Download Cocultivation of myxobacteria with human pathogens An

Cocultivation of myxobacteria with human pathogens
An introduction about Myxobacteria:
Myxobacteria form a large group of gram negative bacteria. Their appearance is rod-shaped
and they live predominantly in soil. Usually they feed on insoluble organic substances. Their
genome size is unusually large for bacteria. In fact the genome of Sorangium cellulosum,
with 13 mio bp, is the largest known prokaryotic genome up to date. Because of its relatively
fast growth and extensive genetic characterization, Myxococcus xanthus is used as a model
organism for the investigation of myxobacteria.
What makes them especially interesting is their multicellular behavior which resembles
eukaryotic slime molds. Therefore it can be said that among the kingdom of bacteria and
archaea, myxobacteria have made the most sophisticated transition into multicellularity. This
special way of behaving will be explained in the following.
Social motility:
As soil living organisms, myxobacteria preferably grow in surfaces where they consume
oxygen from above and nutrients from below. M. xanthus for example has no flagella and
therefore can’t swim in liquid media. While growing on a surface they travel with dynamic
multicellular structures called swarms. The Majority of the cells live within the swarm and
have to compete for nutrients. At the very end of swarm edge one can find a few single cells
though. These cells possess the highest growth rate as no competition for nutrients takes
place.
By using an ability called social motility, myxobacteria facilitate swarm expansion. This
occurs when Type IV pili at the leading cell pole bind to exopolisaccharides (EPS) on
neighboring cells or on the gliding surface. The Binding facilitates the retraction of the pili
which generates a force to propel the cell forward. As a result myxobacteria need to secrete
EPS on a surface in order to move. Cells living in a swarm though can share their EPS as
public goods, which reduces the burden of production for each individual. As a result
maximum swarm expansion rate is achieved at high cell density.
Fruiting body formation:
Upon starvation myxobacteria undergo development of fruiting bodies. Therefore individual
cells arrange themselves into aggregates. Depending on the species the form of these
structures can vary. M. xanthus for example forms dorm shaped mounds whereas
chondromyces crocatus or stigmatella aurantiaca build tree like structures. Aggregation of
cells is regulated by a quorum-like cell-cell signaling called A-Signal which monitors the
colony’s cell density. Activation of developmental genes expression for fruiting body initiation
only occurs when a minimum threshold of the A-Signal is exceeded. The progress is as
follows. At first myxobacterial cells recognizes their kin. After that cells stop their natural
outward spreading and start to migrate inward. In doing that they create a kind of traffic jam
where cells arrange themselves in parallel lines in very close proximity. Afterward they
develop a dome shaped mount consisting of around 100.000 cells. Only a small fraction of
around 1 % of the population differentiates into highly environmentally resistant spores. The
remaining cells differentiate into peripheral rods or lyse. Therefore the population survives
only at the expense of the majority of cells.
Predation:
Myxobacterial swarms are able feed on prey as a collective. When the swarm encounters a
foreign bacterial prey colony it penetrates the colony by secretion of hydrolytic enzymes and
antibiotics to kill and afterwards consume nutrients of the cell lysate. It was shown that
predation efficiency is enhanced by high cell density. Therefore myxobacterial swarms are
described as a wolf pack because of they are capable to work cooperatively as an efficient
predation unit.
Secondary metabolites:
Most myxobacteria produce secondary metabolites which are essential for many bacterial
processes. These natural products possess highly diverse structures and biological activities.
DKxanthene for example was shown to play an important role in fruiting body formation,
whereas Myxovirescin is essential for predation activity.
Difficulties with cultivation and genetic manipulation hinder discovery of the biosynthetic
potential in many species. With the help of genome mining many putative gene clusters for
natural product biosynthesis were revealed.
How to utilize the hidden potential:
Under laboratory conditions myxobacteria doesn’t exploit their full potential. Many natural
compounds of discovered gene clusters remain unknown. Reasons for that can be
suboptimal culture conditions, evolutionary inactivated gene clusters of the absence of
naturally competing bacteria or prey. Strategies to utilize this hidden potential are varying
media components, control of culture parameters during fermentation, UV-mutagenesis or
insertion of promotors upstream the cluster genes.
Cocultivation of myxobacteria with human pathogens:
Other strategies which are part of the upcoming Master thesis are the cultivation on Agar
plates rather than liquid medium and the cocultivation of myxobacteria in the presence of
prey to initiate the expression of silent gene cluster. The aim of the thesis is the discovery of
unknown antibiotics, as well as the extraction, purification and structural analysis of the
compound.
A prospective workflow could include: 1. the observation of the bacterial growth during
cocultivation; 2. performance of metabolite extraction of the agar plate of interest; 3.
Verification of inhibition with raw extract; 4. separation of raw extract via liquid
chromatography, following analysis of fraction via MS; 5. Elucidation of the structure via
MS/MS and NMR; 6. Identification of gene localization by knockout of gene clusters and at
last: 7. gene manipulation for elucidation of biosynthesis and overproduction of identified
compound
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doi:10.1128/AEM.02863-07.
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Cao P, Dey A, Vassallo CN, Wall D. How Myxobacteria Cooperate. J Mol Biol. 2015
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https://en.wikipedia.org/wiki/Myxobacteria