Main research directions

In the Moraru phage lab we are researching viruses infecting bacteria, that is bacteriophages. We are currently running four main lines of research:

• Investigating phages of marine heterotrophic bacteria, with a focus on roseophages

• Developing single-cell methods for the study of phage-host interactions

• Developing bioinformatics tools for the study of phages

• Investigating ssDNA prophages in bacteria and archaea.

Phages of marine heterotrophic bacteria

Within the marine environment our main interest lies in the bacteriophages infecting the Roseobacter Group, or shortly roseophages. Our goals are to gain a deeper understanding of the phylogenetic and genomic diversity of the roseophages, of their phage-host interactions, biogeographic distribution and ecology. Our roseophage isolates infect Sulfitobacter, Lentibacter and Octadecabacter. To answer our research questions, we are using genomic sequencing, sequence database mining, host range assays, phage targeted genomeFISH and different methods for phylogenetic analysis.

This research is funded by the Deutsche Forschungsgemeinschaft. Cristina Moraru is the PI of the B6 Project “Bacteriophages of the Roseobacter Group” within the third phase (2018-2021) of the Collaborative Research Center Roseobacter (TRR51).

Single cell methods for the study of phage-host interactions

Methods measuring bulk parameters, as for example real time PCR or metagenomics, return results averaged across the whole microbial population. By contrast, single cell methods are able to measure intrapopulational variations and thus offer a unique view of the interactions between viruses and their hosts.

Cristina Moraru and collaborators have developed phageFISH and direct-geneFISH. These are single cell methods based on Fluorescence in situ Hybridization (FISH), which use gene targeted polynucleotide probes to detect (viral) genes inside microbial cells. PhageFISH https://doi.org/10.1111/1462-2920.12100 uses an enzymatic signal amplification, to ensure visualization of gene targets with low copy numbers. Because of this, it can only broadly estimate the number of phages in a cell, by measuring the area of the phage signal. Even so, this semi-quantitative measurement was sufficient to classify phage signals in 3 categories: i) new infections; ii) replicating infections and iii) advanced infections (late replication and encapsidation), and thus, to determine the infection phases of the HP1 phage on its host Pseudoalteromonas sp. H100. In direct-geneFISH https://doi.org/10.1111/1462-2920.13432 the polynucleotide probes are labeled with multiple fluorochromes, and no enzymatic signal amplification is required. As a consequence, the fluorescent signal intensity can be used to quantify the per cell target copy number.

In the Moraru lab we are applying a modified version of the direct-geneFISH protocol, which uses genome wide phage probes, to study roseophage-host interactions. In collaboration with other researchers, Cristina Moraru is extending the application of the direct-geneFISH protocol to various microbial systems. For example, working with the group of Alexander Probst (University of Duisburg-Essen) has led to the detection of novel archaeal viruses in samples from the terrestrial deep biosphere. A successful collaboration with Dolors Vaque and Yaiza Castillo de la Pena (Institue for Marine Sciences, Barcelona) has resulted in the development of a modified direct-geneFISH protocol to detect and quantify viruses infecting eukaryotic phytoplankton.

To enable the probe design, Cristina Moraru has developed gene-PROBER (see below).

Bioinformatics tools for the study of phages

ProFi

ProFi predicts prophages of ssDNA viruses in bacteria and archaea.

Go to ProFi website

VIRIDIC

VIRIDIC calculates nucleotide based intergenomic distances for viral genomes.

Go to VIRIDIC website

genePROBER

GenePROBER designs polynucleotide probes for targeting genes, genomic regions or even entire genomes of phages, bacteria or archaea.

Go to gene-PROBER website

CoGePh

CpGePh finds core genes in a set of phage genomes.

Go to CoGePh website

Master and PhD level courses

R programming for (meta)-genomic sequence analysis

Lecturer: Dr. Cristina Moraru

This is a crash course in R and Bioinformatics for the master students in the programs Master in Microbiology and Master in Environmental Sciences at the University of Oldenburg, as well as for microbiology PhD students who need to work with DNA and protein sequence data. The course starts by introducing basic R programing concepts, as for example variables, vectors, lists, data frames, for loops and if else statements. Then it demonstrates how to create custom functions and how to apply them to large data sets using apply and map functions, or for loops, for comparison. The course continues with an introduction in the R packages needed to manipulate strings, data frames and sequence data, as for example stringr. seqinr, tidyr, dplyr, magrittr, purr etc. Then, the students are thought how to integrate different command line bioinformatics tools in R scripts, to create veritable pipelines able to analyze large amount of data. As a practical example, the students will create their own microbial genome annotation pipeline. This pipeline will include the prediction of tRNAs and open reading frames, the translation of genes to proteins, the assignment of gene functions after performing BLASTP searches against the NCBI blast database, and the creation of a genome map. And finally, the course will demonstrate how to create reports using R markdown. Go to stud.IP.

Introduction in sequencing and sequence analysis

Lecturers: Dr. Cristina Moraru and Apl. Prof. Dr. Thorsten Brinkhoff

This course is tailored for the master students in the programs Master in Microbiology and Master in Environmental Sciences at the University of Oldenburg. The theoretical part aims to teach the basic concepts for sequencing and sequence analysis, with a focus on microorganisms. The course will demonstrate how to i) work with Sanger sequencing data by using DNA Baser to analyze chromatograms, quality checking, end trim and assembly of contigs; ii) use sequence databases, as for example the ones from NCBI, InterPro, etc; iii) perform BLAST searches to determine the identity of different microbial strains using 16S rRNA or 18S rRNA genes; iv) perform BLAST searches to identify the function of proteins; v) use Geneious to browse and annotate microbial genomes; vi) construct phylogenetic trees using ARB and rRNA gene databases and vii) design primers and probes. In the practical part of the course, the sequencing of microbial genomes using the Oxford Nanopore next generation sequencing technology will be demonstrated. Go to stud.IP.

Methods in aquatic microbial ecology

Lecturers: Dr. Cristina Moraru, Apl. Prof. Dr. Thorsten Brinkhoff, Prof. Dr. Meinhard Simon

This is a practical course for the master students in the programs Master in Microbiology and Master in Environmental Sciences at the University of Oldenburg. The course teaches various techniques for the analysis of microorganisms in environmental samples, with a focus on the marine environment. During the course, Cristina Moraru is guiding the students through different Fluorescence in situ Hybridization (FISH) protocols. First, 16S rRNA CARD-FISH is used to identify microbial cells in sea water samples. Second, a direct-geneFISH protocol is used to detect bacterial cells infected with viruses and monitor the progress of viral infection. The results are visualized using epifluorescence microscopy. Go to stud.IP.

Microbial diversity

Lecturers: Dr. Cristina Moraru, Apl. Prof. Dr. Thorsten Brinkhoff and colleagues

This is a theoretical course for the master students in the programs Master in Microbiology and Master in Environmental Sciences at the University of Oldenburg. The students are introduced in various aspects of microbial diversity, from viruses, to bacteria and unicellular protists. During the course, Cristina Moraru is lecturing about viruses and the cellular structure of eukaryotes. A broad overview of all virus types is given, from dsDNA viruses to ssDNA and RNA viruses. The focus is on viruses of bacteria and archaea, but also viruses of eukaryotes are touched upon. The main topics are the classification of viruses, their infection strategies and their ecology. Go to stud.IP.

Ecology of marine microbes

Lecturers: Dr. Cristina Moraru, Apl. Prof. Dr. Thorsten Brinkhoff and colleagues

This is a practical course for the master students in the programs Master in Microbiology and Master in Environmental Sciences at the University of Oldenburg. The students will be working in small teams, on various microbial ecology topics. Cristina Moraru will supervise the virus related project. During this project, the students will enrich, isolate and screen viruses infecting marine bacteria. Selected viruses will be genome sequenced using the Oxford Nanopore technology and morphologically characterized by uranyl acetate staining and Transmission Electron Microscopy. Alternatively, the infection cycle will be analyzed for selected virus-host pairs, using phage targeted genomeFISH, in conjunction with phage counts by SybGreen or phage plaques forming units methodologies, bacterial cell counts and so on. Go to stud.IP.

SERVERS:

- KRONOS

- RHEA

- High Performance Computing Cluster (HPC) of the University of Oldenburg

Accessibility of the KRONOS and RHEA Servers (needs user accounts, please contact Matthias Schroeder):

- using MobaXterm

- using RStudio Server from this link: http://kronos.icbm.uni-oldenburg.de:8787/

Data storage on the KRONOS and RHEA Servers:

- either locally in user home folder (limited space, NO backup)

- or in the Bioinf folder, located on a HDD in Uni Oldenburg Central IT, plenty of space available, Backed Up. This folder is mounted in the root of the ARB Server and has the following path: /bioinf

Structure of the Bioinf folder:

- /bionf/data - all common sequence databases are stored here. All users have read access to this folder.

- /bioinf/home - the folders of individual users are stored here. Users should have access only to their own folder here.

- /bioinf/shared - folders dedicated to group sharing.

Accessibility of the HPC (needs an HPC account, please contact central IT services of University of Oldenburg):

- using MobaXterm

New programs:

If you can't find your desired program in the table below, you can contact Matthias Schroeder to arrange their installation.

Existing programs:

NCBI blast databases:

Raw data metagenomes

Principal investigator - Cristina Moraru, PhD

Senior Scientist

Department of The Biology of Geological Processes

Institute for Chemistry and Biology of the Marine Environment

Room W16A-1-112, Carl-von-Ossietzky –Str. 9 -11, D-26111 Oldenburg, Germany

Telephone 0049 441 798 3218

Email: liliana.cristina.moraru( at )uni-oldenburg.de

Member of the Bacterial and Archaeal Viruses Subcommittee of the International Committee on Taxonomy of Viruses (ICTV)

Research interests

PhD students

Vera Bischoff, MSc

Email: vera.bischoff( at )uni-oldenburg.de

Research interests

I'm interested in the diversity of lytic bacteriophages infecting marine bacteria of the Rhodobacteraceae family. My PhD thesis, which is part of the Transregional Collaborative Research Centre SFB/TRR51 “Roseobacter”. During my thesis I have isolated phages from the North Sea infecting the genera Sulfitobacter, Lentibacter and Octadecabacter. I am currently investigating their phylogeny and genetic characteristics, as well as their life cycle and biogeographical distribution.

Falk Zucker, MSc

Email: falk.zucker( at )uni-oldenburg.de

Research interests

I study bacteriophages infecting marine Rhodobacteraceae (the Roseobacter group). In particular those that are capable of integrating themselves into the bacterial chromosome or stay dormant as an extrachromosomal plasmid – also known as temperate bacteriophages.

For my studies I use a number of different techniques, including among others:

• PhageFISH, to enable the detection of phages and prophages in single cells as well as to characterize the phage infection stages.

• Induction of a broad range of bioinformatically predicted prophages via triggering the bacterial SOS response, for example using mitomycin C.

• Nanopore sequencing to detect and verify putative prophage regions among several genomes of the Roseobacter group.

Technical Assistance

Andrea Schlingloff, BSc

Email: andrea.schlingloff( at )uni-oldenburg.de

Responsibilities

Aaron Woolley, MSc

Email: aaron.woolley( at )uni-oldenburg.de

Responsibilities

Ismail Hayani-Aji, BSc

Email: ismail.hayani.aji( at )uni-oldenburg.de

Responsibilities

Former members

Completed master and bachelor studies

Visiting Scientists

Moraru, C., Varsani, A., and Kropinski, A.M. (2020) VIRIDIC – a novel tool to calculate the intergenomic similarities of prokaryote-infecting viruses. bioRxiv. https://doi.org/10.1101/2020.07.05.188268

Castillo, Y.M., Sebastián, M., Forn, I., Grimsley, N., Yau, S., Moraru, C., and Vaqué, D. (2020) Visualization of viral infection dynamics in a unicellular eukaryote and quantification of viral production using virus fluorescence in situ hybridization. Frontiers in microbiology, 11, article 1559. https://doi.org/10.3389/fmicb.2020.01559

Bischoff, V., Zucker, F. and Moraru, C. (early online, 2019): Marine Bacteriophages. In Encyclopedia of Virology 4th edition, Reference Module in Life Sciences, Elsevier. https://doi.org/10.1016/B978-0-12-809633-8.20988-6

Bischoff, V., Bunk, B., Meier-Kolthoff, J.P., Spröer, C., Poehlein, A., Dogs, M., Nguyen, M., Petersen, J., Daniel, R., Overmann, J., Göker, M., Simon, M., Brinkhoff T. and Moraru, C. (2019): Cobaviruses - a new globally distributed phage group infecting Rhodobacteraceae in marine ecosystems. The ISME Journal 13 (6), pp. 1404–1421. https://doi.org/10.1038/s41396-019-0362-7

Jimena Barrero-Canosa, Cristina Moraru (2019): PhageFISH for monitoring phage infections at single cell level. In Martha R. J. Clokie, Andrew M. Kropinski, Rob Lavigne (Eds.): Bacteriophages. Methods and protocols. New York: Humana Press; Springer (Springer protocols, Methods and protocols, volume 1898), pp. 1–26. https://doi.org/10.1007/978-1-4939-8940-9_1

Barrero-Canosa, J., Moraru, C*., Zeugner, Laura; Fuchs, Bernhard M.; Amann, Rudolf* (2017): Direct-geneFISH: a simplified protocol for the simultaneous detection and quantification of genes and rRNA in microorganisms. Environmental Microbiology 19 (1), pp. 70–82. https://doi.org/10.1111/1462-2920.13432

Moraru C. and Allers E. (2014) Fluorescence in situ hybridization (FISH) for the identification and quantification of microorganisms, In Skovhus, T.L., Caffrey, S., and Hubert, C.R.J.. Molecular Methods and Applications in Microbiology (Norfolk, UK: Caister Academic Press).

Allers, E*., Moraru, C.*, Duhaime, M.B., Beneze, E., Solonenko, N., Canosa, J.B., Amann, R., and Sullivan, M.B. (2013). Single-cell and population level viral infection dynamics revealed by phageFISH, a method to visualize intracellular and free viruses. Environmental Microbiology 15: 2306-2318. https://doi.org/10.1111/1462-2920.12100

Moraru, C., and Amann, R. (2012) Crystal ball: Fluorescence in situ hybridization in the age of super-resolution microscopy. Systematic and Applied Microbiology 35: 549-552. https://doi.org/10.1016/j.syapm.2012.10.001

Lenk, S., Moraru, C., Hahnke, S., Arnds, J., Richter, M., Kube, M., Reinhardt, R., Brinkhoff, T., Harder, J., Amann, R., and Musmann, M. (2012) Roseobacter clade bacteria are abundant in coastal sediments and encode a novel combination of sulfur oxidation genes. The ISME Journal 6: 2178-2187. https://dx.doi.org/10.1038%2Fismej.2012.66

Moraru, C., Moraru, G., Fuchs, B.M., and Amann, R. (2011) Concepts and software for a rational design of polynucleotide probes. Environmental Microbiology Reports 3: 69-78. https://doi.org/10.1111/j.1758-2229.2010.00189.x

Petersen, J.M., Zielinski, F.U., Pape, T., Seifert, R., Moraru, C., Amann, R., Hourdez, S., Girguis, P.R., Wankel, S.D., Barbe, V., Pelletier, E., Fink, D., Borowski, C., Bach, W., and Dubilier, N. (2011) Hydrogen is an energy source for hydrothermal vent symbioses. Nature 476: 176-180. https://doi.org/10.1038/nature10325

Moraru, C., Lam, P., Fuchs, B.M., Kuypers, M.M.M. and Amann, R. (2010) GeneFISH – an in situ technique for linking gene presence and cell identity in environmental microorganisms. Environmental Microbiology 12: 3057-3073. https://doi.org/10.1111/j.1462-2920.2010.02281.x https://doi.org/10.1111/j.1462-2920.2010.02281.x

Ploug, H., Musat, N., Adam, B., Moraru, C., Lavik, G., Bergman, B., and Kuypers, M.M.M.. (2010) Carbon and nitrogen fluxes associated with Aphanizomenon sp. in the Baltic Sea. The ISME Journal 4: 1215-1223. https://doi.org/10.1038/ismej.2010.53

Cristina Moraru, PhD

Senior Scientist

Department of The Biology of Geological Processes

Institute for Chemistry and Biology of the Marine Environment

University of Oldenburg

Room W16A-1-112

Carl-von-Ossietzky –Str. 9 -11, D-26111 Oldenburg, Germany

Telephone 0049 441 798 3218

Email: liliana.cristina.moraru( at )uni-oldenburg.de