Domain structure and pore loops in the 2-hydroxycarboxylate transporter family

The 2-hydroxycarboxylate transporter (2HCT) family is a family of bacterial secondary transporters for substrates like citrate, malate and lactate. The family is in class ST[3] of the MemGen classification system that groups membrane proteins in structural classes based on hydropathy profile analysis. The combination of computational analysis of the proteins in class ST[3] and available experimental data on members of the 2HCT family has yielded a detailed structural model of the transporters. The core of the model is formed by two homologous domains with opposite orientation in the membrane. Each domain consists of 5 trans membrane segments and contains a pore loop between the 4th and 5th segment. The two pore loops enter the membrane-embedded part from opposite sides of the membrane (trans pore loops) and are believed to form the translocation pathway in the 3D structure. A genome wide study of the cellular location of the C-terminus of all Escherichia coli membrane proteins [Daley et al., 2005. Science 308:1321-1323] showed that the C-termini of the 19 E. coli proteins in class ST[3] were correctly predicted by the structural model.     

The 2-hydroxycarboxylate transporter family: physiology, structure, and mechanism.

The 2-hydroxycarboxylate transporter family is a family of secondary transporters found exclusively in the bacterial kingdom. They function in the metabolism of the di- and tricarboxylates malate and citrate, mostly in fermentative pathways involving decarboxylation of malate or oxaloacetate. These pathways are found in the class Bacillales of the low-CG gram-positive bacteria and in the gamma subdivision of the Proteobacteria. The pathways have evolved into a remarkable diversity in terms of the combinations of enzymes and transporters that built the pathways and of energy conservation mechanisms. The transporter family includes H+ and Na+ symporters and precursor/product exchangers. The proteins consist of a bundle of 11 transmembrane helices formed from two homologous domains containing five transmembrane segments each, plus one additional segment at the N terminus. The two domains have opposite orientations in the membrane and contain a pore-loop or reentrant loop structure between the fourth and fifth transmembrane segments. The two pore-loops enter the membrane from opposite sides and are believed to be part of the translocation site. The binding site is located asymmetrically in the membrane, close to the interface of membrane and cytoplasm. The binding site in the translocation pore is believed to be alternatively exposed to the internal and external media. The proposed structure of the 2HCT transporters is different from any known structure of a membrane protein and represents a new structural class of secondary transporters.     

Repression of sporulation in Bacillus subtilis by L-malate.

L-Malate repressed sporulation in the wild-type strain of Bacillus subtilis. When 75 mM L-malate was added to the growth medium at the time of inoculation, the appearance of heat-resistant spores was delayed 6 to 8 h. The synthesis of extracellular serine protease, alkaline phosphatase, glucose dehydrogenase, and dipicolinic acid was similarly delayed. Sporulation was not repressed when malate was added to the culture at t4 or later. A mutant was selected for ability to sporulate in the presence of malate. This strain could also sporulate in the presence of glucose. The malate-resistant mutant grew poorly with malate as sole carbon source, although it possessed an intact citric acid cycle, and it showed increased levels of malic enzyme. This indicates a defect in the metabolism of malate in the mutant. A mutant lacking malate dehydrogenase activity was also able to sporulate in the presence of malate. A model for the regulation of sporulation by malate is presented and discussed. Citric acid cycle intermediates other than malate did not affect sporulation. In contrast to previous results, sporulation of certain citric acid cycle mutants could be greatly increased or completely restored by the addition of intermediates after the enzymatic block. The results indicate that the failure of citric acid cycle mutants to sporulate can be adequately explained by lack of energy and lack of glutamate.     

Transmembrane topology of the Acr3 family arsenite transporter from Bacillus subtilis

The transmembrane topology of the Acr3 family arsenite transporter Acr3 from Bacillus subtilis was analysed experimentally using translational fusions with alkaline phosphatase and green fluorescent protein and in silico by topology modelling. Initial topology prediction resulted in two models with 9 and 10 TM helices respectively. 32 fusion constructs were made between truncated forms of acr3 and the reporter genes at 17 different sites throughout the acr3 sequence to discriminate between these models. Nine strong reporter protein signals provided information about the majority of the locations of the cytoplasmic and extracellular loops of Acr3 and showed that both the N- and the C-termini are located in the cytoplasm. Two ambiguous data points indicated the possibility of an alternative 8 helix topology. This possibility was investigated using another 10 fusion variants, but no experimental support for the 8 TM topology was obtained. We therefore conclude that Acr3 has 10 transmembrane helices. Overall, the loops which connect the membrane spanning segments are short, with cytoplasmic loops being somewhat longer than the extracellular loops. The study provides the first ever experimentally derived structural information on a protein of the Acr3 family which constitutes one of the largest classes of arsenite transporters.     

Identification of the L-aspartate transporter in Bacillus subtilis

YveA of Bacillus subtilis, a putative transporter of the amino acid/polyamine/organocation (APC) superfamily, is shown to mediate uptake of both L-aspartate and L-glutamate as well as having sensitivity to L-aspartate hydroxamate. This 14 TMS protein is the primary aspartate uptake system in B. subtilis and serves as the prototype for a new family within the APC superfamily.     

Characterization of the major citrate synthase of Bacillus subtilis.

The major citrate synthase of Bacillus subtilis (CS-II) was purified to near homogeneity and shown to correspond to the product of the citZ gene. Accumulation of CS-II during exponential growth and stationary phases paralleled expression of the citZ gene. The physical and kinetic properties of CS-II were similar to those of citrate synthase enzymes from Bacillus megaterium and from eukaryotic cells but differed from those of citrate synthases from many gram-negative bacteria.     

Evidence that the Bacillus subtilis SpoIIGA protein is a novel type of signal-transducing aspartic protease

The bacterium Bacillus subtilis undergoes endospore formation in response to starvation. sigma factors play a key role in spatiotemporal regulation of gene expression during development. Activation of sigma factors is coordinated by signal transduction between the forespore and the mother cell. sigma(E) is produced as pro-sigma(E), which is activated in the mother cell by cleavage in response to a signal from the forespore. We report that expression of SpoIIR, a putative signaling protein normally made in the forespore, and SpoIIGA, a putative protease, is necessary and sufficient for accurate, rapid, and abundant processing of pro-sigma(E) to sigma(E) in Escherichia coli. Modeling and mutational analyses provide evidence that SpoIIGA is a novel type of aspartic protease whose C-terminal half forms a dimer similar to the human immunodeficiency virus type 1 protease. Previous studies suggest that the N-terminal half of SpoIIGA is membrane-embedded. We found that SpoIIGA expressed in E. coli is membrane-associated and that after detergent treatment SpoIIGA was self-associated. Also, SpoIIGA interacts with SpoIIR. The results support a model in which SpoIIGA forms inactive dimers or oligomers, and interaction of SpoIIR with the N-terminal domain of SpoIIGA on one side of a membrane causes a conformational change that allows formation of active aspartic protease dimer in the C-terminal domain on the other side of the membrane, where it cleaves pro-sigma(E).     

YknWXYZ is an unusual four-component transporter with a role in protection against sporulation-delaying-protein-induced killing of Bacillus subtilis

YknXYZ is the ATP-binding cassette export complex from Bacillus subtilis, where YknX is a membrane fusion protein, YknY is an ATPase, and YknZ is a permease. The yknXYZ genes are arranged into an operon that also includes yknW, encoding a membrane protein with four putative transmembrane segments. Previous studies suggested that the yknWXYZ operon belongs to the σ(w) regulon and protects cells from the endogenous toxin SDP (sporulation-delaying protein) encoded by sdpC. In this study, we investigated the composition and function of YknW and YknXYZ. We report that the yknWXYZ operon is constitutively expressed in growing B. subtilis cells independently from sdpC. Chemical cross-linking in vivo and copurification approaches established that YknX interacts with YknYZ, whereas YknW binds YknXYZ, indicating that all four proteins form a complex in vivo. The complex assembly is modulated by YknW but proceeds in the absence of SdpC. When overproduced alone, YknW provides partial protection against SDP toxin, but all four Ykn proteins are required for full protection against both endogenous and exogenous SDP. We conclude that YknWXYZ is an unusual four-component transporter with a role in the starvation-induced killing of B. subtilis cells.     

CotA of Bacillus subtilis is a copper-dependent laccase

The spore coat protein CotA of Bacillus subtilis displays similarities with multicopper oxidases, including manganese oxidases and laccases. B. subtilis is able to oxidize manganese, but neither CotA nor other sporulation proteins are involved. We demonstrate that CotA is a laccase. Syringaldazine, a specific substrate of laccases, reacted with wild-type spores but not with DeltacotA spores. CotA may participate in the biosynthesis of the brown spore pigment, which appears to be a melanin-like product and to protect against UV light.     

bmr3, a third multidrug transporter gene of Bacillus subtilis.

A third multidrug transporter gene named bmr3 was cloned from Bacillus subtilis. Although Bmr3 shows relatively low homology to Bmr and Blt, the substrate specificities of these three transporters overlap. Northern hybridization analysis showed that expression of the bmr3 gene was dependent on the growth phase.