Antimicrobial activities and diversity of sponge derived microbes

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Title Antimicrobial activities and diversity of sponge derived microbes
Author(s) Flemer, Burkhardt
Publication date 2013
Original citation Flemer, B. 2013. Antimicrobial activities and diversity of sponge
derived microbes. PhD Thesis, University College Cork.
Type of publication Doctoral thesis
Rights © 2013, Burkhardt Flemer
http://creativecommons.org/licenses/by-nc-nd/3.0/
Embargo information No embargo required
Item downloaded http://hdl.handle.net/10468/1192
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University College Cork
Coláiste na hOllscoile Corcaigh

Antimicrobial activities and diversity of sponge derived
microbes

A thesis presented to the National University of Ireland for the
degree of Doctor of Philosophy

Burkhardt Flemer, Dipl. Ing. (FH)

National University of Ireland, Cork
Department of Microbiology

January 2013

Head of Department: Professor Gerald F. Fitzgerald
Supervisors: Professor Alan Dobson, Professor Fergal O’Gara
This thesis is dedicated to my family, my inspiration
DECLARATION 4
ABSTRACT 5
CHAPTER 1 8
INTRODUCTION

BODY PLAN OF MARINE SPONGES 9
Demosponges 9
MICROBIAL SPONGE ASSOCIATES 10
HIGHTHROUGHPUT SEQUENCING AS A TOOL TO ESTIMATE SPONGE- 16
ASSOCIATED MICROBIOTA
Studies employing pyrosequencing for the analysis of sponge-associated 17
microbiota
ESTABLISHMENT AND MAINTENANCE OF SPONGE MICROBE 21
INTERACTIONS
SYMBIONT FUNCTION 22
Carbon metabolism 23
Nitrogen metabolism 23
Sulphur metabolism 25
SPONGE ASSOCIATED FUNGI 26
DEEP SEA SPONGES 28
SECONDARY METABOLITES FROM MARINE SPONGES AND SPONGE 29
DERIVED MICROBES
Potential of sponge-ass. MO for the production of bioactive metabolites 30
Bioactive natural products from marine sponges 32
POLYKETIDE SYNTHASES (PKS) AND NON RIBOSOMAL PEPTIDE 33
SYNTHASES (NRPS)
NRPSs 33
PKSs 34
AIMS AND OVERVIEW OF THE PRESENTED STUDY 36
REFERENCES 36

CHAPTER 2 54
DIVERSITY AND ANTIMICROBIAL ACTIVITIES OF
MICROBES FROM TWO IRISH MARINE SPONGES, SUBERITES
CARNOSUS AND LEUCOSOLENIA SP.

ABSTRACT 55
INTRODUCTION 56
MATERIALS AND METHODS 58
Sponge sampling 58
Isolation of bacteria 58
Deferred antagonism assay 58
Well diffusion assay 59
PCR anplificaton of 16S-rRNA genes 59
Sequencing and phylogenetic analysis of 16S-rRNA gene products 60
RESULTS 61
Isolation of bacteria 61
Antimicrobial activity - deferred antagonism assay 63
Antimicrobial activity - well diffusion assay 68
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DISCUSSION 68
REFERENCES 73
SUPPLEMENTARY FIGURES 78

CHAPTER 3 83
DIVERSITY AND ANTIMICROBIAL ACTIVITIES OF FUNGAL
ISOLATES OBTAINED FROM 12 MARINE SPONGES SAMPLED
IN LOUGH HYNE, IRELAND

ABSTRACT 84
INTRODUCTION 85
MATERIALS AND METHODS 86
Sponge sampling 86
Isolation of fungi 86
Deferred antagonism assay 87
Well diffusion assay 87
Preparation and testing of antibiotic discs 88
Isolation of genomic DNA from fungal isolates and PCR anplificaton of 18S- 89
rRNA genes
Sequencing and phylogenetic analysis of 18S-rRNA gene products 89
Analysis of secondary metabolite genes 90
RESULTS 92
Isolation of fungi 92
Antimicrobial activity - deferred antagonism assay 92
Antimicrobial activity - well diffusion assay and antibiotic discs 97
Secondary metabolite genes in the genome of W9F6 97
DISCUSSION 102
Diversity 102
Bioactivity 104
Secondary metabolite genes 104
CONCLUSION 106
REFERENCES 107

CHAPTER 4 112
ANTIMICROBIAL ACTIVITY AND CHEMICAL CHARAC-
TERIZATION OF A SECONDARY METABOLITE FROM A
SPONGE DERIVED MICROMONOSPORA SP.

ABSTRACT 113
INTRODUCTION 114
MATERIALS AND METHODS 115
Sponge sampling 115
Isolation of bacteria 115
Deferred antagonism assay 115
Well diffusion assay 116
Fermentation and preparation of CFS for the extraction of antimicrobial 116
compounds
Extraction of antimicrobial compounds 117
Liquid-liquid extraction 118
Extraction of microbial pellet 118
Resazurin assay 118
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Thin layer and preparative scale chromatography 119
Analytical procedures 119
RESULTS AND DISCUSSION 120
Scale up 126
Purification of bioactive compounds 129
LC-MS and NMR analysis 129
LC-MS and NMR of fractions 5,6,8a, 8b and 9 134
CONCLUSION 135
REFERENCES 140

CHAPTER 5 142
DISTINCT PROKARYOTIC DIVERSITY AND SPATIAL
DISTRIBUTION OF MICROBES ASSOCIATED WITH DEEP SEA
SPONGES REVEALED BY PYROSEQUENCING

ABSTRACT 143
INTRODUCTION 144
MATERIALS AND METHODS 146
Sponge sampling 146
Metagenomic DNA extraction from seawater and sponge samples 146
PCR amplicon library preparation for pyrosequencing 147
Pyrosequencing data analysis 147
Preparation of Phylogenetic trees 148
RESULTS 149
Sponge sampling 149
Initial analysis 149
Bacterial vs. archaeal diversity 152
Archaeal diversity 152
Bacterial diversity 154
Classification into lower taxonomic levels 155
DISCUSSION 163
Bacterial community 163
Archaeal community 166
Analysis of shared OTUs 167
Spatial distribution of sponge-associated microbes in S. normani 168
Deep water sponges’ microbiota vs. microbiota of shallow water sponges 169
CONCLUSION 171
REFERENCES 172
SUPPLEMENTARY TABLES 178

CHAPTER 6 189
GENERAL DISCUSSION
GENERAL DISCUSSION 190
REFERENCES 199

ACKNOWLEDGEMENTS 204

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I herewith declare that the thesis is my own work and has not been submitted for
another degree, either at University College Cork or elsewhere.

Burkhardt Flemer _____________________________

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ABSTRACT

Marine sponges are the single best marine source for novel secondary metabolites
with bioactivity and play host to a microbiota with a remarkable diversity. In this
study, marine sponges collected in Irish waters were analysed for their associated
microbiota. Bacteria and fungi were isolated from several sponge species and
analysed for their diversity and antimicrobial activity and deep water sponges were
analysed for their associated bacteria and Archaea employing 454 pyrosequencing.

In order to evaluate the diversity and antimicrobial activity of bacteria from the
marine sponges Suberites carnosus (class Demospongiae) and Leucosolenia sp.
(class Calcaraea) collected at Lough Hyne, Ireland, two hundred and thirty seven
bacteria were isolated. Isolates from the phyla Actinobacteria, Bacteroidetes,
Firmicutes and Proteobacteria were obtained. Isolates of the genus Pseudovibrio
were dominant among the bacteria from S. carnosus whereas Pseudoalteromonas
and Vibrio were the dominant genera isolated from Leucosolenia sp. Approximately
50% of the isolates from S. carnosus displayed antibacterial activity and ~15% of the
isolates from Leucosolenia sp. demonstrated activity against the test fungal strains.
The antibacterial activity observed was mostly from Pseudovibrio and
Spongiobacter isolates, while the majority of the antifungal activity was observed
from the Pseudoalteromonas, Bacillus and Vibrio isolates. Overall, both sponges
possess a diverse range of bioactive and potentially novel bacteria. Differences
observed from the sponge-derived groups of isolates in terms of bioactivity suggests
that S. carnosus isolates may be a better source of antibacterial compounds, while
Leucosolenia sp. isolates appear to be a better source of antifungal compounds. Both
sponge species have been evaluated for the first time for their associated microbiota.

12 marine sponge samples from the same site, among them the S. carnosus and
Leucosolenia sp. sample, were analysed for their associated culturable fungi and 71
fungal isolates were obtained. Based on their 18S-rRNA gene sequence the isolates
were found to be very diverse with 22 OTUs determined at a sequence identity of
98.5% and different phylotypes present in some of the OTUs. Together with genera

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or orders typically isolated from marine sponges such as Hypocreales, Pleosporales
and Eurotiales some isolates were representatives of taxa seldomly found in marine
sponges. All isolates were assessed for their antimicrobial activity against 2 Gram
postitive and 1 Gram negative bacterium in the deferred antagonism assay and more
than 60% of the isolates tested positive in at least one assay against at least one of
the test strains. Isolates with clear and/ or broad range activity were also tested in the
well diffusion assay and the disc diffusion assay against the same bacterial as well as
5 fungal test strains (4 yeasts and 1 mycelium forming fungi). Isolates grouping into
three OTUs of the orders Hypocreales, Pleosporales and Eurotiales showed
especially strong and broad range activity. One of the isolates, which was closely
related to Fusarium oxysporum and showed activity against bacteria and fungi, was
investigated for its secondary metabolite genes (NRPS and PKS). At least 5 different
NRPS genes were identified in its genome based on partial gene sequences obtained
from a clone library. The sequence identity to published NRPS sequences of one
gene was as low as 50 % highlighting the likelihood that this isolate may be capable
of producing potentially novel secondary metabolites.

A Micromonospora sp. was isolated from a Haliclona simulans sample collected in
Irish waters. The isolate inhibited the growth of Gram positive bacterial test strains
in three different antimicrobial assays. Several production media were subsequently
employed to optimize the production of the antimicrobial compound(s) and a scale
up culture with the best medium was extracted for its antimicrobial compounds. The
purification of a yellow-orange compound with strong bioactivity against B. subtilis,
S. aureus and P. aeruginosa was facilitated by bio-assay guided chromatography and
the compound was tried to be characterized utilising LC-MS and NMR. By LC-MS
two compounds of bioactivity were detected in the extracts, one with an m/z=617.21
(positive mode) and one with an m/z=619.21. Another compound with little or no
bioactivity was also detected and had an m/z=445.17. Compounds with similar m/z-
values have previously been reported from Streptomyces and Micromonospora
isolates; quinocycline and isoquinocycline A with an m/z=619.21, the compounds
quinocycline/kosinostatin and isoquinocline B with an m/z=617.21 and aglycons of
all derivatives with an m/z=445.17. However, NMR studies of the isolated
compounds did not confirm the presence of known (iso)quinocylines in the extracts

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derived from the here presented Micromonospora isolate and thus the bioactive
compounds could indeed be novel.

Two deep water sponges, Lissodendoryx diversichela and Stelletta normani were
shown to host a remarkably different bacterial and archaeal diversity by application
of 454 Pyrosequencing targeting a region of the 16S-rRNA gene common to
Bacteria and Archaea. The L. diversichela community was dominated by a single γ-
proteobacterial sponge symbiont, closely related to the non-phototrophic,
chemoheterotrophic and aerobic genus Granulosicoccus and uncultured bacteria
previously found in shallow water sponges. The S. normani sample hosted a largely
sponge specific microbial community, even more diverse than has been previously
reported for shallow water sponges. Organisms potentially involved in nitrification
(Crenarchaeota), denitrification (Nitrospira, Acidobacteria and Chromatiales),
sulphate reduction (Desulfovibrio) and secondary metabolite production
(Poribacteria, Chloroflexi and Actinobacteria) were found to be spatially distributed
in the sponge. Whereas reads classified as Crenarchaeota, Nitrospira and
Chromatiales were more often encountered in the cortex, Desulfovibrio and
Chloroflexi featured more heavily in the choanosome. The presence of a large
proportion of unclassified reads in this sample highlights the potentially novel
microbial community associated with this sponge. Even though Archaea were less
abundant in both sponges than Bacteria they made up an important fraction of the
prokaryotic community. While, on the higher taxonomic levels, many similarities
between shallow- and deep water sponge-associated microbial communities were
detected, by comparing shared OTUs from the presented study with shallow-water
derived pyrosequencing reads, a deep sea specific population was implied.

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