Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1.

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest Km values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.

The expression of the serine proteinase gene of Bacillus intermedius in Bacillus subtilis.

The gene encoding for Bacillus intermedius serine proteinase was cloned and the complete nucleotide sequence was determined. Gene expression was explored in the protease-deficient strain Bacillus subtilis AJ73 during different stages of growth. Catabolite repression involved in control of proteinase expression during transition state and onset of sporulation was not efficient at the late stationary phase. Salt stress leads to induction of serine proteinase production during B. subtilis AJ73(pCS9) post-exponential growth. Expression of proteinase in B. subtilis deg-mutants may be controlled by DegU regulator. B. subtilis spo0-mutants failed to accomplish B. intermedius proteinase production. These data suggest complex network regulation of B. intermedius serine proteinase expression, including the action of spo0, degU, catabolite repression and demonstrate changes in control of enzyme biosynthesis at different stages of growth.

Salt stress induction of glutamyl endopeptidase biosynthesis in Bacillus intermedius.

Bacteria from the genus Bacillus have evolved complicated regulatory networks to be protected from various environmental stresses, including sudden increase in salinity. Among these regulatory mechanisms is the DegS-DegU signal transduction system, which controls degradative enzyme synthesis and is involved in sensing salt stress in Bacillus subtilis. We report the study of biosynthesis regulation of Bacillus intermedius glutamyl endopeptidase under salt stress conditions. Salt stress during growth in medium containing 1-2.5 M NaCl, KCl or disodium succinate leads to the induction of glutamyl endopeptidase. Analysis of the regulatory region of the gene for B. intermedius glutamyl endopeptidase revealed the presence of a tentative target sequence for DegU control, AGATN10TTGAG. For the expression of the glutamyl endopeptidase gene, functional DegU protein is required. Thus, we suggest that expression of the gene for B. intermedius glutamyl endopeptidase may be controlled by a regulatory system analogous to DegS-DegU two-component system in B. subtilis.

High-quality draft genome sequence of a new phytase-producing microorganism Pantoea sp. 3.5.1.

Strain 3.5.1 was isolated from soils of the Republic of Tatarstan, Russia, on the basis of presence of a high phytate-degrading activity. Strains with such activities attract special interest because of its potential use as feed additives and natural manures. Strain 3.5.1 harbors a 99 % 16S rRNA nucleotide sequence similarity to different Pantoea species (P. vagans, P. ananatis, P. agglomerans, P. anthophila and Pantoea sp.) and exhibits unique biochemical properties that do not allow strain identification up to species. Moreover, the strain 3.5.1 shows a low ANI and MALDI-TOF Mass Spectrometry scores. Thus, it is likely that the strain 3.5.1 represents a new Pantoea species. Here, we present the genome sequence of Pantoea sp. strain 3.5.1. The 4,964,649 bp draft genome consists of 23 contigs with 4,556 protein-coding and 143 RNA genes. Genome sequencing and annotation revealed two phytase genes and putative regulatory genes controlling its activity.

Bacterial enzymes effectively digest Alzheimer's ß-amyloid peptide.

Aggregated ß-amyloid peptides play key roles in the development of Alzheimer's disease, and recent evidence suggests that microbial particles, among others, can facilitate their polymerization. Bacterial enzymes, however, have been proved to be beneficial in degrading pathological fibrillar structures in clinical settings, such as strepto-kinases in resolving blood-clots. The purpose of this study was to investigate the ability of bacterial substances to effectively hydrolyze ß-amyloid peptides. Degrading products of several proteinases from Bacillus pumilus were evaluated using MALDI-TOF mass-spectrometry, and their toxicity was assessed in vitro using cell-culture assays and morphological studies. These enzymes have proved to be non-toxic and were demonstrated to cleave through the functional domains of ß-amyloid peptide. By yielding inactive fragments, proteinases of Bacillus pumilus may be used as candidate anti-amyloid agents.

Draft Genome Sequence of Bacillus pumilus 7P, Isolated from the Soil of the Tatarstan Republic, Russia.

Here, we present a draft genome sequence of Bacillus pumilus strain 7P. This strain was isolated from soil as an extracellular RNase-producing microorganism. The RNase of B. pumilus 7P is considered to be a potential antiviral and therapeutic antitumor agent, and it might be appropriate for agriculture and academic synthesis of oligoribonucleotides.

A novel secreted metzincin metalloproteinase from Bacillus intermedius.

The mprBi gene from Bacillus intermedius 3-19 encoding a novel secreted metalloproteinase was identified. The mpriBi gene was expressed in an extracellular proteinase-deficient Bacillus subtilis BG 2036 strain and the corresponding protein was characterized biochemically. The 19 kDa MprBi protein was purified to homogeneity and sequenced by mass spectroscopy and Edman degradation methods. Amino acid sequence analysis of MprBi identified an active site motif HEYGHNFGLPHD and a conserved structural component Met-turn, both of which are unique features of the metzincin clan. Furthermore, MprBi harbors a number of distinct sequence elements characteristic of proteinase domains in eukaryotic adamalysins. We conclude that MprBi and similar proteins from other Bacillus species form a novel group of metzincin metalloproteinases in prokaryotes.

Selection of cultivation medium for production of late stationary phase serine proteinases from Bacillus intermedius.

B. intermedius have been shown previously to secrete two serine proteinases: glutamyl endopeptidase 2 and subtilisin 2 during the late stationary phase, with maximal levels of the enzymes activities recorded at the 40th and 44th hours of growth, respectively. In the current study, we analyzed the impact of various culture medium components on biosynthesis of these proteinases. Yeast extract and gelatin did not stimulate the enzymes biosynthesis. However, on the medium containing 0.1% casein subtilisin 2 production increased to reach 140%. Biosynthesis of both serine proteinases, produced by B. intermedius at the late stationary phase, were found to be inhibited by individual amino acids, and to be insensitive to catabolite repression. In order to maximise enzyme production, the presence of Ca(2+) and Mg(2+) at concentration of 5 mM was shown to be necessary. Based on the results of this work, the composition of a complex culture media for the effective production of late stationary phase proteinases by B. intermedius was developed.

Proteinases from Bacillus intermedius secreted in the late stages of sporulation.

BACKGROUND: Proteinases are widely used in various fields of medicine, such as the treatment of burns, purulent wounds, or decubitus ulcers. On the basis of new microbial proteinases produced by nonpathogenic organisms, a new generation of medical preparations can be developed. Representatives of the Bacillus genera are nonpathogenic and are suitable for producing various proteases in large quantities. B. intermedius is shown to produce a set of alkaline proteases at the early and late stationary phase of growth. MATERIAL/METHODS: The activity of alkaline proteinases was determined using synthetic chromogenous substrates Z-Glu-pNA and Z-Ala-Ala-Leu-pNA. To determine beta-galactosidase activity, 2-nitro-beta-D-galactopyranosid was used. Spores were calculated by phase-contrast microscopy. RESULTS: During the late stages of sporulation B. intermedius 3-19 cells were shown to secrete two proteinases into the medium: glutamyl endopeptidase, with maximum activity at 40 hours of growth, and subtilisin, with maximum activity at 44 hours of growth. Evidence for the secretion of these enzymes into the medium was provided by measuring beta-galactosidase activity. CONCLUSIONS: Our results show that proteinases from B. intermedius (glutamyl endopeptidase 2 and subtilisin 2) in the late stationary phase of growth are secreted enzymes. This suggests that these enzymes play a role in sporulation.