similarity of over 75% to the lysyl hydroxylase (lysyl hydroxylase 1) characterized earlier. This similarity is even higher in the carboxyl-terminal end of the molecules. Lysyl hydroxylase 2 contains nine cysteine residues, which are conserved in lysyl hydroxylase 1. Furthermore, the conserved histidines and aspartate residues required for lysyl hydroxylase activity are present in the sequence. Northern analysis identified a transcript of 4.2 kilobases, which was highly expressed in pancreas and muscle tissues. Expression of cDNA in insect cells using a baculovirus vector yielded proteins with lysyl hydroxylase activity and an antiserum against a synthetic peptide of the deduced amino acid sequence recognized proteins with molecular weights of 88 and 97 kDa in homogenates of the transfected cells.