Different Minimal Signal Peptide Lengths Recognized by the Archaeal Prepilin-Like Peptidases FlaK and PibD

In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FlaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis ΔflaK mutant indicated that processing of preflagellins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation.The bacterial type IV prepilin peptidase (TFPP) is a well-characterized enzyme belonging to a family of novel aspartic acid proteases (20). It is responsible for the cleavage of N-terminal signal peptides from prepilins and pseudopilins, prior to their incorporation into the type IV pilus structure (22, 30, 31). The prepilin peptidase is also responsible for the processing of prepilin-like proteins needed for type II secretion (22). In Archaea, the existence of bacterial TFPP-like enzymes has also been reported, and they have been most extensively studied in relation to the assembly of the archaeal flagellum. In the euryarchaeotes Methanococcus maripaludis and Methanococcus voltae, the preflagellin peptidase FlaK was demonstrated to be responsible for cleaving the N-terminal signal peptide from the preflagellin prior to its incorporation into the growing flagellar filament, a step essential to flagellar assembly (6, 7, 26). In Sulfolobus solfataricus, an acidophilic crenarchaeote, the equivalent enzyme, PibD, was also shown to process preflagellins (4). Site-directed mutagenesis of FlaK and PibD demonstrated that both aspartic acid residues that aligned with aspartic acid residues essential for bacterial TFPP activity were also essential in the archaeal enzymes (6, 32), indicating that the two archaeal peptidases belong with the bacterial TFPPs in this novel family of aspartic acid proteases (20). More recently, an additional archaeal TFPP was found to be required for cleavage of the prepilin substrates (33) that are assembled into the unique pili of M. maripaludis (37).The substrate specificity of the archaeal preflagellin peptidase remains an open question. Like prepilin peptidases, FlaK in M. voltae has stringent requirements for the amino acids surrounding the cleavage site of the substrate, especially the −1 glycine, −2 and −3 lysines, and the +3 glycine (numbers given relative to the cleavage site) (35); the last position was conserved in all archaeal flagellins (25). Upon N-terminal sequence alignment of all available archaeal flagellin amino acid sequences at the predicted cleavage site, it was found that most archaeal preflagellin signal peptides are quite conserved in length, with the typical flagellin signal peptide being 11 to 12 amino acids in length (Table ​(Table1).1). It is speculated that while a certain amount of flexibility might exist, some optimum and minimum length probably exists that is crucial for the juxtaposition of the signal peptide and signal peptidase with respect to each other and the membrane (18). A recent study examining possible type IV pilin-like substrates in archaea using the FlaFind program indicated that such substrates may be more widespread than initially thought (33). Since in Methanococcus the pilins are processed by a second TFPP (EppA) (33), it is very possible that the preflagellins might be the only substrates of FlaK in these archaea.

TABLE 1.

N-terminal amino acid alignment of selected archaeal flagellin sequences^a

Reproductive conflict delays the recovery of an endangered social species

1. Evolutionary theory predicts that individuals, in order to increase their relative fitness, can evolve behaviours that are detrimental for the group or population. This mismatch is particularly visible in social organisms. Despite its potential to affect the population dynamics of social animals, this principle has not yet been applied to real-life conservation. 2. Social group structure has been argued to stabilize population dynamics due to the buffering effects of nonreproducing subordinates. However, competition for breeding positions in such species can also interfere with the reproduction of breeding pairs. 3. Seychelles magpie robins, Copsychus sechellarum, live in social groups where subordinate individuals do not breed. Analysis of long-term individual-based data and short-term behavioural observations show that subordinates increase the territorial takeover frequency of established breeders. Such takeovers delay offspring production and decrease territory productivity. 4. Individual-based simulations of the Seychelles magpie robin population parameterized with the long-term data show that this process has significantly postponed the recovery of the species from the Critically Endangered status. 5. Social conflict thus can extend the period of high extinction risk, which we show to have population consequences that should be taken into account in management programmes. This is the first quantitative assessment of the effects of social conflict on conservation.     

Biocoagulation of dairy wastewater by Lactobacillus casei TISTR 1500 for protein recovery using micro-aerobic sequencing batch reactor (micro-aerobic SBR)

This study investigated biocoagulation of dairy process wastewater with a new system of the micro-aerobic sequencing batch reactor (micro-aerobic SBR) at a batch bench scale. Lactobacillus casei TISTR 1500 was inoculated to produce acid coagulants under non-sterile acid conditions. Colloidal proteins were removed by employing a solid–liquid separation step as a pre-treatment. The micro-aerobic SBR process had the efficiencies of organic reduction with 73.6 ± 5.9%, 90.1 ± 1.3%, and 85.7 ± 0.6% of chemical oxygen demand (COD), proteins, and sugars without adding external coagulant, and flocculant, respectively. Sustained acid fermentation was achieved for at least 150 cycles by applying an indigenous fill-react-settle-draw-idle sequence in the micro-aerobic SBR process and the use of different solid retention times at 3, 6, 9, 12 and 15 d, consecutively. The micro-aerobic SBR system was able to support lactic acid bacteria (LAB) growth with long SRT (12 and 15 d), due to at least 3 factors: the large inoculum size employed, relatively high concentration of lactic acid produced, and the change in pH during the restoring stage. Current process offered a possible alternative to the more costly chemical and other biological pre-treatments.     

How species traits and affinity to urban land use control large-scale species frequency

Aim Although urban areas only occupy c. 2.8% of the earth's land surface, urbanization threatens biodiversity as areas of high human population density often coincide with high biodiversity. Therefore, nature conservation should concentrate on both remote areas and densely populated regions. Protecting rare plant species in rural and urban areas can contribute to the protection of biodiversity. We therefore need to understand why species are rare. Studies on causes of rarity often concentrate on either plant traits or extrinsic threats (such as habitat fragmentation or nitrogen enrichment). However, there are only a few studies that combine traits and extrinsic threats, although such analyses might clarify causes of rarity. We assessed how the affinity of vascular plant species to urban land use (‘urbanity’) interacts with plant traits in determining species frequency. Location Germany, resolution c. 12 km × 11 km. Methods Species with a low frequency may be rare because they occur in rare habitats or because of other reasons, although their habitat is frequent. Therefore, we calculated the frequency of species corrected for habitat frequency, i.e. relative species frequency. We explained relative species frequency by the interactions of species traits and species affinity to urban land use using generalized linear models. Simultaneous autoregressive error models controlled for phylogenetic relationships of species. Results Relative species frequency depends on species affinity to urban land use, independent of the different interactions between traits and urbanity used as predictors. The higher the urbanity the higher is species frequency. Urbanity interacts with species preferences towards temperature and soil acidity. Moreover, dispersal, nitrogen preferences and origin explain relative species frequency, amongst others. Main conclusions Many rare species, especially those preferring cool or acidic habitats might already have disappeared from urban areas. Analyses that combine species traits and environmental effects can explain the causes of rarity and help to derive better conservation strategies.     

Improvement of isoflavone aglycones production using β-glucosidase secretory produced in recombinant Aspergillus oryzae

β-Glucosidase (BGL1) from Aspergillus oryzae was efficiently produced in recombinant A. oryzae using sodM promoter-mediated expression system. The yield of BGL1 was 960 mg/l in liquid culture, which is 20-fold higher than the yield of BGL1 produced using the yeast Saccharomyces cerevisiae. Recombinant BGL1 converted isoflavone glycosides into isoflavone aglycones more efficiently than β-glucosidase from almond. In addition, BGL1 produced isoflavone aglycones even in the presence of the insoluble form of isoflavone glycosides.     

The expense for one implantation of a banked bone allograft from a cadaveric donor and the issues affecting current advanced medical treatment in the Japanese orthopaedic field

Demand for banked bone allografts is increasing in Japan; however, there are too few bone banks and the bone bank network is not well-established. One reason for this was lack of funding for banks. Bone banks had to bear all material expenses of banked bone allografts themselves because this was not designated a covered expense. In December 2004, the Japanese government started a new “Advanced Medical Treatment” administration system which allowed an approved institution to charge the expense of authorized advanced medical treatments directly to patients. The treatment named “Cryopreserved allogenic bone and ligamentous tissue retrieved from cadaveric donor” was approved as an advanced medical treatment in March 2007. We present the calculation method and the expense per implantation of a banked bone allograft from a cadaveric donor under this treatment and raise issues which affect this advanced medical treatment and remain to be resolved in the Japanese orthopaedic field.     

Developmental changes in the regulation of calcium-dependent neurite outgrowth

Intracellular calcium ions (Ca²⁺) have an essential role in the regulation of neurite outgrowth, but how outgrowth is controlled remains largely unknown. In this study, we examined how the mechanisms of neurite outgrowth change during development in chick and mouse dorsal root ganglion neurons. 2APB, a potent inhibitor of inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R), inhibited neurite outgrowth at early developmental stages, but not at later stages. In contrast, pharmacological inhibition with Ni²⁺, Cd²⁺, or dantrolene revealed that ryanodine receptor (RyR)-mediated Ca²⁺-induced Ca²⁺ release (CICR) was involved in neurite outgrowth at later stage, but not at early stages. The distribution of IP₃R and RyR in growth cones also changed during development. Furthermore, pharmacological inhibition of the Ca²⁺-calmodulin-dependent phosphatase calcineurin with FK506 reduced neurite outgrowth only at early stages. These data suggest that the calcium signaling that regulates neurite outgrowth may change during development from an IP₃R-mediated pathway to a RyR-mediated pathway.     

Expression and stability of the nontoxic component of the botulinum toxin complex

Clostridium botulinum produces botulinum neurotoxin (BoNT) as a large toxin complex associated with nontoxic-nonhemagglutinin (NTNHA) and/or hemagglutinin components. In the present study, high-level expression of full-length (1197 amino acids) rNTNHA from C. botulinum serotype D strain 4947 (D-4947) was achieved in an Escherichia coli system. Spontaneous nicking of the rNTNHA at a specific site was observed during long-term incubation in the presence of protease inhibitors; this was also observed in natural NTNHA. The rNTNHA assembled with isolated D-4947 BoNT with molar ratio 1:1 to form a toxin complex. The reconstituted toxin complex exhibited dramatic resistance to proteolysis by pepsin or trypsin at high concentrations, despite the fact that the isolated BoNT and rNTNHA proteins were both easily degraded. We provide definitive evidence that NTNHA plays a crucial role in protecting BoNT, which is an oral toxin, from digestion by proteases common in the stomach and intestine.