Deep Mediterranean Prokaryote Communities - Nucleic Acid and Culture Studies

Aims and objectives

1. To identify the elements of prokaryotic communities which play key roles in carbon processing in the deep benthic boundary layer

2. To determine the contribution of these key elements to the overall structure and biodiversity of individual prokaryotic communities and their distribution with depth and geographical location.

3. To determine the degree to which the prokaryotic community is perturbed by organic loading (natural or anthropogenic) and to obtain isolates of active members of the deep ocean prokaryotic community for further study of their  physiology and biotechnological potential.

Methodologies

Analysis of  prokaryotic community structure and biodiversity by methods based on nucleic acid sequences is, at present, the method of choice for microbial ecologists. It can provide a synoptic view of the community, enable inter-community comparisons, and indicate the phylogeny of individual community components. Nucleic-acid based methods have several disadvantages, however: The operational taxonomic units (OTU’s) used to describe the community may be poorly defined. At best they may identify a clone as represented by a specific DNA sequence. The phylogenic position of this sequence as compared with other reported sequences may be determined, but it provides no information about the clone, its physiology , metabolism etc. Typically we will have no idea as to whether the clone is active, inactive, an essential part of the functioning community or redundant.

Pressure-retainer sampler and Pressure vessel used for batch enrichment studies: a) connection for pressurizing pump; b) non-return valve; d) pressure vessel; e) vessel cap; f) air/pressure release valve

Alternative methods of community structure cannot solve this problem: prokaryote identification and determination of function by morphology is not feasible. Cultural methods provide growing clones for further  study, but are generally dismissed as dealing with only a small and possibly unrepresentative fraction of the total community.

Our approach combines nucleic acid based methods together with the use of enrichments and culture based methods, and is intended to identify the key prokaryotic elements involved in carbon cycling and their distribution.  Changes in community structure which occur during sequential batch or semi-continuous enrichments are followed by Degrading Gradient gel Electrophoresis; (DGGE: a synoptic DNA-based method).In this way we can identify the “fitter” clones (those that out-compete the other elements of the community) under the specific enrichment conditions. Changes in “cultivable” bacteria are also monitored by the use of the MPN (most probably number) method using multiwell plates. Unlike conventional plate counts, MPN facilitates the counting and isolation of clones under near-environmental conditions and thus deals with oligotrophs potentially relevant to deep ocean ecosystem functioning rather than unrepresentative copiotrophs. Enrichments under quasi-environmental conditions, combined with culture and DNA based analyses identify potential “keystone” members of the prokaryotic community and define them in terms of their DNA sequence. This information may then be used to determine their contribution to the overall structure and biodiversity of individual prokaryotic communities and their distribution with depth and geographical location. This is achieved by associating them with specific bands and peaks in DGGE “fingerprints” of natural communities and also by the use fluorescent sequence-based probes (FISH).