Propionigenium and Vibrio species identified as possible component causes of shrimp white feces syndrome (WFS) associated with the microsporidian Enterocytozoon hepatopenaei.

White feces syndrome (WFS) in cultivated shrimp is characterized by white shrimp midguts (intestines) and white fecal strings that float as mats on pond surfaces. The etiology of WFS is complex, but one type called EHP-WFS is associated with the microsporidian Enterocytozoon hepatopenaei (EHP). The hepatopancreas (HP), midgut and fecal strings of EHP-WFS shrimp exhibit massive quantities of EHP spores together with mixed, unidentified bacteria. In EHP-WFS ponds, some EHP-infected shrimp show white midguts (WG) and produce white feces while other EHP-infected shrimp in the same pond show grossly normal midguts (NG) and produce no white feces. We hypothesized that comparison of the microbial flora between WG and NG shrimp would reveal probable combinations of microbes significantly associated with EHP-WFS. To test this, we selected a Penaeus vannamei cultivation pond exhibiting severe WFS and used microscopic and microbial profiling analyses to compare WG and NG samples. Histologically, EHP was confirmed in the HP and midgut of both WG and NG shrimp, but EHP burdens were higher and EHP tissue damage was more severe in WG shrimp. Further, intestinal microbiomes in WG shrimp were less diverse and had higher abundance of bacteria from the genera Vibrio and Propionigenium. Propionigenium burden in the HP of WG shrimp (9364 copies/100 ng DNA) was significantly higher (P = 1.1 × 10-5) than in NG shrimp (12 copies/100 ng DNA). These findings supported our hypothesis by revealing two candidate bacterial genera that should be tested in combination with EHP as potential component causes of EHP-WFS in P. vannamei.

Ring assembly of c subunits of F0 F1 -ATP synthase in Propionigenium modestum requires YidC and UncI following MPIase-dependent membrane insertion.

The c subunits of F0 F1 -ATP synthase (F0 c) assemble into a ring structure, following membrane insertion that is dependent on both glycolipid MPIase and protein YidC. We analyzed the insertion and assembly processes of Propionigenium modestum F0 c (Pm-F0 c), of which the ring structure is resistant to SDS. Ring assembly of Pm-F0 c requires P. modestum UncI (Pm-UncI). Ring assembly of in vitro synthesized Pm-F0 c was observed when both YidC and Pm-UncI were reconstituted into liposomes of Escherichia coli phospholipids. Under the physiological conditions where spontaneous insertion had been blocked by diacylglycerol, MPIase was necessary for Pm-F0 c insertion allowing the subsequent YidC/Pm-UncI-dependent ring assembly. Thus, we have succeeded in the complete reconstitution of membrane insertion and subsequent ring assembly of Pm-F0 c.

Functional asymmetry of the F(0) motor in bacterial ATP synthases.

F(1)F(0) ATP synthases use the electrochemical potential of H(+) or Na(+) across biological membranes to synthesize ATP by a rotary mechanism. In bacteria, the enzymes can act in reverse as ATP-driven ion pumps creating the indispensable membrane potential. Here, we demonstrate that the F(0) parts of a Na(+)- and H(+)-dependent enzyme display major asymmetries with respect to their mode of operation, reflected by the requirement of approximately 100 times higher Na(+) or H(+) concentrations for the synthesis compared with the hydrolysis of ATP. A similar asymmetry is observed during ion transport through isolated F(0) parts, indicating different affinities for the binding sites in the a/c interface. Together with further data, we propose a model that provides a rationale for a differential usage of membrane potential and ion gradient during ATP synthesis as observed experimentally. The functional asymmetry might also reflect an important property of the ATP synthesis mechanism in vivo. In Escherichia coli, we observed respiratory chain-driven ATP production at pH 7-8, while P-site pH values < 6.5 were required for ATP synthesis in vitro. This discrepancy is discussed with respect to the hypothesis that during respiration lateral proton diffusion could lead to significant acidification at the membrane surface.

UncI protein can mediate ring-assembly of c-subunits of FoF1-ATP synthase in vitro.

In F(o)F(1)-ATP synthase, multimeric c-subunits are assembled to a ring (c-ring) in the membranes that rotates as protons flow across F(o). We recently reported that assembly of c-ring of Propionigenium modestum in the membranes of Escherichia coli cells required P. modestum UncI, a product of the conserved uncI gene in the F(o)F(1) operon. However, cooperation with endogenous factors in E. coli remained unclear. Here, P. modestum c-subunit was synthesized in vitro in the presence of liposomes. When c-subunit alone was synthesized, it did not form c-ring. However, when c-subunit and P. modestum UncI were synthesized together, c-ring was formed. Fusion of the two kinds of liposomes, one containing only unassembled c-subunit and the other only UncI, resulted in gradual formation of c-ring. Thus, UncI alone can mediate in vitro post-translational c-ring assembly.

Bacterial Na+ - or H+ -coupled ATP synthases operating at low electrochemical potential.

In certain strictly anaerobic bacteria, the energy for growth is derived entirely from a decarboxylation reaction. A prominent example is Propionigenium modestum, which converts the free energy of the decarboxylation of (S)-methylmalonyl-CoA to propionyl-CoA (DeltaG degrees =-20.6 kJ/mol) into an electrochemical Na(+) ion gradient across the membrane. This energy source is used as a driving force for ATP synthesis by a Na(+)-translocating F(1)F(0) ATP synthase. According to bioenergetic considerations, approximately four decarboxylation events are necessary to support the synthesis of one ATP. This unique feature of using Na(+) instead of H(+) as the coupling ion has made this ATP synthase the paradigm to study the ion pathway across the membrane and its relationship to rotational catalysis. The membrane potential (Deltapsi) is the key driving force to convert ion translocation through the F(0) motor components into torque. The resulting rotation elicits conformational changes at the catalytic sites of the peripheral F(1) domain which are instrumental for ATP synthesis. Alkaliphilic bacteria also face the challenge of synthesizing ATP at a low electrochemical potential, but for entirely different reasons. Here, the low potential is not the result of insufficient energy input from substrate degradation, but of an inverse pH gradient. This is a consequence of the high environmental pH where these bacteria grow and the necessity to keep the intracellular pH in the neutral range. In spite of this unfavorable bioenergetic condition, ATP synthesis in alkaliphilic bacteria is coupled to the proton motive force (DeltamuH(+)) and not to the much higher sodium motive force (DeltamuNa(+)). A peculiar feature of the ATP synthases of alkaliphiles is the specific inhibition of their ATP hydrolysis activity. This inhibition appears to be an essential strategy for survival at high external pH: if the enzyme were to operate as an ATPase, protons would be pumped outwards to counteract the low DeltamuH(+), thus wasting valuable ATP and compromising acidification of the cytoplasm at alkaline pH.

Osmomechanics of the Propionigenium modestum F(o) motor.

In Propionigenium modestum, ATP is manufactured from ADP and phosphate by the enzyme ATP synthase using the free energy of an electrochemical gradient of Na+ ions. The P. modestum ATP synthase is a clear member of the family of F-type ATP synthases and the only major distinction is an extension of the coupling ion specificity to H+, Li+, or Na+, depending on the conditions. The use of Na+ as a coupling ion offers unique experimental options to decipher the ion-translocation mechanism and the osmotic and mechanical behavior of the enzyme. The single a subunit and the oligomer of c subunits are part of the stator and rotor, respectively, and operate together in the ion-translocation mechanism. During ATP synthesis, Na+ diffuses from the periplasm through the a subunit channel onto the Na+ binding site on a c subunit. From there it dissociates into the cytoplasm after the site has rotated out of the interface with subunit a. In the absence of a membrane potential, the rotor performs Brownian motions into either direction and Na+ ions are exchanged between the two compartments separated by the membrane. Upon applying voltage, however, the direction of Na+ flux and of rotation is biased by the potential. The motor generates torque to drive the rotation of the gamma subunit, thereby releasing tightly bound ATP from catalytic sites in F(1). Hence, the membrane potential plays a pivotal role in the torque-generating mechanism. This is corroborated by the fact that for ATP synthesis, at physiological rates, the membrane potential is indispensable. We propose a catalytic mechanism for torque generation by the F(o) motor that is in accord with all experimental data and is in quantitative agreement with the requirement for ATP synthesis.