Over
the past 30 years, there has been a dramatic rise in the number of
infections caused by multidrug-resistant bacteria, which have proliferated
due to the misuse and overuse of antibiotics. Over this same time
period, however, there has also been a decline in the number of antibiotics
with novel mechanisms of action coming to market. Therefore, there
is a growing need for an increase in the speed at which new antibiotics
are discovered and developed. Natural products produced by bacteria
have been and continue to be a robust source of novel antibiotics;
however, new and complementary methods for screening large bacterial
libraries for novel antibiotic production are needed due to the current
agar methods being limited in scope, time consuming, and prone to
error. Herein, we describe a rapid, robust, and quantitative high-throughput
liquid culture screening method for antibiotic production by bacteria.
This method has the ability to screen both mono- and coculture mixtures
of bacteria in vitro and be adapted to other phenotypic
natural product analyses. Over 260 bacterial species were screened
in monoculture, and 38 and 34% were found to produce antibiotics capable
of inhibition of Staphylococcus aureus or Escherichia coli, respectively,
with 8 and 4% being classified as strong producers (≥30% growth
inhibition), respectively. Bacteria found to not produce antibiotics
in monoculture were also screened in coculture using an adaptation
of this method. Of the more than 270 cocultures screened, 14 and 30%
were found to produce antibiotics capable of inhibition of S. aureus or E. coli, respectively. Of those bacteria found to produce antibiotics in
monoculture, 43 bacteria were subjected to 16S rRNA sequencing and
found to be majority Pseudomonas (37%), Serratia (19%), and Bacillus (14%) bacteria, but two novel
producers, Herbaspirillum and Kluyvera, were also found.
Many carbon-fixing organisms have evolved CO 2 concentrating mechanisms (CCMs) to enhance the delivery of CO 2 to RuBisCO, while minimizing reactions with the competitive inhibitor, molecular O 2 . These distinct types of CCMs have been extensively studied using genetics, biochemistry, cell imaging, mass spectrometry, and metabolic flux analysis. Highlighted in this paper, the cyanobacterial CCM features a bacterial microcompartment (BMC) called 'carboxysome' in which RuBisCO is co-encapsulated with the enzyme carbonic anhydrase (CA) within a semi-permeable protein shell. The cyanobacterial CCM is capable of increasing CO 2 around RuBisCO, leading to one of the most efficient processes known for fixing ambient CO 2 . The carboxysome life cycle is dynamic and creates a unique subcellular environment that promotes activity of the Calvin-Benson (CB) cycle. The carboxysome may function within a larger cellular metabolon, physical association of functionally coupled proteins, to enhance metabolite channelling and carbon flux. In light of CCMs, synthetic biology approaches have been used to improve enzyme complex for CO 2 fixations. Research on CCMassociated metabolons has also inspired biologists to engineer multi-step pathways by providing anchoring points for enzyme cascades to channel intermediate metabolites towards valuable products.
Bacterial microcompartments (BMCs) are protein-encapsulated compartments found across at least 23 bacterial phyla. BMCs contain a variety of metabolic processes that share the commonality of toxic or volatile intermediates, oxygen-sensitive enzymes and cofactors, or increased substrate concentration for magnified reaction rates. These compartmentalized reactions have been computationally modeled to explore the encapsulated dynamics, ask evolutionary-based questions, and develop a more systematic understanding required for the engineering of novel BMCs. Many crucial aspects of these systems remain unknown or unmeasured, such as substrate permeabilities across the protein shell, feasibility of pH gradients, and transport rates of associated substrates into the cell. This review explores existing BMC models, dominated in the literature by cyanobacterial carboxysomes, and highlights potentially important areas for exploration.
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