The ice-nucleation protein (Inp) is a glycosyl phosphatidylinositol-anchored outer membrane protein found in some Gram-negative bacteria. Using Pseudomonas syringae inp as an anchoring motif, we investigated the functional display of a foreign protein, Zymomonas mobilis levansucrase (LevU), on the surface of Escherichia coli. The cells expressing Inp-LevU were found to retain both the ice-nucleation and whole-cell levansucrase enzyme activities, indicating the functional expression of Inp-LevU hybrid protein on the cell surface. The surface localization was further verified by immunofluorescence microscopy, fluorescence-activated cell sorting flow cytometry and immunogold electron microscopical examination. No growth inhibition or changes in the outer membrane integrity were observed upon the induction of fusion protein synthesis. Viability of the cells was also maintained over 48 hours in the stationary phase. Surface-displayed levansucrases were found to be resistant to the externally added proteases unless the cells were treated with EDTA. When the levansucrase-displayed cells were used as the enzyme source, levan (44 g/L) was efficiently synthesized from sucrose (130 g/L) with 34% (wt/wt) conversion yield, generating glucose (65 g/L) as a by-product.

We have developed a recombinant live oral vaccine using the ice-nucleation protein (Inp) from Pseudomonas syringae to display viral antigens on the surface of Salmonella spp. Fusion proteins containing viral antigens were expressed in the oral vaccine strain, Salmonella typhi Ty21a. Surface localization was verified by immunoblotting and fluorescence-activated cell sorting. The immunogenicity of surface-displayed viral antigens on the recombinant live vaccine strain was assessed in mice inoculated intranasally and intraperitoneally. Inoculation resulted in significantly higher serum antibody level than those induced by viral antigens expressed intracellularly. Thus, this multivalent mucosal live vaccine may provide an effective means for inducing mucosal or systemic immune responses against multiple viral antigens.

N(ε) -lysine acetylation, a reversible and highly regulated PTM, has been shown to occur in the model Gram-negative bacteria Escherichia coli and Salmonella enterica. Here, we extend this acetylproteome analysis to Bacillus subtilis, a model Gram-positive bacterium. Through anti-acetyllysine antibody-based immunoseparation of acetylpeptides followed by nano-HPLC/MS/MS analysis, we identified 332 unique lysine-acetylated sites on 185 proteins. These proteins are mainly involved in cellular housekeeping functions such as central metabolism and protein synthesis. Fifity-nine of the lysine-acetylated proteins showed homology with lysine-acetylated proteins previously identified in E. coli, suggesting that acetylated proteins are more conserved. Notably, acetylation was found at or near the active sites predicted by Prosite signature, including SdhA, RocA, Kbl, YwjH, and YfmT, indicating that lysine acetylation may affect their activities. In 2-amino-3-ketobutyrate CoA ligase Kbl, a class II aminotransferase, a lysine residue involved in pyridoxal phosphate attachment was found to be acetylated. This data set provides evidence for the generality of lysine acetylation in eubacteria and opens opportunities to explore the consequences of acetylation modification on the molecular physiology of B. subtilis.

The bacterial thermoalkalophilic lipases optimally hydrolyze saturated fatty acids at elevated temperatures. They also have significant sequence homology with staphylococcal lipases, and both the thermoalkalophilic and staphylococcal lipases are grouped as the lipase family I.5. We report here the first crystal structure of the lipase family I.5, the structure of a thermoalkalophilic lipase from Bacillus stearothermophilus L1 (L1 lipase) determined at 2.0-Å resolution. The structure is in a closed conformation, and the active site is buried under a long lid helix. Unexpectedly, the structure exhibits a zinc-binding site in an extra domain that accounts for the larger molecular size of the family I. Lipases that hydrolyze emulsion of lipids with long-chain fatty acids are widely distributed in animals, plants, fungi, and bacteria. Of these, microbial lipases from fungi and bacteria have attracted special attention for various industrial applications because most microbial lipases can be extracellularly produced in large quantities, have broad substrate specificity, and are quite stable under non-natural conditions (1, 2).The bacterial thermoalkalophilic lipases, which have important potential in applications for enzymatic processing of lipids at elevated temperatures and in an organic solvent phase (3), are found from various thermophilic bacterial strains including Bacillus stearothermophilus, 1 Bacillus thermocatenulatus (4),Bacillus thermoleovorans (5), B. stearothermophilus, and Bacillus sp. TP10A. 2 These thermoalkalophilic lipases have about 95% amino acid sequence identity among them and show a significant homology of 30 -35% with the mature lipases from other Gram-positive bacteria Staphylococcus strains, some of which are pathogenic, and their lipases are involved in the pathogenic processes as reviewed previously (8). In contrast to the homology with staphylococcal lipases, the thermoalkalophilic lipases exhibit no sequence homology with other microbial lipases (2). They are also characterized by their molecular sizes of 40 -45 kDa that are significantly larger than those of other microbial lipases (usually under 35 kDa). Thus, the thermoalkalophilic lipases and staphylococcal lipases were grouped together as one family of lipases named the lipase family I.5 (1) or Staphylococcus family (2). Although the thermoalkalophilic lipases and staphylococcal lipases are grouped as the same family, they show many differences in biochemical characteristics such as optimal temperature, optimal pH, and substrate specificity. The thermoalkalophilic lipases reach maximum activity at 60 -75°C and pH 8 -10, whereas staphylococcal lipases show low optimal temperatures and pH optimum of 6 -9 (8 -10). In the case of Staphylococcus haemolyticus L62 lipase, the optimum temperature is 28°C and retains more than 30% activity at 4°C (10).

Bacillus thuringiensis subsp tochigiensis HD868 was identified as a bacteriocin producer which exhibited a bactericidal effect against closely related species. This bacteriocin designated as tochicin, was partially purified by 75% ammonium sulfate precipitation followed by subsequent dialysis. This partially purified tochicin showed a narrow antibacterial spectrum of activity against most of 20 typical B. thuringiensis strains and a strain of B. cereus, but not against other bacteria and yeasts tested. The antibacterial activity of tochicin on sensitive indicator cells disappeared completely by proteinase K treatment (1 mg ml-1), which indicates its proteinaceous nature. Tochicin was very stable throughout the range of pH 3.0-9.0 and was relatively heat-stable at 90 degrees C, but bacteriocin activity was not detected after boiling for 30 min. The relationship between cell growth and bacteriocin production was studied in a semi-defined medium. Tochicin activity was detected at the mid-log growth phase, reached the maximum at the early stationary phase, but decreased after the stationary phase. Direct detection of tochicin activity on sodium dodecyl sulfate-polyacrylamide gel suggested it has an apparent molecular mass of about 10.5 kDa. Tochicin exhibited a bactericidal activity against B. thuringiensis subsp thompsoni HD522 in phosphate buffer (pH 7.0).

In Bacillus subtilis, large genomic deletions have been carried out for genome reduction, antibiotic overproduction, and heterologous protein overexpression. In view of the ecofriendliness of B. subtilis, it is critical that engineering preserves its food-grade status and avoids leaving foreign DNA in the genome. Existing methods of generating large genomic deletions leave antibiotic resistance markers or display low mutation efficiency. In this study, we introduced a clustered regularly interspaced short palindromic repeat-derived genome engineering technique to develop a highly efficient method of generating large genomic deletions in B. subtilis without any trace of foreign DNA. Using our system, we produced 38 kb plipastatin-synthesizing pps operon deletion with 80% efficiency. The significant increase in mutation efficiency was due to plasmids-delivered Streptococcus pyogenes-originated SpCas9, target-specific sgRNA and a donor DNA template, which produces SpCas9/sgRNA endonuclease complex continuously for attacking target chromosome until the mutagenic repair occurs. Our system produced single-gene deletion in spo0A (∼100%), point mutation (∼68%) and GFP gene insertion (∼97%) in sigE and demonstrated its broad applicability for various types of site-directed mutagenesis in B. subtilis.

In order to study the physiological role of acetate metabolism inEscherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate,d-lactate, and l-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by thepta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The ptamutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-β-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

An extreme diversity of substrates and catalytic reactions of cytochrome P450 (P450) enzymes is considered to be the consequence of evolutionary adaptation driven by different metabolic or environmental demands. Here we report the presence of numerous natural variants of P450 BM3 (CYP102A1) within a species of Bacillus megaterium. Extensive amino acid substitutions (up to 5% of the total 1049 amino acid residues) were identified from the variants. Phylogenetic analyses suggest that this P450 gene evolve more rapidly than the rRNA gene locus. It was found that key catalytic residues in the substrate channel and active site are retained. Although there were no apparent variations in hydroxylation activity towards myristic acid (C14) and palmitic acid (C16), the hydroxylation rates of lauric acid (C12) by the variants varied in the range of >25-fold. Interestingly, catalytic activities of the variants are promiscuous towards non-natural substrates including human P450 substrates. It can be suggested that CYP102A1 variants can acquire new catalytic activities through site-specific mutations distal to the active site.