Mikrobiologiskt och Tumörbiologiskt Centrum (MTC) / Microbiology and Tumor Biology Center (MTC)
Abstract
One of the most fundamental ways of signal perception and propagation is
mediated by the bacterial two-component system (TCS). These types of
systems mediate bacterial adaptation that may cause disease in a host due
to expression of virulence factors. Understanding the principles behind
the bacterial adaptation process mediated by TCSs may thus aid in the
development of novel types of antibiotics.
In this work we first characterized the impact of the BarA-UvrY TCS when
it comes to adaptation of E. coli to varying carbon sources both in vitro
and in a monkey cystitis infection model. Growth and efficient
utilization of limited nutrients is central for the bacteria when it
comes to the infection process. A bacterium mutated in the BarA-UvrY TCS
is deficient in the ability to switch between different carbon sources
and thus severely attenuated when nutrients are limited and vary over
time. However, when gluconeogenic carbon sources are present in excess
our results indicate that lack of the BarA-UvrY TCS leads to a higher
fitness, probably due to an increased amount of free CsrA protein
promoting gluconeogensis.
To get more insight regarding the molecular function of the BarA-UvrY TCS
we benefited from the ability of UvrY to be activated by acetyl
phosphate. This enabled us to characterize BarA sensor proteins with
mutations in domains believed to be important for sensor signal
propagation and phosphatase activity. The results indicate that the HAMP
domain (histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis
protein, and phosphatase) in BarA is vital for the kinase and phosphatase
switching ability. of the sensor and that the two N-terminal domains of
BarA are both involved in dephosphorylating UvrY. The dephosphorylating
process was also shown to be mediated by a dimer of two BarA sensor
proteins.
The BarA-UvrY TCSs controls the carbon storage regulator (Csr) system
that has earlier been implicated in biofilm formation in both E. coli and
Salmonella. We decided to establish a model for studying the kinetics and
the impact of these and other regulatory systems on early Salmonella
biofilm formation, using a combination of atomic force microscopy and
light microscopy. Following the initial proliferation phase, we observed
a dispersal of the bacteria and formation of microcolonies that
subsequently merged into a confluent biofilm. The dispersal was clearly
distinct from the detachment phase that occurs after formation of the
mature biofilm. Mutations in different global regulatory genes and genes
controlling the production of extracellular polymeric substances (EPS)
had a moderate to severe impact on the ability of Salmonella to form a
biofilm. Loss of the CsrA protein had a drastic effect on cell morphology
and caused a loss of EPS and flagella, unlike loss of the CsrB and CsrC
ncRNAs, which caused an increase in EPS and flagella production.
In the last study the yhdA gene was identified, via a transposon
screening approach, as a factor affecting BarA-UvrY TCS signaling. The
yhdA gene encodes a 646 amino acid protein containing both a GGDEF-like
and an EAL-like domain. These domains are involved in formation and
breakdown of 3',5'-cyclic diguanylic acid (c-di-GMP), a second messenger
important for the switch between the vegetative and sessile phase of the
bacteria. Additional complementation studies using the yhdA gene
expressed in trans, and the ability of UvrY to be activated by acetyl
phosphate in the absence of BarA, suggested that YhdA affects the ability
of the BarA sensor to switch between phosphatase and kinase activity