Marie B. Lund

Diversity, abundance and expression of nitrite reductase ( nirK )-like genes in marine thaumarchaea

Ammonia-oxidizing archaea (AOA) are widespread and abundant in aquatic and terrestrial habitats and appear to have a significant impact on the global nitrogen cycle. Like the ammonia-oxidizing bacteria, AOA encode a gene homologous to copper-containing nitrite reductases (nirK), which has been studied very little to date. In this study, the diversity, abundance and expression of thaumarchaeal nirK genes from coastal and marine environments were investigated using two mutually excluding primer pairs, which amplify the nirK variants designated as AnirKa and AnirKb. Only the AnirKa variant could be detected in sediment samples from San Francisco Bay and these sequences grouped with the nirK from Candidatus Nitrosopumilus maritimus and Candidatus Nitrosoarchaeum limnia. The two nirK variants had contrasting distributions in the water column in Monterey Bay and the California Current. AnirKa was more abundant in the epi- to mesopelagic Monterey Bay water column, whereas AnirKb was more abundant in the meso- to bathypelagic California Current water. The abundance and community composition of AnirKb, but not AnirKa, followed that of thaumarchaeal amoA, suggesting that either AnirKa is not exclusively associated with AOA or that commonly used amoA primers may be missing a significant fraction of AOA diversity in the epipelagic. Interestingly, thaumarchaeal nirK was expressed 10–100-fold more than amoA in Monterey Bay. Overall, this study provides valuable new insights into the distribution, diversity, abundance and expression of this alternative molecular marker for AOA in the ocean.

Diversity and host specificity of the Verminephrobacter–earthworm symbiosis

Symbiotic bacteria of the genus Verminephrobacter (Betaproteobacteria) were detected in the nephridia of 19 out of 23 investigated earthworm species (Oligochaeta: Lumbricidae) by 16S rRNA gene sequence analysis and fluorescence in situ hybridization (FISH). While all four Lumbricus species and three out of five Aporrectodea species were densely colonized by a mono-species culture of Verminephrobacter, other earthworm species contained mixed bacterial populations with varying proportions of Verminephrobacter; four species did not contain Verminephrobacter at all. The Verminephrobacter symbionts could be grouped into earthworm species-specific sequence clusters based on their 16S rRNA and RNA polymerase subunit B (rpoB) genes. Closely related host species harboured more closely related symbionts than did distantly related hosts. Co-diversification of the symbiotic partners could not be demonstrated unambiguously due to the poor resolution of the host phylogeny [based on histone H3 and cytochrome c oxidase subunit I (COI) gene sequence analyses]. However, there was a pattern of symbiont diversification within four groups of closely related hosts. The mean rate of symbiont 16S rRNA gene evolution was determined using a relaxed clock model, and the rate was calibrated with paleogeographical estimates of the time of origin of Lumbricid earthworms. The calibrated rates of symbiont 16S rRNA gene evolution are 0.012-0.026 substitutions per site per 50 million years and thus similar to rates reported from other symbiotic bacteria.

Beneficial effect of Verminephrobacter nephridial symbionts on the fitness of the earthworm Aporrectodea tuberculata.

ABSTRACT Almost all lumbricid earthworms (Oligochaeta: Lumbricidae) harbor species-specific Verminephrobacter (Betaproteobacteria) symbionts in their nephridia (excretory organs). The function of the symbiosis, and whether the symbionts have a beneficial effect on their earthworm host, is unknown; however, the symbionts have been hypothesized to enhance nitrogen retention in earthworms. The effect of Verminephrobacter on the life history traits of the earthworm Aporrectodea tuberculata (Eisen) was investigated by comparing the growth, development, and fecundity of worms with and without symbionts given high (cow dung)- and low (straw)-nutrient diets. There were no differences in worm growth or the number of cocoons produced by symbiotic and aposymbiotic worms. Worms with Verminephrobacter symbionts reached sexual maturity earlier and had higher cocoon hatching success than worms cured of their symbionts when grown on the low-nutrient diet. Thus, Verminephrobacter nephridial symbionts do have a beneficial effect on their earthworm host. Cocoons with and without symbionts did not significantly differ in total organic carbon, total nitrogen, or total hydrolyzable amino acid content, which strongly questions the hypothesized role of the symbionts in nitrogen recycling for the host.

Detection and persistence of fecal Bacteroidales as water quality indicators in unchlorinated drinking water.

The results of this study support the use of fecal Bacteroidales qPCR as a rapid method to complement traditional, culture-dependent, water quality indicators in systems where drinking water is supplied without chlorination or other forms of disinfection. A SYBR-green based, quantitative PCR assay was developed to determine the concentration of fecal Bacteroidales 16S rRNA gene copies. The persistence of a Bacteroides vulgatus pure culture and fecal Bacteroidales from a wastewater inoculum was determined in unchlorinated drinking water at 10 degrees C. B. vulgatus 16S rRNA gene copies persisted throughout the experimental period (200 days) in sterile drinking water but decayed faster in natural drinking water, indicating that the natural microbiota accelerated decay. In a simulated fecal contamination of unchlorinated drinking water, the decay of fecal Bacteroidales 16S rRNA gene copies was considerably faster than the pure culture but similar to that of Escherichia coli from the same wastewater inoculum.

Purifying selection and molecular adaptation in the genome of Verminephrobacter, the heritable symbiotic bacteria of earthworms

While genomic erosion is common among intracellular symbionts, patterns of genome evolution in heritable extracellular endosymbionts remain elusive. We study vertically transmitted extracellular endosymbionts (Verminephrobacter, Betaproteobacteria) that form a beneficial, species-specific, and evolutionarily old (60–130 Myr) association with earthworms. We assembled a draft genome of Verminephrobacter aporrectodeae and compared it with the genomes of Verminephrobacter eiseniae and two nonsymbiotic close relatives (Acidovorax). Similar to V. eiseniae, the V. aporrectodeae genome was not markedly reduced in size and showed no A–T bias. We characterized the strength of purifying selection (ω = dN/dS) and codon usage bias in 876 orthologous genes. Symbiont genomes exhibited strong purifying selection (ω = 0.09 ± 0.07), although transition to symbiosis entailed relaxation of purifying selection as evidenced by 50% higher ω values and less codon usage bias in symbiont compared with reference genomes. Relaxation was not evenly distributed among functional gene categories but was overrepresented in genes involved in signal transduction and cell envelope biogenesis. The same gene categories also harbored instances of positive selection in the Verminephrobacter clade. In total, positive selection was detected in 89 genes, including also genes involved in DNA metabolism, tRNA modification, and TonB-dependent iron uptake, potentially highlighting functions important in symbiosis. Our results suggest that the transition to symbiosis was accompanied by molecular adaptation, while purifying selection was only moderately relaxed, despite the evolutionary age and stability of the host association. We hypothesize that biparental transmission of symbionts and rare genetic mixing during transmission can prevent genome erosion in heritable symbionts.

Evolution of the tripartite symbiosis between earthworms, Verminephrobacter and Flexibacter-like bacteria

Nephridial (excretory organ) symbionts are widespread in lumbricid earthworms and the complexity of the nephridial symbiont communities varies greatly between earthworm species. The two most common symbionts are the well-described Verminephrobacter and less well-known Flexibacter-like bacteria. Verminephrobacter are present in almost all lumbricid earthworms, they are species-specific, vertically transmitted, and have presumably been associated with their hosts since the origin of lumbricids. Flexibacter-like symbionts have been reported from about half the investigated earthworms; they are also vertically transmitted. To investigate the evolution of this tri-partite symbiosis, phylogenies for 18 lumbricid earthworm species were constructed based on two mitochondrial genes, NADH dehydrogenase subunit 2 (ND2) and cytochrome c oxidase subunit I (COI), and compared to their symbiont phylogenies based on RNA polymerase subunit B (rpoB) and 16S rRNA genes. The two nephridial symbionts showed markedly different evolutionary histories with their hosts. For Verminephrobacter, clear signs of long-term host-symbiont co-evolution with rare host switching events confirmed its ancient association with lumbricid earthworms, likely dating back to their last common ancestor about 100 million years (MY) ago. In contrast, phylogenies for the Flexibacter-like symbionts suggested an ability to switch to new hosts, to which they adapted and subsequently became species-specific. Putative co-speciation events were only observed with closely related host species; on that basis, this secondary symbiosis was estimated to be minimum 45 MY old. Based on the monophyletic clustering of the Flexibacter-like symbionts, the low 16S rRNA gene sequence similarity to the nearest described species (<92%) and environmental sequences (<94.2%), and the specific habitat in the earthworm nephridia, we propose a new candidate genus for this group, Candidatus Nephrothrix.

The earthworm-Verminephrobacter symbiosis: an emerging experimental system to study extracellular symbiosis.

Almost all Lumbricid earthworms (Oligochaeta: Lumbricidae) harbor extracellular species-specific bacterial symbionts of the genus Verminephrobacter (Betaproteobacteria) in their nephridia. The symbionts have a beneficial effect on host reproduction and likely live on their hosts waste products. They are vertically transmitted and presumably associated with earthworms already at the origin of Lumbricidae 62–136 million years ago. The Verminephrobacter genomes carry signs of bottleneck-induced genetic drift, such as accelerated evolutionary rates, low codon usage bias, and extensive genome shuffling, which are characteristic of vertically transmitted intracellular symbionts. However, the Verminephrobacter genomes lack AT bias, size reduction, and pseudogenization, which are also common genomic hallmarks of vertically transmitted, intracellular symbionts. We propose that the opportunity for genetic mixing during part of the host—symbiont life cycle is the key to evade drift-induced genome erosion. Furthermore, we suggest the earthworm-Verminephrobacter association as a new experimental system for investigating host-microbe interactions, and especially for understanding genome evolution of vertically transmitted symbionts in the presence of genetic mixing.

Distinct effects of the nephridial symbionts Verminephrobacter and Candidatus Nephrothrix on reproduction and maturation of its earthworm host Eisenia andrei

ABSTRACT Verminephrobacter, the most common specific symbionts in the nephridia (excretory organs) of lumbricid earthworms, have been shown to improve reproduction of the garden earthworm Aporrectodea tuberculata under nutrient limitation. It is unknown how general this beneficial trait is in the Verminephrobacter‐earthworm symbiosis, whether other nephridial symbionts also affect host fitness and what the mechanism of the fitness increase is. Here we report beneficial effects of Verminephrobacter and Candidatus Nephrothrix on life history traits of the compost worm Eisenia andrei, which in addition to these two symbionts also hosts Agromyces‐like bacteria in its mixed nephridial community: while growth was identical between control, Verminephrobacter‐free and aposymbiotic worms, control worms produced significantly more cocoons and offspring than both Verminephrobacter‐free and aposymbiotic worms, confirming the reproductive benefit of Verminephrobacter in a second host with different ecology and feeding behavior. Furthermore, worms with Verminephrobacter and Ca. Nephrothrix, or with only Ca. Nephrothrix present, reached sexual maturity significantly earlier than aposymbiotic worms; this is the first evidence for a beneficial role of Ca. Nephrothrix in earthworms. Riboflavin content in cocoons and whole earthworms was unaffected by the presence or absence of nephridial symbionts, suggesting that nutritional supplementation with this vitamin does not play a major role in this symbiosis.

Biparental transmission of Verminephrobacter symbionts in the earthworm Aporrectodea tuberculata (Lumbricidae)

Abstract Most lumbricid earthworms harbor species‐specific Verminephrobacter symbionts in their excretory organs (nephridia). These symbionts are vertically transmitted via the cocoon, where they colonize the embryos. Despite cospeciation for >100 million years with their hosts, Verminephrobacter lack genome reduction and AT bias typical of evolutionary old, vertically transmitted symbionts, caused by recurring bottlenecks. We hypothesized that biparental symbiont transmission into the cocoon enabled genetic mixing and relieved the bottleneck, and tested biparental transmission experimentally for V. aporrectodeae subsp. tuberculata, the specific symbiont of the earthworm Aporrectodea tuberculata, for which aposymbiotic worm lines are available. Virgin symbiotic and aposymbiotic adult worms were tagged, mated in pairs, separated before the start of cocoon production and their offspring assessed for Verminephrobacter. Specific PCR detected the symbionts in 41.5% of 188 juveniles produced by 20 aposymbiotic worms; fluorescence in situ hybridization showed a patchy but successful colonization of their nephridia. Symbionts were present in the mucus but absent in feed, soil, and spermatophora/nephridia of the aposymbiotic partner, suggesting symbiont transfer via mucus during mating. These results are consistent with the hypothesis that genome evolution in Verminephrobacter is distinct from other vertical‐ly transmitted symbionts due to genetic mixing during transmission, partially facilitated by biparental transmission.

Earthworm ecology affects the population structure of their Verminephrobacter symbionts

Earthworms carry species-specific Verminephrobacter symbionts in their nephridia (excretory organs). The symbionts are vertically transmitted via the cocoon, can only colonize the host during early embryonic development, and have co-speciated with their host for about 100 million years. Although several studies have addressed Verminephrobacter diversity between worm species, the intra-species diversity of the symbiont population has never been investigated. In this study, symbiont population structure was examined by using a multi-locus sequence typing (MLST) approach on Verminephrobacter isolated from two contrasting ecological types of earthworm hosts: the high population density, fast reproducing compost worms, Eisenia andrei and Eisenia fetida, and the low-density, slow reproducing Aporrectodea tuberculata, commonly found in garden soils. Three distinct populations were investigated for both types and, according to MLST analysis of 193 Verminephrobacter isolates, the symbiont community in each worm individual was very homogeneous. The more solitary A. tuberculata carried unique symbiont populations in 9 out of 10 host individuals, whereas the symbiont populations in the social compost worms were homogeneous across host individuals from the same population. These data suggested that host ecology shaped the population structure of Verminephrobacter symbionts. The homogeneous symbiont populations in the compost worms led to the hypothesis that Verminephrobacter could be transferred bi-parentally or via leaky horizontal transmission in high-density, frequently mating worm populations.