Studying marine virus communities and distribution in their natural communities.

Marine viruses influence host community composition, however, the effect of viruses on communities of phytoplankton is sparsely studied. Natural algal-virus communities which infect and lyse marine primary producers are an abundant and active part of the marine ecosystem.

 

 To understand the dynamics & effects of phytoplankton viruses, the genetic composition of virus communities needs to be understood. Specifically viruses infecting the marine phytoplankton Micromonas pusillla lead to the development of the degenerate algal-virus-specific (AVS) PCR primers AVS1 and AVS2, which amplify a 700 base pair fragment of algal-virus DNA polymerase genes.

 Phylogenetic analysis of amplified DNA polymerase fragments showed that cultured algal-viruses formed a monophyletic group, compared to double-stranded DNA viruses.

 Algal-virus specific primers were used to amplify unknown algal virus DNA polymerase fragments from natural virus communities, showing that algal-virus diversity can be studied using molecular techniques. 

 DGGE analysis of PCR products that have been amplified bu AVS PCR primers can be used to examine natural algal-virus community diversity, for example, the banding patterns (fingerprints) in this experiment of samples taken from one location (Salmon Inlet) were identical and easily distinguished from other fingerprints; these samples were collected at the same time and location, but at different depths. However, the temperature and salinity at this site did not vary much with depth, indicating that the water and viruses were mixed well.

 On the opposite end of the spectrum, samples taken from a different location (Pendrell Sound) have differing banding patterns at the different depths. This is due to the salinity at depth being double that of surface salinity, meaning that the water and viruses here were had stratification.

 The DNA polymerase sequences derived from the PCR and DGGE were closely related to known algal-viruses, and remarkably, some 98% of sequences from samples taken at the Southern Ocean were identical to those from coastal British Columbia, despite how different the environments are.

 This paper showed how PCR and DGGE can be used to recover and identify unknown algal-virus DNA polymerase sequences from the natural environment, and that similar sequences can be recovered from different areas worldwide. 

 I found this paper interesting as it shows how the curiosity sparked by viruses infecting a specific species of marine phytoplankton lead to the development of new primers, and then onto being able to study marine virus community diversity in natural communities.

Sequence analysis of marine virus communities reveals that groups of related algal viruses are widely distributed in nature by Steven M. Short and Curtis A.Suttle, 2002

...here's the link if you'd like to read it http://ukpmc.ac.uk/articles/PMC123764/pdf/1471.pdf

Factors influencing viral distribution and abundance along a latitudinal transect of the North Atlantic Ocean at different depths.

It is estimated that there are 10³⁰ viruses in the ocean (approximately 10 times as many as bacteria). Viruses are not just abundant in number they are also genetically diverse.  They influence the composition of marine communities and play an important role in biochemical cycles. Moreover viruses control microbial mortality and may help maintain diversity.

The study by De Corte et al. (2012) looked at the factors controlling viral distribution, abundance and production to assess the potential variations in the relationship between viruses and prokaryotes across a latitudinal transect in the North Atlantic Ocean.

This study is important because currently most published material on virus-prokaryote interactions in marine environments focus on near shore waters whereas in this case a wider area over 4 North Atlantic provinces in different pelagic zones are used.  

Many factors may influence distribution of viruses throughout the ocean but in the end viral distribution depends on the availability of suitable host therefore both prokaryote production and abundance were measured as well as viral production and abundance. The aforementioned factors were assessed throughout the water column and at different depth to determine the potential variations in interactions. Samples were obtained from 24 depths at 33 stations from 5 different pelagic zones. Prokaryote and viral abundance was measured using flow cytometry standard procedure. Samples were stained with SBYR Green I (a fluorescent dye) after being shock frozen with N₂. Viral production was measured using the dilution approach. Prokaryote production was measured using an assay for radioactively labelled leucine incorporation. Radio activity of the samples and a blank were measured and after basic calculations the results were converted into the incorporation rate.

Results showed a significant decrease in abundance of viruses and prokaryotes with depth over all stations (abundance was negatively related to depth) Prokaryote production also decreased with depth. Lytic viral production decreased and lysogenic production stayed the same. Multivariate regression analysis was used to predict the factors explaining the variability of viral abundance between the different depths. Taking the whole data set into consideration the variation was mainly explained by prokaryote abundance, temperature and latitude which together accounted for 73% of total variation. Prokatyote abundance alone accounted for 46% of the variation in abundance.  Temperature and prokaryotic abundance were seen to be the main factors influencing the differences in viral abundance between the 4 provinces, 2 mainly influenced by temperature and 2 by viral abundance. Based on the above results by De Corte et al. (2012) concluded that virus-host interactions significantly change in different oceanic provinces in response to biological factors i.e. host availability but also to chemical and physical factors in the environment.

De Corte, Daniele Sintes, Eva Yokokawa, Taichi Reinthaler, Thomas Herndl, Gerhard J (2012) Links between viruses and prokaryotes throughout the water column along a North Atlantic latitudinal transect. The ISME Journal (2012) 6, 1566–1577.

http://www.nature.com/ismej/journal/v6/n8/full/ismej2011214a.html

Species-Specific Bacterial Symbionts in Hydra: Are Particular Microbial Communities Selected for and Maintained by the Host?

Cnidarians have a simple tissue grade organisation with a limited number of cell- and tissue-types, and mucus as the sole physical barrier between epithelial tissue and a habitat now known to support large numbers of microbes, including potential pathogens.  Without the benefit of defensive phagoctytes, what evidence exists for the selection of particular symbiotic bacteria within the Cnidarian surface tissue epithelium?

Interested in the evolution of metazoan microbial communities, Fraune and Bosche (2007) compared the microbiota of two closely related basal metazoan species: Hydra oligactis and Hydra vulgaris.  In order to examine microbiota evolution over time, hydra polyps were obtained both direct from the wild, and from cultures maintained in laboratories for over 30 years.  Microbial communities were surveyed via various means, including molecular methods (Restriction Fragment Length Polymorphism (RELP) analysis of 16S rRNA), Transmission Election Microscopy (TEM), phase contrast microscopy, and Fluorescent In-Situ Hybridisation (FISH).

RELP patterns indicated that the microbial communities of the two Hydra species were markedly different:  almost all lab-cultured H. oligactus samples shared one RELP pattern, whereas 16 different patterns were observed in H. vulgaris samples; this is a surprising result when considering the uniformity of living conditions (e.g. food, temperature) which these individuals experienced over the proceeding decades, and the authors cite selective constraints as being the probable cause (although care should be taken when interpreting such results, see below).  This result was echoed when wild polyps of both species where analysed: one dominant RELP pattern observed in H. oligactus samples (though different to that of cultured samples), and more diversity observed in the patterns of H. vulgaris samples.  It appears interspecies differences between wild and cultured polyps are far less pronounced than that between the microbial communities of the different hydra species.

Furthermore, species-specific phylotypes were observed via phylogenetic analysis of RELP derived sequences (phylotypes being defined as sequences of ≥97% similarity).  A particularly interesting α-proteobacteria phylotype specific only to H. oligactus was confirmed as a bacterial endosymbiont (via TEM and FISH techniques), previously unreported as extant in Hydra. Again, species’ differences were found to be larger than interspecies:  although no H. vulgaris samples were found to contain any bacterial endosymbiont, all cultured H. oligactus polyp epithelial cells and c. 20% of wild H. oligactus polyp cells were observed as containing this type of α-proteobacteria phylotype.  The authors conclude that the stark differences between the microbial communities of the two species of Hydra, together with the maintenance of a specific type of microbiota over time, suggests that selective pressures applied by the Hydra epithelium help to structure these microbial communities.

This study provides compelling evidence that cnidarian hosts do indeed have a role in shaping their microbiotas; some other investigations have found similarly species-specific microbial communities in other Anthozoans, including some analyses of the threatened stony schleractinians that build coral reefs.  However, observations of such clear cut species-specific differences in anthozoan microbiotas are not always evident, with many studies providing conflicting results; different molecular methods have their own advantages and disadvantages in the assessment of microbial diversity (e.g. the variability inherent to the RELP technique), which must be kept in mind when such investigations are interpreted. 

In addition, various abiotic and biotic factors have been suggested as having an important role in the structuring of microbial communities, including location, temperature, disease, and the influence of bacteriophage; these factors, alongside punitive selective pressures from the host itself, should be considered when designing further investigations into interactions between microbial communities and the metazoans which they inhabit.

Fraune, S. and Bosch, T. C. G. (2007) Long-term maintenance of species-specific bacterial microbiota in the basal metazoan Hydra. PNAS 104, pp 13146-13151.

http://www.pnas.org/content/104/32/13146

Analysis of the CtrA Pathway in Magnetospirillum has revealed an ancestral role in motility in the Alphaproteobacteria.

What is its role to magnetotactic bacteria?

Bacteria use a variety of mechanisms to inhabit a large diversity of environments. One of these organisms is the Magnetotactic Bacteria (MB) which has the ability to navigate along the earth’s magnetic field to find hypoxic environments. This is due to the presence of an organelle called the magnetosome. Magnetosome’s are held together in chains in sub cellular compartments, providing the cell with the capability to align in magnetic fields.

Regulation of developmental events across the cell cycle in Alphaproteobacteria has been investigated most thoroughly on Caulobacter crescentus. The execution of cell division requires the timed coordination of key events, this requires a regulatory network. At the core of this network is the response regulator CtrA. In C. crescentus CtrA is involved in regulation of gene expression and many more key processes. CtrA has been shown to have various roles in the cell division cycles of other bacteria in the phylum. The diverse roles of CtrA in species of Alphaproteobacteria has raised questions as most components have been proven to be important but yet play vastly different roles in the cells of different species.

In this study Greene et al 2012 looked at the role of the CtrA regulatory network and how sub cellular compartments are effected during cell division, which is conserved throughout the Alphaproteobacteria, on magnetotactic bacterium Magnetospirillum magneticum strain AMB-1.

Genetic analysis of this species has identified a genomic region that is essential for magnetosome formation which is now termed the magnetosome island (MAI). Whilst progress has been made into uncovering genes that are essential in the steps of magnetosome formation, the integration of these processes is poorly understood.

Greene et al 2012 took a targeted approach to investigate the regulation of CtrA in the context of a microorganism possessing intracellular organelles by creating Mutants of the AMB-1 with deletions to CtrA and DivK, CtrA’s negative regulator. They found that these control genes were not essential for keeping the cell viable, but the mutants had motility defects indicating that control genes play an important role in cell motility. A ctrA deletion in a highly motile MIA deletion strain stopped motility, whilst a deletion of divK induced motility in a previously nonmotile wild-type of AMB-1 (WT).  

This study also found that CtrA’s activity in AMB-1 is controlled in a manner similar to that of C.crescentus. Greene and colleagues results suggest that the phosphorylation state of CtrA is essential for motility in AMB-1 and that swimming behaviour observed in the divK deletion strain potentially comes from an increased phosphorylated and active CtrA.

Most species of magnetotactic bacteria swim constitutively when grown in culture. Interestingly, in this study, Greene et al found that the vast majority of wild-type AMB-1 cells in culture were non motile. This is in stark contrast to other published studies in which entire populations of AMB-1 cells were found to be swimming. It is possible that differences in growth mediums or environmental conditions caused the differences seen in past studies. However, it is also likely that motile cells were selected for using a magnetically guided swimming-based procedure termed the “racetrack” assay. This is a common practice among some groups working with magnetotactic bacteria. In the case of AMB-1, such a procedure might select for genetic variants that would give rise to constitutively swimming strains. Given Greene et al’s hypothesis explaining the evolutionary benefits of magnetosome chain formation, the lack of motility in AMB-1 was surprising. Wild-type AMB-1 failed to swim even with increased or decreased iron or oxygen concentrations. Another potential reason could be that the isolation and current growth conditions were enough to satisfy the nutritional and energetic requirements of AMB-1 in this experiment, thus negating any reason to devote energy toward movement.

The common feature of the CtrA pathways in many alphaproteobacteria is its role in motility and cell division. Genetic analyses in C. crescentus, and now M. magneticum AMB-1 have shown the involvement of CtrA in the regulation of motility. Greene and co conducted an analysis of the phylogenetic relationships among CtrA alleles from organisms in which its biological role has been investigated and suggested that regulation of motility by CtrA is an ancestral trait. In light of the studies findings, Greene et al put forward that the ancestral alphaproteobacterium was motile and during evolution, acquired regulation of motility by the response regulator CtrA. The results of the study presented here are the most comprehensive experimental examination of a CtrA regulon in an organism. They have provided a glimpse into the evolution and divergent specialisation of this important regulator and provide the basis for future detailed mechanistic studies into its function in AMB-1.

I have decided to review this paper because I have a keen interest in MB and wanted to gain a better understanding of how these organisms work. I also wanted to increase awareness of MB. I feel this paper makes a good contribution to the field but given the long history of this area, i don’t think my review sufficiently cover’s its scope. I suggest that readers interested in gaining a better understanding of the research into MB should go and do some more digging.

S.E. Greene. M. Brilli. B Emanuele. E. Biondi, and A. Komeilia (2012) Analysis of the CtrA Pathway in Magnetospirillum Reveals an ancestral Role in Motility in Alphaproteobacteria. Journal of bacteriology June 2012 Volume 194 Number 11:. 2973–2986

http://jb.asm.org/content/194/11/2973.full.pdf+html

Slim pickings in the deep results in novel symbiosis

The deep sea is characterised by a scarcity of organic material available its inhabitants. As a result organisms of the deep have evolved novel ways of solving this problem. One of which being the symbiosis seen in hydrothermal vents communities where the conversion of inorganic compounds by chemolithotrophic bacteria inhabiting vestimentiferan tube worms is the source of primary production driving the community. In many cases the symbiotic relationship between invertebrates and microorganisms involves the production of organic material by symbiont autotrophy, as in the example highlighted. The study of Godfreddi et al (2005) shows a novel symbiosis between a polycheate worm, Osedax frankpressi, and its symbiotic bacteria of the family Oceanospirillales, where the degradation of whale bone is the result of the heterotrophic lifestyle of Oceanospirillales producing fatty acids for acquisition by the host.

In February 2002 dense assemblages of invertebrates were discovered on a Grey whale carcus (Eschrichtius robustus) at 2891m depth in the Monterey canyon, California. Between 2002-2004 two polychaete worms were collected Osedax frankpressi and O. rubiplumus, from the Siboglidinae family, the deep sea tube worms (determined by morphological and phylogenetic analysis). The Osedax are mouth less, gutless worms that do not possess a trophosome like other Siboglinids, but instead possess elaborate posterior root like extrusions that contain bacteriocytes (50µm in diameter) which house the pleomorphic rod shaped bacteria. 16s ribosomal DNA sequencing revealed the identity of bacteria as being members of the Oceanospirillales, of the family γ-proteobacteria. In O.frankpressi the dominate phylotype (Osedax SYM_1) consisted of 89% of clones and O.rubiplumas dominant phylotype (Osedax SYM_2) consisted of 81%. The fact that the symbionts show phylogenetic relationships to the genus Oceanospirralis is highly significant because this group is involved in the heterotrophic degradation of organic compounds. Evidence for the distribution of Osedax SYM_1  in O.frankpressi came from in-situ FISH microscopy where an oligonucleotide probe (SYM 435_1) hybridized strongly with the symbiont and determined dense populations in the ovisac and root tissue, confirmed by TEM.  After Godfreddi and colleagues determined the phylogentics and the presence of Osedax SYM_1 as the dominant phylotypes in O.frankpressi, investigation continued into the role of the endosymbiont. The dense population in of the symbionts and the lack of trophosome lead Godfreddi and colleagues to suggest that this this symbiosis is nutritionally beneficial to the worm. Evidence for this came from bulk stable carbon and nitrogen values, which showed similarities in worm symbiont/symbiont free tissue and whale bone, suggesting a potentially heterotrophic reliance on bone for nutrition. Heterotrophy of symbionts was further supported by the absence of RuBPCo gene. RuBPCo is a key microbial enzyme in the autotrophic calvin benson cycle, commonly observed in chemolithotrophic bacteria. Examination of O.frankpressi tissue revealed the presence of the bacterial specific biomarker vaccenic acid, a common product of fatty acid metabolism and eicosapentaenic acid (involved in the maintenance of membrane fluidity) which lead the authors to the conclusion that the symbionts were responsible for the synthesis of these compounds which were transferred to the host. The composition of wax etsers in the tissue conformed in carbon number and bond position of vaccenic acid as shown by C¹³ analysis providing further evidence for the synthesis of compounds by Osedax SYM_1.

In conclusion, this study highlights the possible heterotrophic symbiosis between Osedax frankpressi  with Osedax SYM_1. The study conclusively shows the symbiont is a member of the heterotrophic Oceanospirillales and its position and density in host. Similar stable Carbon and nitrogen analysis between whale bone and host, lack of RuBPCo gene and presence of bacterial specific biomarker Vaccinnic acid in host tissue provide evidence for the heterotrophic lifestyle of the endosymbiont and acquisition of symbiont compounds by the host.  Questions still remain into the mechanisms bone degradation and transport to and from the bacteria, as well as in the host.

Godfreddi S.K, Orphan.V.J, Rouse.G.W, Janke. L, Tsegeria. E, Kendru.T, Lee.R Vrijenhoek.R.,(2005). Evolutionary innovation: A bone eating marine symbiosis. Environmental microbiology. (7) 9. 1369-1378.

   

 http://www.mbari.org/staff/vrijen/pdfs/goffredi_2005em.pdf   

Anti-Biofilm Compounds Derived from Marine Sponges… The “Helper Drugs” Against Antibiotic Resistance!

Bacterial biofilms are surface-attached communities of microorganisms; protected from external assault by an extracellular matrix of polysaccharide, protein and nucleic acids. When in this biofilm state, bacteria are significantly more resistant to antibiotics and have utilized this line of defense to infiltrate every environment, including the human body. This is of particular concern as it is estimated that up to 80% of all microbial infections are biofilm based. A prominent problem is the Biofilm infections of indwelling medical devices i.e. catheters, as the infections are almost impossible to eradicate.

In order to find anti-biofilm agents, natural products have been extracted from marine organisms. Sponges have been found to possess anti-biofilm properties, however only two classes of sponge metabolites have been found to possess non-bactericidal biofilm modulators: the terpenoids and the pyrrole-imidazoles. Because of this, there is now an increased effort towards the development of small synthetic molecules that will inhibit and/or disperse bacterial biofilms.

Stowe et al. (2011) give a very descriptive account on biofilm formation and the role it plays in the natural phenomenon of biofouling. The paper discusses the implications biofilm has on industry and medicine and the anti-biofilm agents that are derived from marine sponges. These being: the terpenes and their derivatives and the pyrrole-imidazole alkaloids (PIAs): oroidin, screptin and bromoageliferin. These were extracted from several families of sponge, with a particular focus of bromopyrrole derivatives from the Agelasidae family. Natural products PIAs have been found to be very toxic agents and have microbicidal properties; working against microorganism and higher organisms. Due to these properties, Stowe et al. looked to synthesise from these natural molecules anti-biofilm compounds that were non-microbicidal.  

Stowe et al. were able to develop several successful anti-biofilm compounds that had non-microbicidal properties. In particular, two bromoageliferin derivatives: trans-bromoageliferin (TAGE) and cis-bromoageliferin (CAGE). The compounds were tested for anti-biofilm activity against Pseudomonas aeruginosa using crystal violet assays. They were found to inhibit biofilm formation. To support TAGE’s role as a non-microbicidal biofilm modulator, fluorescence-based experiments with confocal laser scanning microscopy (CLSM) and flow cytometry were used to visualize the phenotypic effects of the molecule. CLSM demonstrated that 100 μM TAGE affected the biofilm structure of P. aeruginosa.

Stowe et al. then combined these anti-biofilm compounds with conventional antibiotics and found that the results of which, resensitized previously resistant bacterial strains; this allowed the commercial drug to resume activity. In particular the combination of the compound and antibiotic were able to resensitize drug-resistant methicillin-resistant Straphylococcus aureus (MRSA) and multi-drug resistant Acinetobacter baumannii (MDRAB).   

These “Helper Drugs” derived from marine sponges will most likely act as adjuvants to conventional antibiotics against antibiotic resistance. However, toxicity tests must be carried out to establish the effect these anti-biofilm compounds have on bacteria in their native host, for example: gut bacteria, before they can be tested on animal models and developed for medicinal use. I chose to review this article because the results obtained have contributed significantly to our knowledge of bacteria bio-film structure and offered promising solutions to the current anti-biotic resistance crisis.  

Stowe, S.D., Richards, J.J., Tucker A.T., Thompson, R., Melander, C., and Cavanagh, J. (2011)

'Anti-Biofilm Compounds Derived from Marine Sponges', Marine Drugs, 9, 2010-2035. 

http://www.mdpi.com/1660-3397/9/10/2010

Winter-summer comparison of Antarctic bacterioplankton: WHO is there and WHAT are they doing?

Summer in Antarctica is characterized by continuous high solar irradiance, water column stratification and intense primary production from phytoplankton. As winter moves in, sea ice forms and the marine habitat is plunged into darkness; the lack of sun light and lowering temperatures cause the water column to mix and primary production from photosynthesis virtually ceases. Previous studies have identified that these seasonal changes in conditions correspond to a drastic change in microbial abundance and activity, as well as a shift in microbial diversity. To date research efforts to identify the main players in the different microbial communities have been limited. Furthermore there has been no specific objective to analyse the metabolic capabilities of the different communities. In particular there is a lack of information on winter communities due to the harsh conditions and thick sea ice which hampers sampling efforts. The objective of the paper reviewed was therefore, to better define the community diversity and genome-encoded capabilities of Antarctic bacterioplankton in both winter and summer climates.

Grzymski et al., (2012) use a bombardment of metagenomic techniques in order to gather the desired information about each microbial community; allowing them to observe data on organisms which may not be culturable. Genetic parameters directly measured included subunit ribosomal RNA (SS rRNA) sequences as well as genomic end sequences; this was achieved using a standardised DNA/RNA extraction kit and following instructions from the Joint Genome Institute. The genomic information was then subjected to a variety of phylogenetic statistical analyses and comparisons using genomic databases, allowing data such as predicted genome size, GC content and specific functional genes to be investigated.

The paper identifies many different clades in each community using the SSrRNA which enabled them to produce phylogenetic trees. Overall it was found that the winter community had a significantly higher phylogenetic and functional diversity. The general unique features of the winter community were the presence of chemolithoautotrophic bacteria and archaea (who obtain energy from the oxidation of inorganic compounds and carbon from the fixation of carbon dioxide). Interestingly the summer community showed no archaea at all. The authors were able to come to these conclusions from a wide range of interesting and detailed data, not all of which can be summarised here. I found the specific functional gene search particularly interesting; this showed that there is a greater metabolic and functional diversity in winter. For example, the winter community contained more post-translational modification genes, indicating that a higher amount of protein re-folding occurs in winter. This is likely due to protein damage in the more stressful lower temperatures and oligotrophic condition.

The observation of high levels of chemoloithoautotrophy in winter is novel in Antarctic waters. Given the huge size of the Southern Ocean this discovery, and its potential inferences, is particularly important. More work is needed in order to find out if this process is widespread in the Southern Ocean as it may be an important carbon sink which has previously been unaccounted for in carbon budget studies. It may also go some way to explaining anomalies in inorganic nitrogen content, possibly changing the way we understand the global carbon and nitrogen cycles.

I decided to review this paper in order to better understand metagenomic techniques and their applications following my earlier confession that I didn’t have a good grasp of it all. I feel this paper communicated their huge array of results clearly which helps overall understanding and I strongly recommend any other bloggers in the same position give this a read.

Grzymski, J. J., Riesenfeld, C. S., Williams, T. J., Dussaq, A. M., Ducklow, H., Erickson, M., Cavicchioli, R., et al. (2012). A metagenomic assessment of winter and summer bacterioplankton from Antarctica Peninsula coastal surface waters. The ISME journal, 6(10), 1901–15.

http://www.nature.com/ismej/journal/v6/n10/full/ismej201231a.html