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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.
