Synthesis of cobalamin de novo by Salmonella enterica serovar Typhimurium strain LT2 and the absence of this ability in Escherichia coli present several problems. This large synthetic pathway is shared by virtually all salmonellae and must be maintained by selection, yet no conditions are known under which growth depends on endogenous B 12 . The cofactor is required for degradation of 1,2-propanediol and ethanolamine. However, cofactor synthesis occurs only anaerobically, and neither of these carbon sources supports anaerobic growth with any of the alternative electron acceptors tested thus far. This paradox is resolved by the electron acceptor tetrathionate, which allows Salmonella to grow anaerobically on ethanolamine or 1,2-propanediol by using endogenously synthesized B 12 . Tetrathionate provides the only known conditions under which simple cob mutants (unable to make B 12 ) show a growth defect. Genes involved in this metabolism include the ttr operon, which encodes tetrathionate reductase. This operon is globally regulated by OxrA (Fnr) and induced anaerobically by a two-component system in response to tetrathionate. Salmonella reduces tetrathionate to thiosulfate, which it can further reduce to H 2 S, by using enzymes encoded by the genes phs and asr. The genes for 1,2-propanediol degradation (pdu) and B 12 synthesis (cob), along with the genes for sulfur reduction (ttr, phs, and asr), constitute more than 1% of the Salmonella genome and are all absent from E. coli. In diverging from E. coli, Salmonella acquired some of these genes unilaterally and maintained others that are ancestral but have been lost from the E. coli lineage.Virtually all Salmonella isolates synthesize B 12 de novo under anaerobic conditions (27,34,43). The ability to synthesize and import B 12 requires more than 35 known genes (48)-approaching 1% of the genome. However, mutations that eliminate B 12 synthesis from otherwise wild-type strains cause no growth defect under the standard aerobic or anaerobic lab conditions used thus far. Since evolutionary maintenance of such a large fraction of the genome requires selection, it seems inescapable that natural conditions must exist under which endogenously synthesized B 12 is important to growth of salmonellae. Salmonella enterica serovar Typhimurium makes B 12 de novo only in the absence of oxygen (27). Degradation of ethanolamine or 1,2-propanediol requires B 12 and provides a carbon and energy source, but growth on these compounds has been observed only under aerobic conditions requiring exogenous B 12 (28,46). These paradoxical aspects of B 12 metabolism have been reviewed (47).The B 12 paradox may be resolved by the finding, described here, that the electron acceptor tetrathionate supports anaerobic use of ethanolamine or 1,2-propanediol as the sole carbon and energy source by using endogenously synthesized B 12 . Under anaerobic conditions, tetrathionate supports considerably better growth on these carbon sources than the other alternative electron acceptors tested. Tetrathionate plu...

Disulfide-coupled refolding reactions of five omega-conotoxins, Ca2+ channel antagonists derived from marine snails of the genus Conus, were examined. These peptides are 23-26 amino acid residues long, and the native conformation of each is stabilized by three disulfide bonds. Although the primary structures of the peptides show only limited sequence similarity, the patterns of disulfides and three-dimensional conformations are very similar. Refolding of the reduced proteins was promoted by the disulfide form of glutathione (GSSG) in the presence of reduced glutathione (GSH). All five of the peptides examined were able to refold to the native conformation, as judged by reversed-phase HPLC behavior, with efficiencies of 16% for omega-MVIIC, 28% for omega-MVIID, and 50% for omega-MVIIA, omega-GVIA, and omega-SVIA. The refolded form of omega-MVIIA was further shown to have biological activity indistinguishable from that of the native form, as well as the same rate of reductive unfolding in the presence of dithiothreitol. The overall folding rate and efficiency of omega-MVIIA was found to be quite sensitive to the thiol-disulfide redox potential, with optimum rates and yields obtained in the presence of GSSG and GSH at concentrations similar to those believed to be present in the endoplasmic reticulum. The folding efficiency of this peptide was greatly reduced by the addition of 8 M urea, indicating that formation of the correct disulfides is determined largely by noncovalent interactions, as opposed to steric constraints arising from the spacing between Cys residues. These results demonstrate that the mature forms of at least some omega-conotoxins contain sufficient information to direct correct folding and disulfide formation, in spite of their small size and limited sequence conservation.

BackgroundBovine tuberculosis (bTB), caused by Mycobacterium bovis, is an important livestock disease raising public health and economic concerns around the world. In New Zealand, a number of wildlife species are implicated in the spread and persistence of bTB in cattle populations, most notably the brushtail possum (Trichosurus vulpecula). Whole Genome Sequenced (WGS) M. bovis isolates sourced from infected cattle and wildlife across New Zealand were analysed. Bayesian phylogenetic analyses were conducted to estimate the substitution rate of the sampled population and investigate the role of wildlife. In addition, the utility of WGS was examined with a view to these methods being incorporated into routine bTB surveillance.ResultsA high rate of exchange was evident between the sampled wildlife and cattle populations but directional estimates of inter-species transmission were sensitive to the sampling strategy employed. A relatively high substitution rate was estimated, this, in combination with a strong spatial signature and a good agreement to previous typing methods, acts to endorse WGS as a typing tool.ConclusionsIn agreement with the current knowledge of bTB in New Zealand, transmission of M. bovis between cattle and wildlife was evident. Without direction, these estimates are less informative but taken in conjunction with the low prevalence of bTB in New Zealand’s cattle population it is likely that, currently, wildlife populations are acting as the main bTB reservoir. Wildlife should therefore continue to be targeted if bTB is to be eradicated from New Zealand. WGS will be a considerable aid to bTB eradication by greatly improving the discriminatory power of molecular typing data. The substitution rates estimated here will be an important part of epidemiological investigations using WGS data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3569-x) contains supplementary material, which is available to authorized users.

The peptide Ca2+ channel antagonists found in the venoms of Conus snails, omega-conotoxins, are synthesized as precursors that include a leader peptide, presumed to direct the polypeptide to the endoplasmic reticulum, and a propeptide of unknown function. In addition, the precursors are synthesized with a C-terminal Gly residue that is posttranslationally converted to a terminal amide group. In order to determine whether the precursor sequences contain information that helps direct folding of the mature sequences, the disulfide-coupled folding of mature omega-conotoxin MVIIA was compared with that of two putative precursor forms: pro-omega-MVIIA-Gly, which contains the propeptide and the C-terminal Gly residue, and omega-MVIIA-Gly, which differs from the mature form only at the C-terminus. The three forms folded with similar kinetics, but the folding efficiency of omega-MVIIA-Gly was greater than 80%, versus approximately 50% for both mature omega-MVIIA and the form containing the propeptide. The enzyme protein disulfide isomerase was found to catalyze disulfide formation and folding of all three forms similarly. The affinity of omega-MVIIA-Gly for receptors in chick brain synaptosomes was approximately 10-fold lower than that of the mature peptide, and the N-terminal propeptide of pro-omega-MVIIA-Gly was found to decrease binding further, by approximately 100-fold. These results suggest that the omega-conotoxins do not rely on the propeptide region of their precursors to facilitate folding. Rather, the mature sequence contains most of the information required to specify the native disulfide pairings and three-dimensional conformation. The C-terminal Gly may enhance the folding efficiency by forming interactions that stabilize the native conformation with respect to other disulfide-bonded forms.

MotA and MotB are membrane proteins that form the stator of the bacterial flagellar motor. Each motor contains several MotA 4 MotB 2 complexes, which function independently to conduct protons across the membrane and couple proton flow to rotation. The mechanism of rotation is not understood in detail but is thought to involve conformational changes in the stator complexes driven by proton association/dissociation at a critical Asp residue of MotB (Asp 32 in the protein of Escherichia coli). MotA has four membrane segments and MotB has one. Previous studies using targeted disulfide crosslinking showed that the membrane segments of the two MotB subunits are together at the center of the complex, surrounded by the TM3 and TM4 segments of the four MotA subunits. Here, the cross-linking studies are extended to TM1 and TM2 of MotA, using Cys residues introduced in several positions in the segments. The observed patterns of disulfide cross-linking indicate that the TM2 segment is positioned between segments TM3 and TM4 of the same subunit, where it could contribute to the proton-channel-forming part of the structure. TM1 is at the interface between TM4 of its own subunit and the TM3 segment of another subunit, where it could stabilize the complex. A structural model based on the cross-linking results shows unobstructed pathways reaching from the periplasm to the Asp 32 residues near the inner ends of the MotB segments. The model indicates a close proximity for certain conserved, functionally important residues. The results are used to develop an explicit model for the proton-induced conformational change in the stator.The bacterial flagellar motor is an ion-fueled machine capable of turning at hundreds of revolutions per second (1-3). In most species, the motor can turn either CW or CCW, and reversals in direction are the basis of regulated movement such as chemotaxis (4,5). Although more than two-dozen proteins are needed for assembly and operation of the flagellum, only five are thought to function closely in rotation. FliG, FliM, and FliN form the "switch complex" on the rotor that determines the direction of rotation (6,7). MotA and MotB are membrane proteins that form the stator, functioning to conduct protons across the membrane and couple proton flow to rotation (8-12) (Figure 1, left). While structural studies of the rotor proteins are fairly advanced (13-16), less is known about the structure of the stator. The stator complexes have subunit composition MotA 4 MotB 2 (17,18). MotA has four TM segments, relatively large domains in the cytoplasm, and only short segments in the periplasm (9,19). MotB has a short segment in the cytoplasm, a single TM segment, and a large periplasmic domain that includes features believed to bind peptidoglycan (10,20,21 In a current model for the rotation mechanism, torque is produced as the stator undergoes conformational changes driven by proton association/dissociation at Asp 32 (23). In support of this model, mutations that neutralized the charge of Asp 32 (e.g., replace...

Although it contains only 25 amino acid residues, omega-conotoxin MVIIA folds into a well-defined three-dimensional structure that is stabilized by 3 disulfide bonds. To assess the contributions of the disulfides to folding and stability, three analogues, each with one pair of disulfide-bonded Cys residues replaced with Ala, were prepared and characterized. The analogues also contained a C-terminal Gly residue that is believed to be present when the peptide folds in vivo and has been shown previously to stabilize the native structure. Circular dichroism spectra and biological assays of the analogues indicated that removing any one of the disulfides greatly destabilized the native conformation. The two disulfides in each analogue were also reduced much more rapidly than in the native form with three disulfides. When the analogues were fully reduced and allowed to form disulfides in the presence of oxidized and reduced glutathione, the native disulfides were not formed in preference to non-native disulfides, further indicating that the forms with two-native disulfides are not significantly stabilized by noncovalent interactions. However, the measured equilibrium constants for disulfide formation indicate that forming any two of the three native disulfides leads to an effective concentration of approximately 25-50 M for the two remaining thiols. The two-disulfide analogues thus appear to represent a stage of folding in which the polypeptide is constrained to a distribution of relatively compact conformations that greatly favor formation of the third disulfide and the final folded structure.

The ability to DNA fingerprint Mycobacterium bovis isolates helped to define the role of wildlife in the persistence of bovine tuberculosis in New Zealand. DNA fingerprinting results currently help to guide wildlife control measures and also aid in tracing the source of infections that result from movement of livestock. During the last 5 years we have developed the ability to distinguish New Zealand (NZ) M. bovis isolates by comparing the sequences of whole genome sequenced (WGS) M. bovis samples. WGS provides much higher resolution than our other established typing methods and greatly improves the definition of the regional localization of NZ M. bovis types. Three outbreak investigations are described and results demonstrate how WGS analysis has led to the confirmation of epidemiological sourcing of infection, to better definition of new sources of infection by ruling out other possible sources, and has revealed probable wildlife infection in an area considered to be free of infected wildlife. The routine use of WGS analyses for sourcing new M. bovis infections will be an important component of the strategy employed to eradicate bovine TB from NZ livestock and wildlife.

Mutants of Salmonella enterica lacking polyphosphate kinase (ppk) grow poorly in the presence of the weak organic acids acetate, propionate, and benzoate. This sensitivity is corrected by methionine and seems to result from destabilization of MetA (homoserine transsuccinylase), the first enzyme in methionine biosynthesis. The MetA protein is known to be sensitive to thermal inactivation, and ppk mutants are more sensitive to heat-induced methionine auxotrophy. Peroxide increases the sensitivity of ppk mutants to both heat and acid and may oxidatively damage (carbonylate) destabilized MetA. While acid appears to impair methionine biosynthesis, it leads to derepression of MetA and may inhibit growth by causing toxic accumulation of denatured protein. This is supported by the observation that the overexpression of MetA in ppk mutants causes acid sensitivity that is not corrected by methionine. We propose that polyphosphate acts as a chemical chaperone that helps refold MetA and/or may stimulate proteolysis of toxic denatured protein. The instability of MetA protein may provide a metabolic fuse that blocks growth under conditions that denature proteins; the sensitivity of this fuse is modulated by polyphosphate.The acid sensitivity of ppk mutants was noted in the course of work on ethanolamine metabolism. The ethanolamine operon (eut) encodes five proteins that resemble components of carboxysomes, organelles thought to concentrate CO 2 (30). Certain mutants lacking carboxysomes grow better with a high concentration of CO 2 (5%), and this growth is prevented by ppk mutations (19). It seemed possible that the ppk mutant phenotype was due to reduced internal pH, an expected side effect of high CO 2 . Consistent with this possibility, ppk mutants were also sensitive to several weak organic acids, suggesting that polyphosphate protects cells against reduced internal pH.Polyphosphate (polyP) is important for growth and survival of both bacterial and eukaryotic species, but a single precise role has been difficult to define (reviewed in references 12 and 31). PolyP is synthesized in response to high salt, nitrogen limitation, phosphate limitation, and amino acid starvation and can act as a storage reservoir for phosphate and high-energy phosphate bonds. The negative charges on this polymer buffer cellular compartments and chelate various cations (Mn, Mg, Ca, Zn, Fe, Cu, and Cd). Lack of polyP reduces expression of the transcription factor RpoS (57) and impairs induction of the SOS system of DNA repair (61). PolyP stimulates ATP-dependent proteolysis of certain ribosomal proteins after a shift from rich to minimal media and thereby provides bacterial cells with a source of amino acids during adaptation to growth in minimal medium (32, 33).Weak organic acids, such as acetate and propionate, impose a stress and are used commercially to inhibit bacterial growth. In nature, these acids are fermentation by-products produced by many bacteria (reviewed in references 2, 34, and 50). It is not entirely clear how acids inhibit growth...