Peter Deines

Impact of Violacein-Producing Bacteria on Survival and Feeding of Bacterivorous Nanoflagellates

ABSTRACT We studied the role of bacterial secondary metabolites in the context of grazing protection against protozoans. A model system was used to examine the impact of violacein-producing bacteria on feeding rates, growth, and survival of three common bacterivorous nanoflagellates. Freshwater isolates of Janthinobacterium lividum and Chromobacterium violaceum produced the purple pigment violacein and exhibited acute toxicity to the nanoflagellates tested. High-resolution video microscopy revealed that these bacteria were ingested by the flagellates at high rates. The uptake of less than three bacteria resulted in rapid flagellate cell death after about 20 min and cell lysis within 1 to 2 h. In selectivity experiments with nontoxic Pseudomonas putida MM1, flagellates did not discriminate against pigmented strains. Purified violacein from cell extracts of C. violaceum showed high toxicity to nanoflagellates. In addition, antiprotozoal activity was found to positively correlate with the violacein content of the bacterial strains. Pigment synthesis in C. violaceum is regulated by an N-acylhomoserine lactone (AHL)-dependent quorum-sensing system. An AHL-deficient, nonpigmented mutant provided high flagellate growth rates, while the addition of the natural C. violaceum AHL could restore toxicity. Moreover, it was shown that the presence of violacein-producing bacteria in an otherwise nontoxic bacterial diet considerably inhibited flagellate population growth. Our results suggest that violacein-producing bacteria possess a highly effective survival mechanism which may exemplify the potential of some bacterial secondary metabolites to undermine protozoan grazing pressure and population dynamics.

Sponge‐specific clusters revisited: a comprehensive phylogeny of sponge‐associated microorganisms

Marine sponges often contain diverse and abundant communities of microorganisms including bacteria, archaea and eukaryotic microbes. Numerous 16S rRNA-based studies have identified putative sponge-specific microbes that are apparently absent from seawater and other (non-sponge) marine habitats. With more than 7500 sponge-derived rRNA sequences (from clone, isolate and denaturing gradient gel electrophoresis data) now publicly available, we sought to determine whether the current notion of sponge-specific sequence clusters remains valid. Comprehensive phylogenetic analyses were performed on the 7546 sponge-derived 16S and 18S rRNA sequences that were publicly available in early 2010. Overall, 27% of all sequences fell into monophyletic, sponge-specific sequence clusters. Such clusters were particularly well represented among the Chloroflexi, Cyanobacteria, Poribacteria, Betaproteobacteria and Acidobacteria, and in total were identified in at least 14 bacterial phyla, as well as the Archaea and fungi. The largest sponge-specific cluster, representing the cyanobacterium Synechococcus spongiarum, contained 245 sequences from 40 sponge species. These results strongly support the existence of sponge-specific microbes and provide a suitable framework for future studies of rare and abundant sponge symbionts, both of which can now be studied using next-generation sequencing technologies.

'Sponge-specific' bacteria are widespread (but rare) in diverse marine environments.

Numerous studies have reported the existence of sponge-specific 16S ribosomal RNA (rRNA) gene sequence clusters, representing bacteria found in sponges but not detected in other environments, such as seawater. The advent of deep-sequencing technologies allows us to examine the rare microbial biosphere in order to establish whether these bacteria are truly sponge specific, or are more widely distributed but only at abundances below the detection limit of conventional molecular approaches. We screened >12 million publicly available 16S rRNA gene pyrotags derived from 649 seawater, sediment, hydrothermal vent and coral samples from temperate, tropical and polar regions. We detected 77 of the 173 previously described sponge-specific clusters in seawater or other non-sponge samples, albeit generally at extremely low abundances. Sequences representing the candidate phylum ‘Poribacteria’, previously thought to be largely restricted to sponges, were recovered from 46 (out of 411) seawater and 41 (out of 129) sediment samples. While the presence of an organism does not imply that it is active in situ, our results do suggest that many ‘sponge-specific’ bacteria occur more widely outside of sponge hosts than previously thought.

A new coupon design for simultaneous analysis of in situ microbial biofilm formation and community structure in drinking water distribution systems

This study presents a new coupon sampling device that can be inserted directly into the pipes within water distribution systems (WDS), maintaining representative near wall pipe flow conditions and enabling simultaneous microscopy and DNA-based analysis of biofilms formed in situ. To evaluate this sampling device, fluorescent in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analyses were used to investigate changes in biofilms on replicate coupons within a non-sterile pilot-scale WDS. FISH analysis demonstrated increases in bacterial biofilm coverage of the coupon surface over time, while the DGGE analysis showed the development of increasingly complex biofilm communities, with time-specific clustering of these communities. This coupon design offers improvements over existing biofilm sampling devices in that it enables simultaneous quantitative and qualitative compositional characterization of biofilm assemblages formed within a WDS, while importantly maintaining fully representative near wall pipe flow conditions. Hence, it provides a practical approach that can be used to capture the interactions between biofilm formation and changing abiotic conditions, boundary shear stress, and turbulent driven exchange within WDS.

Chloroflexi bacteria are more diverse, abundant, and similar in high than in low microbial abundance sponges

Some marine sponges harbor dense and phylogenetically complex microbial communities [high microbial abundance (HMA) sponges] whereas others contain only few and less diverse microorganisms [low microbial abundance (LMA) sponges]. We focused on the phylum Chloroflexi that frequently occurs in sponges to investigate the different associations with three HMA and three LMA sponges from New Zealand. By applying a range of microscopical and molecular techniques a clear dichotomy between HMA and LMA sponges was observed: Chloroflexi bacteria were more abundant and diverse in HMA than in LMA sponges. Moreover, different HMA sponges contain similar Chloroflexi communities whereas LMA sponges harbor different and more variable communities which partly resemble Chloroflexi seawater communities. A comprehensive phylogenetic analysis of our own and publicly available sponge-derived Chloroflexi 16S rRNA gene sequences (>xa0780 sequences) revealed the enormous diversity of this phylum within sponges including 29 sponge-specific and sponge-coral clusters (SSC/SCC) as well as a supercluster consisting of >xa0250 sponge-derived and a single nonsponge-derived 16S rRNA gene sequence. Interestingly, the majority of sequences obtained from HMA sponges, but only a few from LMA sponges, fell into SSC/SCC clusters. This indicates a much more specific association of Chloroflexi bacteria with HMA sponges and suggests an ecologically important role for these prominent bacteria.

Gut microbiome of the critically endangered New Zealand parrot, the kakapo (Strigops habroptilus).

The kakapo, a parrot endemic to New Zealand, is currently the focus of intense research and conservation efforts with the aim of boosting its population above the current ‘critically endangered’ status. While virtually nothing is known about the microbiology of the kakapo, given the acknowledged importance of gut-associated microbes in vertebrate nutrition and pathogen defense, it should be of great conservation value to analyze the microbes associated with kakapo. Here we describe the first study of the bacterial communities that reside within the gastrointestinal tract (GIT) of both juvenile and adult kakapo. Samples from along the GIT, taken from the choana (≈throat), crop and faeces, were subjected to 16 S rRNA gene library analysis. Phylogenetic analysis of >1000 16 S rRNA gene clones, derived from six birds, revealed low phylum-level diversity, consisting almost exclusively of Firmicutes (including lactic acid bacteria) and Gammaproteobacteria. The relative proportions of Firmicutes and Gammaproteobacteria were highly consistent among individual juveniles, irrespective of sampling location, but differed markedly among adult birds. Diversity at a finer phylogenetic resolution (i.e. operational taxonomic units (OTUs) of 99% sequence identity) was also low in all samples, with only one or two OTUs dominating each sample. These data represent the first analysis of the bacterial communities associated with the kakapo GIT, providing a baseline for further microbiological study, and facilitating conservation efforts for this unique bird.

Phenotypic variation in Pseudomonas sp CM10 determines microcolony formation and survival under protozoan grazing

Abstract We investigated the survival mechanism of the bacterium Pseudomonas sp. CM10 in the presence of a flagellate predator. The bacterium had been isolated from a continuous culture containing bacterivorous nanoflagellates. On agar plates, we found intraclonal dimorphism of Pseudomonas sp. CM10 colonies at high frequencies: The primary mucoid colony type generated a secondary non-mucoid form. Unlike the repeated generation of non-mucoid colonies from mucoid clones, we did not observe the occurrence of mucoid forms in non-mucoid populations. In semicontinuous and batch cultures, we investigated the ability of the two morphs to survive predation by the bacterivorous flagellate Ochromonas sp. under conditions of growth and starvation. In predator-free cultures, populations of both variants were unicellular but differed in some phenotypic characteristics such as cell motility and hydrophobicity. Grazing treatments revealed that the non-mucoid morph was reduced severely whereas the primary mucoid type survived due to the formation of inert suspended microcolonies stabilized by an extracellular matrix. Effectiveness and competitive trade-offs of microcolony formation were revealed by a competition experiment with the bacterium Pseudomonas putida MM1: Pseudomonas sp. CM10 was displaced in predator-free cultures but outgrew the defenseless and monomorphic competitor under flagellate grazing pressure. We conclude that intraclonal polymorphism may regulate the ability of Pseudomonas sp. CM10 to survive in situations of severe protistan grazing. The formation of inert microcolonies, however, is suggested to be detrimental to rapid growth and dispersal.

Site-specific methane production and subsequent midge mediation within Esthwaite Water, UK

Previous analyses of larval chironomid δ13C have suggested that methanotrophic contribution to biomass is site-specific within lakes. We determined larval biomass, larval and methane gas stable carbon isotopes, and potential methane production from the sediments underlying different water column depths in Esthwaite Water, UK. Methane production increased whereas larval δ13C values typically became lighter with increasing lake depth. Reduced methane production at 15 m depth and correspondingly less13C-depleted larvae in the second year of study suggests that the rate of methane production influenced larval assimilation of methane-derived biomass. Larval distribution and other site-specific parameters, combined with two-source mixing models, were used to estimate potential methane-mediation via the abundant chironomid biomass to higher predators.

Temporal molecular and isotopic analysis of active bacterial communities in two New Zealand sponges

The characterization of changes in microbial communities is an essential step towards a better understanding of host-microbe associations. It is well established that sponges (phylum Porifera) harbour a diverse and abundant microbial community, but it is not known whether these microbial communities change over time. Here, we followed two sponge species (Ancorina alata and Tethya stolonifera) over a 2-year sampling period using RNA (16S rRNA)-based amplicon pyrosequencing and bulk stable isotope analysis (δ(13) C and δ(15)N). A total of 4468 unique operational taxonomic units (OTUs) was identified, which were affiliated with 26 bacterial phyla. Bacterial communities of both sponge species were remarkably stable throughout the monitoring period, driven by a small number of OTUs that dominated their respective communities. Variability of sponge-associated bacterial communities was driven by OTUs that were low in abundance or transient over time. Stable isotope analysis provided evidence of both bacteria- and host-derived nutrients and their variability throughout the season. While δ(15) N values were similar, significant differences were found in δ(13) C of sponge tissue, indicative of a varying reliance on particulate organic matter as a carbon source. Further temporal studies, such as those undertaken here, will be highly valuable to identify which members of a sponge bacterial community are truly symbiotic in nature.

Bacterial water quality and network hydraulic characteristics: a field study of a small, looped water distribution system using culture-independent molecular methods

Aims:u2002 To determine the spatial and temporal variability in the abundance, structure and composition of planktonic bacterial assemblages sampled from a small, looped water distribution system and to interpret results with respect to hydraulic conditions.