Marine sponge-associated planctomycetes and actinobacteria

Izumi, Hiroshi (2012). Marine sponge-associated planctomycetes and actinobacteria PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland.

       
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Author Izumi, Hiroshi
Thesis Title Marine sponge-associated planctomycetes and actinobacteria
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
Publication date 2012-07
Thesis type PhD Thesis
Supervisor John A. Fuerst
Total pages 176
Total colour pages 30
Total black and white pages 146
Language eng
Subjects 0605 Microbiology
060504 Microbial Ecology
060501 Bacteriology
060599 Microbiology not elsewhere classified
Formatted abstract
Marine sponges have been an abundant source of natural products with pharmaceutical importance, and they have therefore been investigated for the discovery of new drug candidates. In recent decades, there has been growing research interest in the association of sponges with their microbial associates. These sponge-associated microorganisms are known for high level of diversity and unique association with the sponge hosts. It has been hypothesized that some of natural products isolated from sponges are indeed synthesized by their microbial associates, shifting research interest from the host sponges to their microbial associates for the discovery of new drug candidates.
     Actinobacteria are known to synthesize a wide range of bioactive compounds including antibiotics and anti-cancer agents. In this study, actinobacteria associated with marine sponges were studied as a model to examine the biological roles of their bioactive compounds in the sponge-associated microbial community. Eleven isolates of Mycobacterium species as well as an antimycobacterial Salinispora arenicola strain were cultured from the sponge Amphimedon queenslandica. This S. arenicola strain was confirmed to produce the antimycobacterial antibiotics in the rifamycin class and the strain displayed inhibitory effects against representatives from two of three Mycobacterium phylotype groups. Evidence for antagonism of sponge-derived Salinispora against sponge-derived Mycobacterium strains from the same sponge specimen and production of antimycobacterial antibiotics by this Salinispora strain suggest that the synthesis of such antibiotics may have functions in competition between sponge microbial community members. In addition to these Mycobacterium strains isolated from A. queenslandica, a Mycobacterium strain that is closely related to Mycobacterium tuberculosis was cultured from the sponge Fascaplysinopsis sp. The close phylogenetic relationship of this strain to M. tuberculosis was demonstrated by analysis of 16S rRNA, rpoB, and hsp65 genes.
    The members of phylum Planctomycetes are distinct from other bacteria in several unusual features including intracellular compartmentalization and a lack of peptidoglycan in their cell walls. Planctomycetes are ubiquitous in natural environments and have been reported to occur in marine sponges. To study the association of planctomycetes with marine sponges, the diversity of planctomycetes occurring in the sponge Niphates sp. was investigated using culture-dependent and culture-independent approaches. The culture-dependent approach resulted in the isolation of a large number of diverse planctomycetes including some novel lineages of Planctomycetes. Culture-independent clone library approach identified unique planctomycete sequences that are closely related to sequences retrieved exclusively from other sponges and corals. Examination of the sponge tissue using a transmission electron microscope reveals that the tissue of the sponge Niphates sp. was almost devoid of microbial cells. These results might have implications for the association of planctomycetes with the less known group of sponges called low microbial abundance (LMA) sponges, which do not harbor dense microbial communities within their tissue unlike their well-studied counterparts, high microbial abundant (HMA) sponges.
     The genome sequencing of the marine planctomycete Rhodopirellula baltica revealed the presence of genes encoding for polyketide synthases (PKSs), non-ribosomal peptide synthases (NRPSs), and halogenases in its genome. PKS, NRPS, and halogenase genes are known to be involved in the biosynthesis of natural products. To investigate the potential of planctomycetes to produce natural products, PKS, NRPS, and halogenase genes of planctomycetes including sponge isolates were analyzed using phylogenetic and bioinformatic approaches. Planctomycete isolates were screened for the presence of PKS, NRPS, and halogenase genes via PCR and the diversity of these genes was examined. The result of phylogenetic analysis reveals that PKS and NRPS genes of planctomycete isolates are remarkably diverse, and, in some cases, these genes are more closely related to those of non-planctomycete species including cyanobacteria. However, halogenase genes of planctomycete isolates are more specific to planctomycetes. The high level of diversity of PKS and NRPS genes and relationships with non-planctomycete genes suggest that horizontal gene transfer of PKS and NRPS genes between planctomycetes and other members of the microbial community might be common phenomena. Bioinformatic analysis of PKSs and NRPSs of planctomycetes reveals that these PKSs and NRPSs are distinct from those of actinobacteria and have unique features, suggesting planctomycetes might produce natural products with novel chemistries.
     Recently, it was shown that the planctomycete Gemmata obscuriglobus is able to uptake large proteins in a process similar to eukaryotic endocytosis. This ability of G. obscuriglobus to uptake macromolecules from external milieu might have implications for a novel mode of nutrition. It is hypothesized that this endocytotic mode of nutrition might be distributed among other planctomycetes and be relevant to the interaction of planctomycetes with the sponge host because sponges are known to contain numerous types of macromolecules. A diverse collection of planctomycetes including sponge isolates were tested for their ability to uptake macromolecules including GFP and fluorescent dextran. Results indicate that ability to uptake dextran is distributed among planctomycetes, while ability to uptake GFP is limited to G. obscuriglobus and a closely related strain. Interestingly, presence and absence of mineral salts dictate the ability of planctomycetes strains to uptake dextran. Marine strains require salt ions for uptake, while uptake by freshwater strains was inhibited by the presence of salts as supplied by artificial seawater. The ability of planctomycetes to uptake fluorescent dextran was applied to select planctomycetes from mixed cultures containing non-planctomycete bacteria using a fluorescence-activated cell sorting (FACS) technique. The result demonstrated that planctomycete cells can be enriched from the mixed cultures, suggesting this novel FACS method might be able to be applied to select planctomycete cells from natural environments such as the sponge-associated microbial community.
Keyword Marine sponge
Actinobacteria
Planctomycetes
Natural product biosynthesis genes
Endocytosis

 
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Created: Tue, 20 Nov 2012, 18:39:59 EST by Hiroshi Izumi on behalf of Scholarly Communication and Digitisation Service