Carboxysomes-one of the most
studied bacterial microcompartments-are found in all cyanobacteria and in some
chemoautotrophs, and are the sites of CO2 fixation. They encapsulate
the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO). This “enzyme
is crucial for carbon assimilation by these bacteria because it catalyzes the
fixation of inorganic carbon onto the organic acceptor molecule” (Menon,
Balaraj). The carboxysome shell plays a pivotal role in increasing the efficiency
of this enzyme by acting as a gate in allowing some metabolites to enter
freely, while slowing the diffusion of others out of the shell. “Bacterial microcompartments
have been credited with enhancing the activity of the enzyme they contain by
providing a unique environment with optimized substrate concentrations or pH”
(Menon, Balaraj). It has been long held the view that bacterial
microcompartments have a lower pH compared to their exterior, and this paper
presents data that contradicts this model. The way that this was tested was by
fusing a pH sensitive green fluorescent protein (GFP) to RubisCO.

 

The first step consisted of
inserting a pH-sensitive GFP gene (gfp) into a RubisCO subunit gene (cbbs) and
replacing wild-type cbbs in H.
neapolitanus, an autotroph, by homologous recombination. This fusion “gave
rise to the pH-sensitive GFP-tagged RubisCO package inside the mutant
carboxysomes” (Menon, Balaraj). A growth curve measuring the optical density at
OD 600 was generated and it was observed that the mutant grew slower compared
to the wild-type at ambient CO2 levels, but their growth rates were
rather the same when supplemented with 5% CO2. In addition, confocal
microscopy was performed, and the images obtained of the fluorescent region
were comparable with carboxysome clusters seen in previous published literature.
Both of these initial experiments showcased that the H. neapolitanus mutant construction was successful.

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The subsequent step consisted
of characterizing the mutant strain. A carboxysome isolation was performed and
via sucrose density gradient it was possible to distinguish the mutant strain,
which had a heavy fluorescent band, from the wild-type, which had a
whitish-blue band. The polypeptide composition of the purified mutant and
wild-type strains were determined by means of SDS-PAGE and immunoblot analysis,
and showed that the GFP-cbbs fusion protein replaces the wild-type cbbs in
mutant carboxysome.

 

Knowing the specific activity
of RubisCO in the purified mutant, a correlation between pH and fluorescence
was attempted to be established. Purified mutant carboxysomes were suspended in
buffers of different pH values and showed that an increase in pH caused a
change in the loss of fluorescence, as previous published data had shown.

 

The last step consisted of
performing in vitro and in vivo experiments to verify whether the carboxysome
shell would be able to keep a pH gradient. Through a pH calorimetric assay, it
was shown that the carboxysome shell is permeable to protons and there is not a
pH difference between the bacterial microcompartment and the cytoplasm. The
results presented in this paper demonstrate that the pH in the carboxysome is comparable
to its exterior.

 

Studying the processes
surrounding and within microcompartments is of vital importance to understanding
how and why some pathogens such as Salmonella
enterica can proliferate in adverse environments, as well as studying shell
permeability, which can be potential drug targets.

 

 

 

 

 

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