Search

Escherichia coli is able to utilize the mixture of carbon sources and produce molecular hydrogen (H 2 ) via formate hydrogen lyase (FHL) complexes. In current work role of transcriptional activator of formate regulon FhlA in generation of fermentation end products and proton motive force, N'N'-dicyclohexylcarbodiimide (DCCD)-sensitive ATPase activity at 20 and 72 hr growth during utilization of mixture of glucose, glycerol, and formate were investigated. It was shown that in fhlA mutant specific growth rate was ~1.5 fold lower compared to wt, while addition of DCCD abolished the growth in fhlA but not in wt. Formate was not utilized in fhlA mutant but wt cells simultaneously utilized formate with glucose. Glycerol utilization started earlier (from 2 hr) in fhlA than in wt. The DCCD-sensitive ATPase activity in wt cells membrane vesicles increased ~2 fold at 72 hr and was decreased 70% in fhlA. Addition of formate in the assays increased proton ATPase activity in wt and mutant strain. FhlA absence mainly affected the ΔpH but not ΔΨ component of Δp in the cells grown at 72 hr but not in 24 hr. The Δp in wt cells decreased from 24 to 72 hr of growth ~40 mV while in fhlA mutant it was stable. Taken together, it is suggested that FhlA regulates the concentration of fermentation end products and via influencing F O F 1 -ATPase activity contributes to the proton motive force generation., (© 2021 International Union of Biochemistry and Molecular Biology.)

Filamentous phages are nonlytic viruses that specifically infect bacteria, establishing a persistent association with their host. The phage particle has no machinery for generating energy and parasitizes its host's existing structures in order to cross the bacterial envelope and deliver its genetic material. The import of filamentous phages across the bacterial periplasmic space requires some of the components of a macrocomplex of the envelope known as the Tol system. This complex uses the energy provided by the proton motive force (pmf) of the inner membrane to perform essential and highly energy-consuming functions of the cell, such as envelope integrity maintenance and cell division. It has been suggested that phages take advantage of pmf-driven conformational changes in the Tol system to transit across the periplasm. However, this hypothesis has not been formally tested. In order to decouple the role of the Tol system in cell physiology and during phage parasitism, we used mutations on conserved essential residues known for inactivating pmf-dependent functions of the Tol system. We identified impaired Tol complexes that remain fully efficient for filamentous phage uptake. We further demonstrate that the TolQ-TolR homologous motor ExbB-ExbD, normally operating with the TonB protein, is able to promote phage infection along with full-length TolA. IMPORTANCE Filamentous phages are widely distributed symbionts of Gram-negative bacteria, with some of them being linked to genome evolution and virulence of their host. However, the precise mechanism that permits their uptake across the cell envelope is poorly understood. The canonical phage model Fd requires the TolQRA protein complex in the host envelope, which is suspected to translocate protons across the inner membrane. In this study, we show that phage uptake proceeds in the presence of the assembled but nonfunctional TolQRA complex. Moreover, our results unravel an alternative route for phage import that relies on the ExbB-ExbD proteins. This work provides new insights into the fundamental mechanisms of phage infection and might be generalized to other filamentous phages responsible for pathogen emergence., (Copyright © 2020 American Society for Microbiology.)

Blue light has been shown to elicit a tumbling response in Escherichia coli , a nonphototrophic bacterium. The exact mechanism of this phototactic response is still unknown. Here, we quantify phototaxis in E. coli by analyzing single-cell trajectories in populations of free-swimming bacteria before and after light exposure. Bacterial strains expressing only one type of chemoreceptor reveal that all five E. coli receptors (Aer, Tar, Tsr, Tap, and Trg) are capable of mediating responses to light. In particular, light exposure elicits a running response in the Tap-only strain, the opposite of the tumbling responses observed for all other strains. Therefore, light emerges as a universal stimulus for all E. coli chemoreceptors. We also show that blue light exposure causes a reversible decrease in swimming velocity, a proxy for proton motive force. This result is consistent with a previously proposed hypothesis that, rather than sensing light directly, chemoreceptors sense light-induced perturbations in proton motive force, although other factors are also likely to contribute. IMPORTANCE Our findings provide new insights into the mechanism of E. coli phototaxis, showing that all five chemoreceptor types respond to light and their interactions play an important role in cell behavior. Our results also open up new avenues for examining and manipulating E. coli taxis. Since light is a universal stimulus, it may provide a way to quantify interactions among different types of receptors. Because light is easier to control spatially and temporally than chemicals, it may be used to study swimming behavior in complex environments. Since phototaxis can cause migration of E. coli bacteria in light gradients, light may be used to control bacterial density for studying density-dependent processes in bacteria., (Copyright © 2019 American Society for Microbiology.)

In Escherichia coli F(1)F(o) ATP synthase, gammaT273 mutants that eliminate the ability to form a hydrogen bond to betaV265 were incapable of ATP synthase-dependent growth and ATPase-dependent proton pumping, had very low rates of ATPase activity catalyzed by purified F(1), and had significantly decreased sensitivity to inhibition by Mg(2+)-ADP-AlF(n) species, while gammaT273D and gammaT273N mutants which maintained or increased the hydrogen bond strength maintained or increased catalytic activity. The betaP262G mutation that increases the potential flexibility of the rigid sleeve that surrounds the gamma subunit C-terminus also virtually eliminated ATPase activity and susceptibility to Mg(2+)-ADP-AlF(n) inhibition. The gammaE275 mutants that retained the ability to form the betaV265 hydrogen bond had higher ATPase activity than those that eliminated the hydrogen bond. These results provide evidence that the ability to form hydrogen bonds between betaV265 and the gamma subunit C-terminus contributes significantly to the rate-limiting step of catalysis and to the ability of the F(1)F(o) ATP synthase to use a proton gradient to drive ATP synthesis. The loss of activity observed with betaP262G may result from increased flexibility conferred by glycine that decreases the efficiency of communication between the gamma subunit-betaV265 hydrogen bonds and the Walker B aspartate at the catalytic site. The partial loss of coupling observed with gammaT273 mutants that eliminate the betaV265 hydrogen bond is consistent with participation of this hydrogen bond in the escapement mechanism for ATP synthesis in which interactions between the gamma subunit and (alphabeta)(3) ring prevent rotation until the empty catalytic site binds substrate.

Defects of respiratory chain protein complexes and the ATP synthase are becoming increasingly implicated in human disease. Recently, mutations in the ATPase 6 gene have been shown to cause several different neurological disorders. The product of this gene is homologous to the a subunit of the ATP synthase of Escherichia coli. Here, mutations equivalent to those described in humans have been introduced into the a subunit of E. coli by site-directed mutagenesis, and the effects of these mutations on the ATPase activity, ATP synthesis and ability of the enzyme to pump protons studied in detail. The effects of the mutations varied considerably. The mutation L262P (9185 T-C equivalent) caused a 70% loss of ATP synthesis activity, reduced DCCD sensitivity, and lowered proton pumping activity. The L207P (8993 T-C equivalent) reduced ATP synthesis by 50%, affected DCCD sensitivity, while proton pumping was only marginally affected when measured by the standard AMCA quenching assay. The other mutations studied affected the functioning of the ATP synthase much less. The results confirm that modeling of these point mutations in the E. coli enzyme is a useful approach to determining how alterations in the ATPase 6 gene affect enzyme function and, therefore, how a pathogenic effect can be exerted.