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Identification of bacterial genes required for diatom-bacteria interactions

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Torres-Monroy,  Ingrid
IMPRS MarMic, Max Planck Institute for Marine Microbiology, Max Planck Society;

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Torres-Monroy, I. (2013). Identification of bacterial genes required for diatom-bacteria interactions. PhD Thesis, Jacobs University, Bremen / Germany.


Cite as: http://hdl.handle.net/21.11116/0000-0001-C6ED-5
Abstract
Aggregate formation in form of marine snow is an essential mechanism in the oceans that mediates the sinking of organic carbon to depth. Interactions between bacteria and diatom play an important role during this process by inducing secretion of different polymers, which increase the size of marine aggregates. Not much is known about the molecular mechanisms responsible for the diatom-bacteria interaction. To address this issue, a bilateral model system consisting of Marinobacter adhaerens HP15 and the diatom Thalassiosira weissflogii has been established. This bacterium specifically attaches to T. weissflogii cells thereby increasing its aggregation and inducing an increased formation of transparent exopolymeric particles. A genetic tool system was developed for M. adhaerens HP15, in which successful expression of reporter genes revealed useful tools for gene expression analyses. In addition, several bacterial genes potentially important during the interaction have been investigated. However, genes specifically expressed in vivo are still unknown. In this work we identified bacterial genes that are induced during the interaction with T. weissflogii by two different approaches. First, an In vivo expression technology (IVET) screening was conducted, constructing a promoter-trap vector containing a fusion between a promoterless selection marker gene and a reporter gene. Generation of a library of plasmids carrying genomic fragments upstream of the fusion and its subsequent transformation into a selection marker mutant allowed the selection of bacterial promoters specifically expressed during the interactions with T. weissflogii. Second, bacterial proteins expressed in response to the presence of T. weissflogii were identified by comparison of protein profiles of bacterial cultures incubated with or without diatom cells and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). By these approaches several genes expressed during the co-cultivation with diatoms were identified. Sequence analyses of these genes showed that they are required for central intracellular metabolism, cell envelope structure, nutrient scavenging, regulation, chemotaxis, secretion, stress response and DNA transfer. These genes may play an important role during the interaction between M. adhaerens HP15 and T. weissflogii. The results obtained in this study contribute to a better understanding of the molecular and biochemical mechanisms responsible for diatom-bacteria interactions. Additionally, the tight adherence (tad) gene locus present on the 187-kb HP15 plasmid and consisting of 9 genes (flp, rcpCA, tadZABCDG), was analyzed. This locus is found in several Gram-negative bacteria encoding for a type IVb fimbrial low-molecularweight (Flp) pilus, which plays a role in adherence and biofilm formation. The identification of a constitutively active promoter upstream the flp gene of M. adhaerens HP15 suggested the tad locus is transcribed. A flp-rcpCA deletion mutant was generated and analyzed in terms of its motility and attachment to abiotic and biotic surfaces. Under the experimental conditions tested, the mutant did neither show a phenotype in terms of surface attachment nor motility. However, the preliminary results of the current study encourage an in-depth analysis of the role of the tad locus in M. adhaerens HP15.