The Replicative DnaE Polymerase of Bacillus subtilis Recruits the Glycolytic Pyruvate Kinase (PykA) When Bound to Primed DNA Templates.

The glycolytic enzyme PykA has been reported to drive the metabolic control of replication through a mechanism involving PykA moonlighting functions on the essential DnaE polymerase, the DnaC helicase and regulatory determinants of PykA catalytic activity in Bacillus subtilis. The mutants of this control suffer from critical replication and cell cycle defects, showing that the metabolic control of replication plays important functions in the overall rate of replication. Using biochemical approaches, we demonstrate here that PykA interacts with DnaE for modulating its activity when the replication enzyme is bound to a primed DNA template. This interaction is mediated by the CAT domain of PykA and possibly allosterically regulated by its PEPut domain, which also operates as a potent regulator of PykA catalytic activity. Furthermore, using fluorescence microscopy we show that the CAT and PEPut domains are important for the spatial localization of origins and replication forks, independently of their function in PykA catalytic activity. Collectively, our data suggest that the metabolic control of replication depends on the recruitment of PykA by DnaE at sites of DNA synthesis. This recruitment is likely highly dynamic, as DnaE is frequently recruited to and released from replication machineries to extend the several thousand RNA primers generated from replication initiation to termination. This implies that PykA and DnaE continuously associate and dissociate at replication machineries for ensuring a highly dynamic coordination of the replication rate with metabolism.

Ferric quinate (QPLEX) inhibits the interaction of major outer membrane protein (MOMP) with the Lewis b (Leb) antigen and limits Campylobacter colonization in broilers.

Campylobacter jejuni colonizes hosts by interacting with Blood Group Antigens (BgAgs) on the surface of gastrointestinal epithelia. Genetic variations in BgAg expression affects host susceptibility to C. jejuni. Here, we show that the essential major outer membrane protein (MOMP) of C. jejuni NCTC11168 binds to the Lewis b (Leb) antigen on the gastrointestinal epithelia of host tissues and this interaction can be competitively inhibited by ferric quinate (QPLEX), a ferric chelate structurally similar to bacterial siderophores. We provide evidence that QPLEX competitively inhibits the MOMP-Leb interaction. Furthermore, we demonstrate that QPLEX can be used as a feed additive in broiler farming to significantly reduce C. jejuni colonization. Our results indicate that QPLEX can be a viable alternative to the preventative use of antibiotics in broiler farming to combat C. jejuni infections.

Pyruvate kinase, a metabolic sensor powering glycolysis, drives the metabolic control of DNA replication.

BACKGROUND: In all living organisms, DNA replication is exquisitely regulated in a wide range of growth conditions to achieve timely and accurate genome duplication prior to cell division. Failures in this regulation cause DNA damage with potentially disastrous consequences for cell viability and human health, including cancer. To cope with these threats, cells tightly control replication initiation using well-known mechanisms. They also couple DNA synthesis to nutrient richness and growth rate through a poorly understood process thought to involve central carbon metabolism. One such process may involve the cross-species conserved pyruvate kinase (PykA) which catalyzes the last reaction of glycolysis. Here we have investigated the role of PykA in regulating DNA replication in the model system Bacillus subtilis. RESULTS: On analysing mutants of the catalytic (Cat) and C-terminal (PEPut) domains of B. subtilis PykA we found replication phenotypes in conditions where PykA is dispensable for growth. These phenotypes are independent from the effect of mutations on PykA catalytic activity and are not associated with significant changes in the metabolome. PEPut operates as a nutrient-dependent inhibitor of initiation while Cat acts as a stimulator of replication fork speed. Disruption of either PEPut or Cat replication function dramatically impacted the cell cycle and replication timing even in cells fully proficient in known replication control functions. In vitro, PykA modulates activities of enzymes essential for replication initiation and elongation via functional interactions. Additional experiments showed that PEPut regulates PykA activity and that Cat and PEPut determinants important for PykA catalytic activity regulation are also important for PykA-driven replication functions. CONCLUSIONS: We infer from our findings that PykA typifies a new family of cross-species replication control regulators that drive the metabolic control of replication through a mechanism involving regulatory determinants of PykA catalytic activity. As disruption of PykA replication functions causes dramatic replication defects, we suggest that dysfunctions in this new family of universal replication regulators may pave the path to genetic instability and carcinogenesis.

Inhibition of extracellular proteases improves the production of a xylanase in Parageobacillus thermoglucosidasius.

BACKGROUND: Parageobacillus thermoglucosidasius is a thermophilic and ethanol-producing bacterium capable of utilising both hexose and pentose sugars for fermentation. The organism has been proposed to be a suitable organism for the production of bioethanol from lignocellulosic feedstocks. These feedstocks may be difficult to degrade, and a potential strategy to optimise this process is to engineer strains that secrete hydrolases that liberate increased amounts of sugars from those feedstocks. However, very little is known about protein transport in P. thermoglucosidasius and the limitations of that process, and as a first step we investigated whether there were bottlenecks in the secretion of a model protein. RESULTS: A secretory enzyme, xylanase (XynA1), was produced with and without its signal peptide. Cell cultures were fractionated into cytoplasm, membrane, cell wall, and extracellular milieu protein extracts, which were analysed using immunoblotting and enzyme activity assays. The main bottleneck identified was proteolytic degradation of XynA1 during or after its translocation. A combination of mass spectrometry and bioinformatics indicated the presence of several proteases that might be involved in this process. CONCLUSION: The creation of protease-deficient strains may be beneficial towards the development of P. thermoglucosidasius as a platform organism for industrial processes.