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Identity and activity of marine microbial populations as revealed by single cell techniques [Elektronische Ressource] / vorgelegt von Cecilia Alonso

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Identity and activity of marine microbial populationsas revealed by single cell techniquesCecilia Alonso Este trabajo está dedicado a mis padres que hicieron todo lo posible -a veces también lo imposible- para ayudarme a encontrar mi camino. Identity and activity of marine microbial populations as revealed by single cell techniquesDissertation zur Erlangung des Grades eines Doktors der Naturwissenschaften-Dr.rer.nat.-Dem Fachbereich Biologie/Chemie der Universität Bremen vorgelegt von Cecilia AlonsoBremen, 2005Die vorliegende Doktorarbeit wurde in der Zeit von April 2002 bis Juli 2005 am Max Planck Institut für marine Mikrobiologie in Bremen angefertigt.Gutachter: Dr. Rudolf AmannDr. Jakob PernthalerDr. Victor SmetacekDr. Daniel CondeTag des Promotionskolloqiums: 14. Oktober 2005 “Tengo una banda amiga que me aguanta el corazón” La Vela Puerca More than in any time, I have to say that this work was achieved with the help of many people. Thanks to my supervisor, Jakob Pernthaler, for all the enthusiasm, creativity and hard work that he dedicated for this thesis. Thanks for the freedom of work that you gave me, for being open to discuss any crazy idea, and for being always there when I needed you.

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Published 01 January 2005
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Identity and activity of marine microbial populations
as revealed by single cell techniques
Cecilia Alonso























Este trabajo está dedicado a mis padres que hicieron todo lo posible -a veces
también lo imposible- para ayudarme a encontrar mi camino.



















Identity and activity of marine microbial populations as
revealed by single cell techniques
Dissertation zur Erlangung des Grades eines
Doktors der Naturwissenschaften
-Dr.rer.nat.-
Dem Fachbereich Biologie/Chemie der
Universität Bremen vorgelegt von
Cecilia Alonso
Bremen, 2005Die vorliegende Doktorarbeit wurde in der Zeit von April 2002 bis Juli 2005
am Max Planck Institut für marine Mikrobiologie in Bremen angefertigt.
Gutachter:
Dr. Rudolf Amann
Dr. Jakob Pernthaler
Dr. Victor Smetacek
Dr. Daniel Conde
Tag des Promotionskolloqiums: 14. Oktober 2005
“Tengo una banda amiga que me aguanta el corazón”
La Vela Puerca

More than in any time, I have to say that this work was achieved with the help of many
people.

Thanks to my supervisor, Jakob Pernthaler, for all the enthusiasm, creativity and hard
work that he dedicated for this thesis. Thanks for the freedom of work that you gave me,
for being open to discuss any crazy idea, and for being always there when I needed you.

Thanks to Rudi Amann- our “big boss”- for his support during this time. Thank you for
making possible that I came here before we knew if we would have funds to complete
the Ph.D. I very much appreciate the spirit that you promote on us by being open to
collaboration with so many people from different grounds.

Many thanks to all the Mollies! The present ones, and the ones that were here during my
stay. Thank you for the very nice working atmosphere, for the tips exchanged, for the
seminars, and of course for the breakfasts and afternoon cakes J. I learned a lot from
you.

Thanks to the students that helped me during this work: Citlali Guerra and Jacob Jacob.
It was very important for me to count on you. Thank you for taking your work with such
responsibility and enthusiasm.

Many thanks to Thierry Lombardot, Federico Battistoni and Marc Mußmann for hunting
errors and horrors in the “this one is really the last” version of the thesis.

Thanks to the members of the Thesis Committee: Rudi Amann, Jakob Pernthaler, Victor
Smetacek and Daniel Conde. Thank you for following this work. I enjoyed very much the
fruitful discussions that we had. They were really interesting and productive for me, and
they gave me a perspective of the work. Thank you also for dedicating your precious
time for the evaluation of this thesis.

This time was not only intense in terms of working, but also in life. Many thanks to my
beloved people, that supported me and accompanied me in these years.

Thanks to my parents Elsa and Hugo, to Gloria, to my aunts, uncles and cousins that
supported me , shared my happiness and sadness, and always reminded me that I am
among them despite the distance.

Thanks to my old friends: Luisa, Lucía, Mariana, el Dele, Hugo, Ana Laura, Andrés,
Ramiro, el Martínez, Laura, María José, Gerardo, Pedro, Sara, Jesús, Sa lvador, Nélida,
Alicia, Inés, Ricardo. Thank you for still holding the net regardless the time and space
differences. Thank you for being my ultimate refuge and for sharing and helping me
understanding my own changes.

Thank you to the new friends I had th e chance to meet here: “the witches” Ayse and
Meral. Thank you for sharing the recipes for daily magic and for holding my heart as
yours. Juan and Laura, my spanish sister souls, thank you for your support, your
enthusiasm for making crazy plans (that sometimes we even realize!), and the fresh
energy that you give me.

Thanks to the “comunidad uruguayense en Bremen” ;-): Fefe, Claudia and Elsa. It is
great to have you close. Thank you for accompanying me and supporting me in
everything. Thank you for helping me in finding the perspective during the hard times.

Thanks to Thierry for the nice time together and for all the support in these last days.

Thanks to the families Lombardot and Saglam for adopting me as their own child.

Thanks to the Microtropien/nnes for giving me the “Hakuna matata” spirit that I needed
so much in these last months. Thank you Mounira, Rabi and Pascal, I learned much
from you.

Thanks to my friends from the Lagerhaus: Nedim, Bülent, Fuad, Bilal, Mehmet and Salim
for being my second home. Thank you for the music and the spinach soup. Thank you
for building such a nice space for meeting and for stimulating the “kulturelle Vielfalt.”

To all of you: Danke, Thank you, Merci, Tesekkürler, Sipas, Gracias!

Table of Contents

Summary.................................................................................................................................... 9

Introduction.............................13

Identity and activity of marine prokaryotes.................................................................14
Single cell approaches to address cellular activities...............15
FISH as activity indicator......................................16
Vital stains for assessing membrane integrity .....................................................................17
Redox dyes for detection of respiratory activity...18
Presence of a nucleoid body19
Detection of growing cells ....................................20
Microautoradiography for tracking substrate uptake ...........................................................21

What are active bacterioplankton cells?......................................26

Results and Discussion.......................................................................27

1. Development of a MARFISH protocol.......................................28
Microautoradiography...........................................................................28
Fluorescence in situ hybridization and catalyzed reporter deposition.31
MARFISH..............................................................33
Protocol development...........................................34
Final protocol for MARFISH performance after incubation with radiolabeled substrates...37
First field tests......................................................38
Comparison with a standard protocol..................40
The automation of counting..................................................................41
Evaluation of reproducibility of the technique......44
Further applications..............................................45
Conclusions..........................................................46

2. Facultative anaerobic metabolism among bacteria in the water column .......47
Introduction...........................................................................................47
Methodology.........................................................48
Results..................................48
Discussion............................51

3. Concentration dependent substrate uptake ...........................................................53
Introduction................................................................53
Methodology.........................54
Results..................................................................55
Discussion............................61

Conclusions and Outlook....................................63

References...............................................................................................67

Publications............................................................85


























Summary


Summary

In most aquatic habitats the mere quantification of bacterial taxa does not appear to provide
sufficient information about their ecological role. Consequently, there is a need for in situ
approaches that allow simultaneous microbial identification and an estimate of microbial
activity. These approaches should optimally provide a resolution at the level of single
populations or even cells as bulk activity measurements seldom correlate with total
abundances of bacteria and specific microbial populations may mediate central
biogeochemical processes. At the end of the 1990’s, a methodological approach was
developed to track substrate uptake by specific prokaryotic groups. This was achieved by the
combination of microautoradiography and fluorescence in situ hybridization (MARFISH).
However, the original MARFISH method had several drawbacks for its application in marine
samples. The first aim of this study was to overcome these limitations by introducing three
major modifications that rendered the method more sensitive, accurate, and suitable for high-
throughput sample processing. In the second half of this work this improved protocol was
employed for two studies on the ecology of particular picoplankton populations in the coastal
North Sea. In the first application the potential for anaerobic metabolism of pelagic bacteria
was investigated. It has been suggested that in coastal environments the potential for
anaerobic metabolism might be a common feature of bacterioplankton, but no direct
evidence had been provided to support this hypothesis. Incorporation of glucose under
anoxic conditions was found in Alphaproteobacteria, Gammaproteobacteria and the
Cytophaga-Flavobacteria. Moreover, specific populations of copiotrophic bacteria
(Alteromonas, Pseudoalteromonas) showed preferential glucose incorporation under anoxic
conditions. In a second application, concentration-dependent uptake of glucose and leucine
was assayed before and during a spring phytoplankton bloom. Coastal pelagic environments
are characterized by concentration gradients of dissolved organic carbon, and by
pronounced seasonal differences in substrate availability for the picoplankton. Microbial taxa
that co-exist in such habitats might thus differ in their ability to incorporate substrates at
various concentrations. Our results supported this hypothesis. Three patterns were observed
for monomer uptake: high numbers of active cells regardless the substrate concentration
(Roseobacter), preference for a specific concentration (SAR11 bacteria), and increasing
numbers of active cells with increasing substrate concentration (SAR86, DE2 cluster of
Bacteroidetes, and Euryarchaeota).