Gareth Lloyd-Jones

Biodiversity of Active and Inactive Bacteria in the Gut Flora of Wood-Feeding Huhu Beetle Larvae (Prionoplus reticularis)

ABSTRACT Huhu grubs (Prionoplus reticularis) are wood-feeding beetle larvae endemic to New Zealand and belonging to the family Cerambycidae. Compared to the wood-feeding lower termites, very little is known about the diversity and activity of microorganisms associated with xylophagous cerambycid larvae. To address this, we used pyrosequencing to evaluate the diversity of metabolically active and inactive bacteria in the huhu larval gut. Our estimate, that the gut harbors at least 1,800 phylotypes, is based on 33,420 sequences amplified from genomic DNA and reverse-transcribed RNA. Analysis of genomic DNA- and RNA-derived data sets revealed that 71% of all phylotypes (representing 95% of all sequences) were metabolically active. Rare phylotypes contributed considerably to the richness of the community and were also largely metabolically active, indicating their participation in digestive processes in the gut. The dominant families in the active community (RNA data set) included Acidobacteriaceae (24.3%), Xanthomonadaceae (16.7%), Acetobacteraceae (15.8%), Burkholderiaceae (8.7%), and Enterobacteriaceae (4.1%). The most abundant phylotype comprised 14% of the active community and affiliated with Dyella ginsengisoli (Gammaproteobacteria), suggesting that a Dyella-related organism is a likely symbiont. This study provides new information on the diversity and activity of gut-associated microorganisms that are essential for the digestion of the nutritionally poor diet consumed by wood-feeding larvae. Many huhu gut phylotypes affiliated with insect symbionts or with bacteria present in acidic environments or associated with fungi.

Sphingobium scionense sp. nov., an aromatic hydrocarbon-degrading bacterium isolated from contaminated sawmill soil.

This study characterized strain WP01(T), a Gram-staining-negative, rod-shaped, aerobic bacterium isolated from a polycyclic aromatic hydrocarbon-contaminated soil in New Zealand. Strain WP01(T) shared many characteristics of the genus Sphingobium: the predominant respiratory quinone (89 %) was ubiquinone with ten isoprene units (Q-10); the major fatty acids were C(18 : 1)omega7c, C(16 : 1)omega7c, C(16 : 0) and C(14 : 0) 2-OH; spermidine was the major polyamine; the DNA G+C content was 63.8 mol%; and the Sphingobium-specific 16S rRNA signatures were conserved. A point of difference from other species of the genus Sphingobium was that strain WP01(T) reduced nitrate to nitrite. The polar lipid pattern consisted of the predominant compounds diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol and sphingoglycolipids. 16S rRNA gene sequence analysis showed that, amongst the recognized species of the genus Sphingobium, strain WP01(T) was most similar to Sphingobium yanoikuyae GIFU 9882(T) and Sphingobium amiense YT(T) (>97 % 16S rRNA gene sequence similarities). The low DNA-DNA relatedness values between strain WP01(T) and S. yanoikuyae GIFU 9882(T) (46.6 %) and S. amiense DSM 16289(T) (25.6 %) indicated no relatedness at the species level. On the basis of these characteristics, it is concluded that strain WP01(T) should be considered as representing a novel species within the genus Sphingobium, for which the name Sphingobium scionense sp. nov. is proposed. The type strain is WP01(T) (=DSM 19371(T)=ICMP 13533(T)).

Bacterial community composition of a wastewater treatment system reliant on N2 fixation

The temporal stability and change of the dominant phylogenetic groups of the domain bacteria were studied in a model plant-based industrial wastewater treatment system showing high levels of organic carbon removal supported by high levels of N2 fixation. Community profiles were obtained through terminal restriction fragment length polymorphism analysis and cloning of 16S rRNA amplicons followed by sequencing. Bacterial community profiles showed that ten common terminal restriction fragments made up approximately 50% of the measured bacterial community. As much as 42% of the measured bacterial community could be monitored by using quantitative PCR and primers that targeted three dominant operational taxonomic units. Despite changes in wastewater composition and dissolved oxygen levels, the bacterial community composition appeared stable and was dominated by α-Proteobacteria and β-Proteobacteria, with a lesser amount of the highly diverse bacterial phylum Bacteroidetes. A short period of considerable change in the bacterial community composition did not appear to affect treatment performance indicating functional redundancy in this treatment system.

Stable isotope probing: Technical considerations when resolving 15N-labeled RNA in gradients

RNA based stable isotope probing (SIP) facilitates the detection and identification of active members of microbial populations that are involved in the assimilation of an isotopically labeled compound. (15)N-RNA-SIP is a new method that has been discussed in recent literature but has not yet been tested. Herein, we define the limitations to using (15)N-labeled substrates for SIP and propose modifications to compensate for some of these shortcomings. We have used (15)N-RNA-SIP as a tool for analysing mixed bacterial populations that use nitrogen substrates. After incubating mixed microbial communities with (15)N-ammonium chloride or (15)N(2) we assessed the fractionation resolution of (15)N-RNA by isopycnic centrifugation in caesium trifluoroacetate (CsTFA) gradients. We found that the more isotopic label incorporated, the further the buoyant density (BD) separation between (15)N- and (14)N-RNA, however it was not possible to resolve the labeled from unlabeled RNA definitively through gradient fractionation. Terminal-restriction fragment length polymorphism (T-RFLP) analysis of the extracted RNA and fluorescent in situ hybridisation (FISH) analysis of the enrichment cultures provided some insight into the organisms involved in nitrogen fixation. This approach is not without its limitations and will require further developments to assess its applicability to other nitrogen-fixing environments.

Composition of nifH in a wastewater treatment system reliant on N2 fixation

High levels of nitrogen fixation have been observed in the wastewaters of pulp and paper mills. In this study, we show that nitrogen fixation in a model pulp and paper wastewater treatment system is supported by a high density of nifH sequences that are of low diversity. Quantitative PCR revealed a ratio of nifH to 16S rDNA of 1.14 ± 0.76 which shows that very high levels of the nifH gene were enriched to support the high rates of nitrogen fixation that occur in this wastewater. Changes in wastewater composition and dissolved oxygen levels did not affect the nifH levels and allowed stable wastewater treatment. The nifH sequences identified display a similar profile to those seen in forest soil environments where nifH sequences derived from α-proteobacteria and β-proteobacteria are also prevalent.

Identifying diazotrophs by incorporation of nitrogen from 15N2 into RNA.

The diversity and abundance of active diazotrophs was investigated in a New Zealand pulp and paper wastewater by enrichment with 15N2. Purified 15N-RNA was analysed by reverse transcription, molecular cloning and sequence analysis of 16S rRNA to reveal a diverse community of bacteria as indicated by a Shannon Weaver Index value of > 2.8. The major class represented in the enriched culture were the γ-Proteobacteria at 85% with a secondary group of the phylum Firmicutes present at 8.2%, the remaining sequences were affiliated with the α- and β-Proteobacterial classes (1.4% and 4.3%, respectively). Three dominant genera, Aeromonas, Pseudomonas and Bacillus, were identified by comparison with published sequences and phylogenetic analysis. To confirm that representatives of the taxonomic groups identified from the active enriched nitrogen-fixing community were capable of fixing nitrogen Aeromonas and Pseudomonas species were cultivated and shown to possess nifH genes. In wastewater, fluorescence in situ hybridisation probing revealed that the dominant nitrogen-fixing population identified in this study were present in the population, but at lower levels. The population is, therefore, reliant on a small sub-population of diazotrophs to supply the communitys nitrogen needs above that already present in the wastewater.

The bacterial microbiota of Stolotermes ruficeps (Stolotermitidae), a phylogenetically basal termite endemic to New Zealand.

Stolotermes ruficeps is a widespread, primitive, lower termite occupying dead and decaying wood of many tree species in New Zealands temperate forests. We identified core bacterial taxa involved in gut processes through combined DNA- and RNA (cDNA)-based pyrosequencing analysis of the 16S nucleotide sequence from five S. ruficeps colonies. Most family and many genus-level taxa were common to S. ruficeps colonies despite being sampled from different tree species. Major taxa identified were Spirochaetaceae, Elusimicrobiaceae and Porphyromonadaceae. Others less well known in termite guts were Synergistaceae, Desulfobacteraceae, Rhodocyclaceae, Lachnospiraceae and Ruminococcaceae. Synergistaceae, Lachnospiraceae and Spirochaetaceae were well represented in the RNA data set, indicating a high-protein synthesis potential. Using 130,800 sequences from nine S. ruficeps DNA and RNA data sets, we estimated a high level of bacterial richness (4024 phylotypes at 3% genetic distance). Very few abundant phylotypes were site-specific; almost all (95%) abundant phylotypes, representing 97% of data set sequences, were detected in at least two S. ruficeps colonies. This study of a little-researched phylogenetically basal termite identifies core bacteria taxa. These findings will extend inventories of termite gut microbiota and contribute to the understanding of the specificity of termite gut microbiota.

Heterologous hybridisation to a Pinus microarray: profiling of gene expression in Pinus radiata saplings exposed to ethephon

BackgroundThe design, construction and application of a Pinus microarray platform are described. The oligonucleotide microarray was developed using publicly available Pinus cDNA sequences mostly derived from Pinus taeda to test whether heterologous hybridisation of microarray probes will generate useful data when hybridised with cRNA constructed from the dominant New Zealand forestry species Pinus radiata.MethodsA comprehensive consensus sequence collection of Pinus cDNA sequences was collated into a non-redundant database used for automated design of 60-mer oligonucleotide microarray probes. The microarray slides, manufactured by Agilent Technologies (Palo Alto, California), were used to monitor gene expression in an induction experiment using 2-chloroethylphosphonic acid, common name ethephon and the active ingredient of the plant growth regulator Ethrel® (Bayer Crop Science). The transcriptomes from tissues of 2-year old Pinus radiata saplings +/− ethephon treatment were compared by hybridisation onto the Pinus microarray slides.ResultsStatistically significant differentially expressed genes identified by heterologous hybridisation to the Pinus microarray following ethephon induction included the up-regulation of genes in the xylem that were related to the metabolism of phenylpropanoids and flavonoids, and also defence responses, specifically against fungal/insect attack and oxidative stress. Bark, mucilaginous xylem and xylem generated largely mutually exclusive cohorts of genes and Gene Ontology (GO) classes. The results are also interpreted in reference to gross and microscopic morphological changes. Samples of gene responses were validated by quantitative RT-PCR.ConclusionThese results confirm the successful development of a Pinus microarray and demonstrate the utility of the microarray for transcriptomic research in Pinus radiata through heterologous hybridisation.

Formation of Poly-ß-hydroxybutyrate from Polycyclic Aromatic Hydrocarbons by Sphingobium scionense sp. WP01

The present study is the first report of the conversion of two simple polycyclic aromatic hydrocarbons, biphenyl and naphthalene, into the aliphatic polymer poly-¦Â-hydroxybutyrate by a single bacterium. The bacterium we have identified as Sphing obium scionense sp. WP01T (=DSM19371T=ICMP 13533T) was originally isolated from contaminated soil containing oil residues and Pinus radiata sap and sawdust. We have shown that Sphing obium scionense sp.WP01T is able to accumulate poly-¦Â-hydroxybutyrate from naphthalene and biphenyl when these are provided as sole carbon sources under nitrogen limited conditions. The generation of PHA from aromatic hydrocarbons as a feedstock provides a route for converting waste polluting hydrocarbons into useable biological polyesters.

Versatile catechol dioxygenases in Sphingobium scionense WP01T

The objective was to understand the roles of multiple catechol dioxygenases in the type strain Sphingobium scionense WP01T (Liang and Lloyd-Jones in Int J Syst Evol Microbiol 60:413–416, 2010a) that was isolated from severely contaminated sawmill soil. The dioxygenases were identified by sequencing, examined by determining the substrate specificities of the recombinant enzymes, and by quantifying gene expression following exposure to model priority pollutants. Catechol dioxygenase genes encoding an extradiol xylE and two intradiol dioxygenases catA and clcA that are highly similar to sequences described in other sphingomonads are described in S. scionense WP01T. The distinct substrate specificities determined for the recombinant enzymes confirm the annotated gene functions and suggest different catabolic roles for each enzyme. The role of the three enzymes was evaluated by analysis of enzyme activity in crude cell extracts from cells grown on meta-toluate, benzoate, biphenyl, naphthalene and phenanthrene which revealed the co-induction of each enzyme by different substrates. This was corroborated by quantifying gene expression when cells were induced by biphenyl, naphthalene and pentachlorophenol. It is concluded that the ClcA and XylE enzymes are recruited in pathways that are involved in the degradation of chlorinated aromatic compounds such as pentachlorophenol, the XylE and ClcA enzymes will also play a role in degradation pathways that produce alkylcatechols, while the three enzymes ClcA, XylE and CatA will be simultaneously involved in pathways that generate catechol as a degradation pathway intermediate.