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STRUCTURE AND FUNCTION OF MICROBIAL COMMUNITIES PROCESSING DISSOLVED ORGANIC MATTER IN MARINE ENVIRONMENTS by Hila Elifantz A dissertation submitted to the Faculty of the University of Delaware in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Marine Studies Fall 2006 Copyright 2006 Hila Elifantz All Rights Reserved UMI Number: 3247689 UMI Microform 3247689 Copyright 2007 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.

Box 1346 Ann Arbor, MI 48106-1346 STRUCTURE AND FUNCTION OF MICROBIAL COMMUNITIES PROCESSING DISSOLVED ORGANIC MATTER IN MARINE ENVIRONMENTS by Hila Elifantz Approved: __________________________________________________________ Nancy M. Dean of the College of Marine and Earth Studies Approved: __________________________________________________________ Daniel Rich, Ph. Provost I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Professor in charge of dissertation I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy.

Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Signed: __________________________________________________________ Thomas Hanson, Ph. Member of dissertation committee I certify that I have read this dissertation and that in my opinion it meets the academic and professional standard required by the University as a dissertation for the degree of Doctor of Philosophy. Member of dissertation committee ACKNOWLEDGMENTS I would like to thank my advisor David Kirchman, for guiding me in the marine microbial world and for his patience with my writing.

My committee; David Hutchins, Thomas Hanson, and Byron Crump for their constructive suggestions regarding my research and my manuscripts along the way. I would also like to thank Steve Wilhelm from the University of Tennessee for the chlorophyll data. I would like to thank the members of Kirchman lab. Liying Yu , Lisa Waidner, Rex Malmstrom, Paul Jones, Vanessa Michelou, Barbara Campbell, Matthew Cottrell, Tiffany Straza, Glen Christman, Dawn Castle and Katie Preen.

All were very helpful in collecting samples, lab work, and in discussing ideas for this study and the results that came out of it. I would like to thank Coren Milbury, Robin Varney, and Nathan Campbell for accommodating my samples on the sequencer. Also thank to the people in Marsh, Warner, Targett, Cary, Sharp, and Hanson labs for letting me use various instruments in their labs. Thanks also for the CMES stuff, especially Peggy Conlon, Paul Dumigan, Lisa Perelly, Doris Manship, Susan Wedeman, Thanks for all my friends in CMS for making the time here enjoyable.

Special thanks to Elif Demir, Karen Pelletreau, Tracy Szela, Rick and Linda Rouf for their support throughout and especially at the end when it seemed that I will never finish. Finally, thank you my friends and family in Israel, for your support from far and the patience. iv TABLE OF CONTENTS LIST OF TABLES………………………………………………………………. vii LIST OF FIGURES…………………………………………………………….

ASSIMILATION OF POLYSACCHARIDES AND GLUCOSE BY MAJOR BACTERIAL GROUPS IN THE DELWARE ESTUARY……………… 13 Abstract…………………………………………………………………. 14 Materials and Methods…………………………………………………. 16 Preparation of 3H-EPS……………………………………. 16 Sample collection and preparation………………………….

17 FISH and microautoradiography analysis………………………. 17 Results…………………………………………………………………… 19 Dominant bacterioplankton groups assimilating glucose and EPS……………………………………………………. 19 Relationships between abundance and DOM assimilation……… 21 The importance of EPS and glucose assimilation to a phylogenetic group…………………………………………. 22 Cell volumes of active and inactive bacteria……………….

DISSOLVED ORGANIC MATTER ASSIMILATION BY HETEROTROPHIC BACTERIAL GROUPS IN THE WESTERN ARCTIC OCEAN………………………………………………………… 41 Abstract…………………………………………………………………. 41 Introduction……………………………………………………………… 42 v Materials and Methods…………………………………………………. 45 Sample collection and uptake of 3H-DOM……………………… 45 FISH and microautoradiography analysis………………………. 47 Results…………………………………………………………………… 47 Abundance of bacterial groups………………………………….

47 Fraction of cells assimilating DOM components………………. 48 Fraction of phylogenetic groups assimilating DOM components. 49 Contribution of phylogenetic groups to DOM uptake…………. 50 Relationships between abundance and DOM assimilation……… 52 Discussion……………………………………………………………….

DIVERSITY AND ABUNDANCE OF GLYCOSYL HYDROLASES FAMILY 5 IN THE NORTH ATLANTIC OCEAN……………………. 70 Introduction……………………………………………………………… 71 Materials and Methods…………………………………………………. 75 PCR primers design……………………………………………. 75 GH5 libraries construction……………………………………….

76 GH5 libraries sequencing………………………………………. 79 Results…………………………………………………………………… 80 Degenerate GH5 primer design…………………………………. 80 Diversity analysis of GH5 clone libraries …………………. 81 Abundance of GH5 in the North Atlantic Ocean……………….

114 vi LIST OF TABLES Table 2.1 Averaged cell volumes of EPS assimilating and non- assimilating bacterial cells……………………………………… 33 Table 2.2 Averaged cell volumes of glucose assimilating and non- assimilating bacterial cells……………………………………….1 Relative abundance of bacteria and major groups in the Western Arctic Ocean……………………………………….1 GH5 clusters as classified by amino acids similarity…………… 98 Table 4.2 Accession numbers of sequences used in Figure 4.3 Sub-groups of cloned GH5 from the MAB and SAR libraries as appeared in the GH5 gene tree (Figure 4. 100 vii LIST OF FIGURES Figure 2.1 Percent cell contributing to DOM assimilation verses percent silver grain area around these cells in each phylogenetic group………………………………………… 35 Figure 2.2 Composition of bacterial communities assimilating A) glucose and B) EPS in the Delaware Estuary………….3 SAR11 clade abundance and contribution to glucose and EPS assimilation in the Delaware Estuary…………….4 Contribution of phylogenetic groups to A) glucose and B) EPS assimilation as a function of group abundance…….5 Fraction of bacteria in group assimilating glucose vs. the fraction of bacteria in group assimilating EPS………….6 Relative abundance of each bacterial group as a function of the fraction of bacteria in phylogenetic group assimilating A) glucose and B) EPS ….1 Sampling sites in the Western Arctic Ocean……………… 64 Figure 3.2 Fraction of all assimilating cells as a function of exposure time to photographic emulsion…………………………….3 Fraction of bacteria in group that assimilated DOM. A) Cytophaga-like bacteria; B) Alpha-proteobacteria; C) Gamma-proteobacteria…………………………………….4 A) Average percent assimilation of DOM by bacterial group as a function of location.

B) Total number of cells in bacterial groups in summer 2004………………………….5 The contribution to DOM assimilation by specific bacterial groups……………………………………………………… 68 Figure 3.6 DOM assimilation as a function of bacterial group's abundance………………………………………………….1 Sampling location along the North Atlantic Ocean……….2 Phylogenetic tree of glycosyl hydrolases family 5……… 102 Figure 4.3 Number of operational taxonomic units as a function of number of clones at 97% similarity. A) Nucleic acids; B) Amino acids……………………………………………….4 Neighbor-joining GH5-like gene tree…………………….5 Percent of clones in each GH5 library that were grouped in the same operational taxonomic unit with sequences from the SSMMC……………………………………………….6 Alignment of the amino acid sequence of CelZ from Erwinia chrysanthemi and the GH5 consensus from MAB and SAR libraries………………………………………….7 Abundance of GH5 genes and chlorophyll concentration along a transect in the North Atlantic Ocean……………… 107 Figure 4.8 Abundance of GH5 genes and chlorophyll concentration in a depth profile from the Sargasso Sea……………………. 108 ix ABSTRACT Heterotrophic bacteria are important participants in the global carbon cycle as they process about 50% of the fixed carbon in the oceans. Since the amount of primary production in the ocean is equal to that in terrestrial environments, the need to understand these processes is obvious.

Various measurements are used to assess the amount of carbon that is processed through bacteria, including bacterial production, bacterial respiration, dissolved organic matter (DOM) concentrations and fluxes. However, none of these methods reveal which bacterial groups use specific compounds in the DOM pool. The aim of this dissertation was to address some of these issues. Microautoradiography combined with fluorescence in situ hybridization (Micro- FISH) was used to evaluate which bacterial groups assimilate common DOM components, and whether this assimilation can be related to other environmental factors.

In the Delaware Estuary the assimilation of glucose and extracellular polymeric substances (EPS) was dominated by the abundant bacterial groups. In the freshwater end of the estuary Actinobacteria and Betaproteobacteria were the dominant groups, while in the saline part of the estuary Alphaproteobacteria and Cytophaga-like bacteria contributed the most to DOM uptake. Only 35-50% of the assimilation could be explained by bacterial group abundance. In addition, groups that x had more assimilating cells were not necessarily more abundant.

Therefore, it seems that the bacterial community in the Delaware Estuary might be affected equally by "bottom- up" (DOM availability) and "top-down" (predation and viral lysis) factors. In contrast, the oligotrophic environment of the Western Arctic Ocean displayed a different pattern. Most of the assimilation (90-99%) could be explained by abundance in the Arctic Ocean. These data suggest that the microbial community in this environment is mostly controlled by DOM availability.

Perhaps the most interesting observation of this study was the level of activity detected in the Arctic environment. Up to 50% of all prokaryotes assimilated amino acids, followed by EPS and proteins. Glucose, however, was the least assimilated compound. The contribution to assimilation of DOM was different among the bacterial groups.

The contribution to DOM assimilation by Cytophaga-like bacteria decreased between the Chukchi Sea shelf and the Canada Basin. In contrast, Alphaproteobacteria contributed the most in the slope region. This group also contributed more to the assimilation of low molecular weight (LMW) DOM, while Cytophaga-like bacteria contributed more to the assimilation of high molecular weight (HMW) DOM. The last part of this dissertation dealt with the molecular mechanism of the degradation of polysaccharides, which is an important part of the labile DOM pool in the marine environment.

The Carbohydrate-Active enZymes (CAZy) and the Sargasso Sea whole-shotgun databases were searched for the common endoglucanases in the marine environment. One sub-group of glycosyl hydrolases, family 5 (GH5), was identified as a potential important enzyme in the marine environment. Two GH5 gene-libraries were xi constructed from the Mid-Atlantic Bight and the Sargasso Sea. The two libraries were different from each other.

The sequences in these libraries were also different from sequences known from cultured bacteria. Translation of the amplified fragment indicated that the important residues for activity are present, and therefore these are potentially active endoglucanases. GH5 abundance in surface water of the North Atlantic, as measured with quantitative PCR (Q-PCR), fluctuated between 10 copies to 200 copies per nanogram DNA. The abundance of GH5 correlated to chlorophyll concentrations in the eastern part of the sampled region and in one depth profile.

The current study added to the growing information regarding the composition of bacterial community in aquatic environments and the role of specific bacterial groups in DOM assimilation. In particular, this study was the first to unfold the relation between structure and function of the bacterial community in the Arctic Ocean, the only cold environment studied in that aspect to date. The molecular study of GH5 revealed the potential of the community for polysaccharides degradation, however, more need to be done to broaden our understanding of the mineralization of these compounds in the marine environment. xii Chapter 1 INTRODUCTION Due to their sheer size, the oceans are responsible for half of global biological carbon fixation (C), estimated at 2.1 Gt annually (Field et al.

This fixed carbon is only a small fraction compared to the global oceanic dissolved organic carbon (DOC) pool, about 685 Gt C, which is similar in size to the atmospheric CO2 pool (Hansell and Carlson 1998). While it seems that there is a balance between the oceans and the atmosphere, it is yet unclear how the rising concentration of CO2 in the atmosphere will affect this balance, and how the ocean C cycle affects this process. The fixed C in the oceans can be either decomposed through the microbial loop, transferred to higher trophic levels via the food chain, or transported into the ocean interior via particulate organic mater (POM) and zooplankton migration (Azam 1998; Carlson 2002; Ducklow et al. In addition to the microbial loop, the other process considered to be important is the carbon biological pump, which sequesters carbon into the seafloor and out of the global cycle.

However, the microbial loop is not separated from the rest of the food web, as bacteria can be grazed upon by microzooplankon (Sherr et al.