Bioinformatics and experimental studies on the structural roles of a surface-exposed α-helix at the C-terminal domain of Chondroitinase ABC I.

A series of single and double mutants generated on residues of a surfaced-exposed helix at the C-terminal domain of chondroitinase ABC I (cABC I) from proteus vulgaris. M886A, G887E, and their respective double mutant, MA/GE were inspired by the sequence of a similar helix segment in 30S ribosomal protein S1. Additionally, M889I, Q891K, and the corresponding double mutant, MI/QK, were made regarding the sequence of a similar helix in chondroitin lyase from Proteus mirabilis. Circular dichroism spectra in the far-UV region, demonstrate that the ordered structure of wild-type (WT), and double mutants are the same; however, the helicity of the ordered structures in MI/QK is higher than that of the WT enzyme. When compared with the single mutants, the double mutants showed higher activity, and that the activity of MI/QK is higher than that of the WT enzyme. Heat-induced denaturation experiments showed that the stability of the tertiary structure of double mutants at moderate temperatures is higher compared with the WT, and single mutants. It concluded that this helix can be considered as one of the hot spots region that can be more manipulated to obtain improved variants of cABC I.

Biochemical characterization of a surfactant-stable keratinase purified from Proteus vulgaris EMB-14 grown on low-cost feather meal.

OBJECTIVES: The bioaccumulation of keratinous wastes from poultry and dairy industries poses a danger of instability to the biosphere due to resistance to common proteolysis and as such, microbial- and enzyme-mediated biodegradation are discussed. RESULTS: In submerged fermentation medium, Proteus vulgaris EMB-14 utilized and efficiently degraded feather, fur and scales by secreting exogenous keratinase. The keratinase was purified 14-fold as a monomeric 49 kDa by DEAE-Sephadex A-50 anion exchange and Sephadex G-100 size-exclusion chromatography. It exhibited optimum activity at pH 9.0 and 60 °C and was alkaline thermostable (pH 7.0-11.0), retaining 87% of initial activity after 1 h pre-incubation at 60 °C. The Km and Vmax of the keratinase with keratin azure were respectively 0.283 mg/mL and 0.241 U/mL/min. Activity of P. vulgaris keratinase was stimulated by Ca2+, Mg2+, Zn2+, Na+ and maintained in the presence of some denaturing agents, except ß-mercaptoethanol while Cu2+ and Pb2+ showed competitive and non-competitive inhibition with Ki 6.5 mM and 17.5 mM, respectively. CONCLUSION: This purified P. vulgaris keratinase could be surveyed for the biotechnological transformation of bioorganic keratinous wastes into valuable products such as soluble peptides, cosmetics and biodegradable thermoplastics.

Structural basis of transcriptional regulation by the HigA antitoxin.

Bacterial toxin-antitoxin systems are important factors implicated in growth inhibition and plasmid maintenance. Type II toxin-antitoxin pairs are regulated at the transcriptional level by the antitoxin itself. Here, we examined how the HigA antitoxin regulates the expression of the Proteus vulgaris higBA toxin-antitoxin operon from the Rts1 plasmid. The HigBA complex adopts a unique architecture suggesting differences in its regulation as compared to classical type II toxin-antitoxin systems. We find that the C-terminus of the HigA antitoxin is required for dimerization and transcriptional repression. Further, the HigA structure reveals that the C terminus is ordered and does not transition between disorder-to-order states upon toxin binding. HigA residue Arg40 recognizes a TpG dinucleotide in higO2, an evolutionary conserved mode of recognition among prokaryotic and eukaryotic transcription factors. Comparison of the HigBA and HigA-higO2 structures reveals the distance between helix-turn-helix motifs of each HigA monomer increases by ~4 Å in order to bind to higO2. Consistent with these data, HigBA binding to each operator is twofold less tight than HigA alone. Together, these data show the HigB toxin does not act as a co-repressor suggesting potential novel regulation in this toxin-antitoxin system.

Proteome-wide identification of epitope-based vaccine candidates against multi-drug resistant Proteus mirabilis.

Proteus mirabilis is one of the important pathogens of urinary tract and exhibits resistance to multiple drugs. Development of vaccine tends to be the most promising and cost-effective remedy against the said pathogen. Herein, we implement a combinatorial approach for screening proteins harboring potential broad-spectrum antigenic epitopes in the proteome of P. mirabilis. The targets are host non-homologous, essential and virulent, and have localization in the extracellular and outer membrane. Immuno-informatics revealed antigenic, surface exposed and broad-spectrum B-cell derived T-cell epitopes for three membrane usher family candidates: AtfC, PMI2533 and PMI1466, which could evoke a substantial immune response. Protein-protein interactions of targeted three proteins have shown their involvement in biologically significant pathways indispensable for the growth and survival of the pathogen. The antigenic epitopes are conserved among all completely annotated strains and docked deeply in the binding cavity of the most prevalent allele-DRB1*0101 in human population. Future work is necessary to characterize the shortlisted proteins and epitopes for immune protection in animal models.

Porous silicon nanoparticle as a stabilizing support for chondroitinase.

Chondroitinase ABCI (cABCI) from Proteus vulgaris is a drug enzyme that can be used to treat spinal cord injuries. One of the main problems of chondroitinase ABC1 is its low thermal stability. The objective of the current study was to stabilize the enzyme through entrapment within porous silicon (pSi) nanoparticles. pSi was prepared by an electrochemical etch of p-type silicon using hydrofluoric acid/ethanol. The size of nanoparticles were determined 180nm by dynamic light scattering and the mean pore diameter was in the range of 40-60nm obtained by scanning electron microscopy. Enzymes were immobilized on porouse silicon nanoparticles by entrapment. The capacity of matrix was 35µg enzyme per 1mg of silicon. The immobilized enzyme displayed lower Vmax values compared to the free enzyme, but Km values were the same for both enzymes. Immobilization significantly increased the enzyme stability at various temperatures (-20, 4, 25 and 37°C). For example, at 4°C, the free enzyme (in 10mM imidazole) retained 20% of its activity after 100min, while the immobilized one retained 50% of its initial activity. Nanoparticles loading capacity and the enzyme release rate showed that the selected particles could be a pharmaceutically acceptable carrier for chondroitinase.

Production of Omega-3 Fatty Acid Ethyl Esters from Menhaden Oil Using Proteus vulgaris Lipase-Mediated One-Step Transesterification and Urea Complexation.

An organic solvent-stable lipase from Proteus vulgaris K80 was used to produce the omega-3 polyunsaturated fatty acid ethyl esters (ω-3 PUFA EEs). First, the lyophilized recombinant lipase K80 (LyoK80) was used to perform the transesterification reaction of menhaden oil and ethanol. LyoK80 produced the ω-3 PUFA EEs with a conversion yield of 82 % in the presence of 20 % water content via a three-step ethanol-feeding process; however, in a non-aqueous condition, LyoK80 produced only a slight amount of the ω-3 PUFA EEs. To enhance its reaction properties, the lipase K80 was immobilized on a hydrophobic bead to derive ImmK80; the biochemical properties and substrate specificity of ImmK80 are similar to those of LyoK80. ImmK80 was then used to produce ω-3 PUFA EEs in accordance with the same transesterification reaction. Unlike LyoK80, ImmK80 achieved a high ω-3 PUFA EE conversion yield of 86 % under a non-aqueous system via a one-step ethanol-feeding reaction. The ω-3 PUFA EEs were purified up to 92 % using a urea complexation method.

A structural view of the dissociation of Escherichia coli tryptophanase.

Tryptophanase (Trpase) is a pyridoxal 5'-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein-protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one group, residues 15 and 59 that are located along the molecular axis R (also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (also termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.

Structure of the alanopine-containing O-polysaccharide and serological cross-reactivity of the lipopolysaccharide of Proteus vulgaris HSC 438 classified into a new Proteus serogroup, O76.

The O-polysaccharide was isolated by mild acid hydrolysis of the lipopolysaccharide of Proteus vulgaris HSC 438, and the following structure was established by chemical methods and one- and two-dimensional (1)H and (13)C NMR spectroscopy: →3)-ß-d-Quip4NAlo-(1→3)-α-d-Galp6Ac-(1→6)-α-d-Glcp-(1→3)-α-l-FucpNAc-(1→3)-ß-d-GlcpNAc-(1→, where d-Qui4N stands for 4-amino-4,6-dideoxy-d-glucose and Alo for N-((S)-1-carboxyethyl)-l-alanine (alanopine); only about half of the Gal residues are O-acetylated. This structure is unique among the Proteus O-polysaccharides, and therefore it is proposed to classify P. vulgaris HSC 438 into a new Proteus serogroup, O76. A serological cross-reactivity of HSC 438 O-antiserum and lipopolysaccharides of some other Proteus serogroups was observed and accounted for by shared epitopes on the O-polysaccharides or lipopolysaccharide core regions, including that associated with d-Qui4NAlo.

Substrate promiscuity in DNA methyltransferase M.PvuII. A mechanistic insight.

M.PvuII is a DNA methyltransferase from the bacterium Proteus vulgaris that catalyzes methylation of cytosine at the N4 position. This enzyme also displays promiscuous activity catalyzing methylation of adenine at the N6 position. In this work we use QM/MM methods to investigate the reaction mechanism of this promiscuous activity. We found that N6 methylation in M.PvuII takes place by means of a stepwise mechanism in which deprotonation of the exocyclic amino group is followed by the methyl transfer. Deprotonation involves two residues of the active site, Ser53 and Asp96, while methylation takes place directly from the AdoMet cofactor to the target nitrogen atom. The same reaction mechanism was described for cytosine methylation in the same enzyme, while the reversal timing, that is methylation followed by deprotonation, has been described in M.TaqI, an enzyme that catalyzes the N6-adenine DNA methylation from Thermus aquaticus. These mechanistic findings can be useful to understand the evolutionary paths followed by N-methyltransferases.

Carbon nanoparticles-assisted mediator-less microbial fuel cells using Proteus vulgaris.

Recently mediator-less microbial fuel cells (MFCs) are attracting great interest among researchers due to their potential applications to electricity generation as well as wastewater treatment. Common mediator-less MFCs employ electroactive bacteria called exoelectrogens to directly transfer electrons to the anode from the bacteria. However, exoelectrogens are rather limited in number and thus may not find general use for practical purposes. Here we showed our results in which mediator-less MFCs could be developed from Gram-negative non-exoelectrogens. By using carbon nanoparticles as a conductive medium to immobilize bacteria, it was possible to generate appreciable electricity from Proteus vulgaris without exogenous mediators. Maximum power density of 269 mW m(-2) and cell voltage of ca. 400 mV were obtained using glucose as a substrate. Power generation was attributed to direct electron transfer and to self-produced mediators, both of which were assisted by carbon nanoparticles. Bacillus subtilis, a Gram-positive bacterium, in the meantime, did not produce appreciable electricity.

Stereospecificity of isotopic exchange of C-α-protons of glycine catalyzed by three PLP-dependent lyases: the unusual case of tyrosine phenol-lyase.

A comparative study of the kinetics and stereospecificity of isotopic exchange of the pro-2R- and pro-2S protons of glycine in (2)H(2)O under the action of tyrosine phenol-lyase (TPL), tryptophan indole-lyase (TIL) and methionine γ-lyase (MGL) was undertaken. The kinetics of exchange was monitored using both (1)H- and (13)C-NMR. In the three compared lyases the stereospecificities of the main reactions with natural substrates dictate orthogonal orientation of the pro-2R proton of glycine with respect to the cofactor pyridoxal 5'-phosphate (PLP) plane. Consequently, according to Dunathan's postulate with all the three enzymes pro-2R proton should exchange faster than does the pro-2S one. In fact the found ratios of 2R:2S reactivities are 1:20 for TPL, 108:1 for TIL, and 1,440:1 for MGL. Thus, TPL displays an unprecedented inversion of stereospecificity. A probable mechanism of the observed phenomenon is suggested, which is based on the X-ray data for the quinonoid intermediate, formed in the reaction of TPL with L-alanine. The mechanism implies different conformational changes in the active site upon binding of glycine and alanine. These changes can lead to relative stabilization of either the neutral amino group, accepting the α-proton, or the respective ammonium group, which is formed after the proton abstraction.

Structure of a glucosyl phosphate-containing O-polysaccharide of Proteus vulgaris O42.

An O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Proteus vulgaris O42 and studied by sugar and methylation analyses along with 1H, 13C and 31P NMR spectroscopy. The following structure of the polysaccharide having a linear pentasaccharide phosphate repeating unit was established: -->3)-alpha-L-FucpNAc4Ac-(1-->4)-alpha-D-Glcp-1-P-(O-->4)-alpha-D-GlcpNAc-(1-->3)-alpha-L-FucpNAc4Ac-(1-->3))-alpha-D-GlcpNAc6Ac-(1--> where the degree of O-acetylation is approximately 80% on GlcNAc and approximately 40% on each of the FucNAc residues. A weak serological cross-reaction of anti-P. vulgaris O42 serum with the lipopolysaccharide of P. vulgaris O39 was observed and accounted for by the sharing of a disaccharide fragment of the O-polysaccharides.

Structure of a new ribitol teichoic acid-like O-polysaccharide of a serologically separate Proteus vulgaris strain, TG 276-1, classified into a new Proteus serogroup O53.

An unusual ribitol teichoic acid-like O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide from a previously non-classified Proteus vulgaris strain TG 276-1. Structural studies using chemical analyses and 2D (1)H and (13)C NMR spectroscopy showed that the polysaccharide is a zwitterionic polymer with a repeating unit containing 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose (D-FucNAc4N) and two D-ribitol phosphate (D-Rib-ol-5-P) residues and having the following structure:[formula: see text] where the non-glycosylated ribitol residue is randomly mono-O-acetylated. Based on the unique O-polysaccharide structure and the finding that the strain studied is serologically separate among Proteus bacteria, we propose to classify P. vulgaris strain TG 276-1 into a new Proteus serogroup, O53.

Structures and serology of the O-antigens of Proteus strains classified into serogroup O17 and former serogroup O35.

INTRODUCTION: Bacteria of the genus Proteus are facultative pathogens which commonly cause urinary tract infections. Based on the serological specificity of the O-chain polysaccharide of the lipopolysaccharide (O-polysaccharide, O-antigen), strains of P. mirabilis and P. vulgaris have been classified into 60 serogroups. Studies on the chemical structure and serological specificity of the O-antigens aim at the elucidation of the molecular basis and improvement of the serological classification of these bacteria. MATERIALS AND METHODS: The O-polysaccharide was prepared by acetic acid degradation of the lipopolysaccharide isolated from dried bacterial mass of each strain by hot phenol/water extraction. (1)H- and (13)C-NMR spectroscopy was used for structural studies. Serological studies were performed with rabbit O-antisera using enzyme immunosorbent assay, passive hemolysis test, and the inhibition of reactions in these assays as well DOC-PAGE and Western blot. RESULTS: Four Proteus strains belonging to serogroups O17 and O35 were found to possess similar O-polysaccharide structures, in particular having the same carbohydrate backbone built up of tetrasaccharide repeating units. However, they differ in the presence or absence of additional substituents, such as phosphoethanolamine in P. mirabilis O17 and glucose in P. penneri O17, as well as in the pattern and degree of O-acetylation of various monosaccharide residues. Serological studies also showed close relationships between the O-antigens studied. CONCLUSIONS: Based on these data it is proposed to reclassify strain P. mirabilis PrK 61/57, formerly representing the O35 serogroup, into the serogroup O17 in the Kauffman-Perch classification system of Proteus.

Characterization and serological classification of O-specific polysaccharide of Proteus mirabilisTG 276-90 from Proteus serogroup O34.

INTRODUCTION: Gram-negative bacteria of the genus Proteus from the family Enterobacteriaceae are currently divided into the five species P. mirabilis, P. vulgaris, P. penneri, P. hauseri, and P. myxofaciens and three unnamed Proteus genomospecies 4, 5, and 6. They are important facultative human and animal pathogens which, under favorable conditions, cause mainly intestinal and urinary tract infections, sometimes leading to serious complications such as acute or chronic pyelonephritis and the formation of bladder and kidney stones. In this study we report on the serological properties of the lipopolysaccharide (LPS) of P. mirabilis TG 276-90, whose O-polysaccharide chemical structure was described earlier. MATERIALS AND METHODS: LPS and alkali-treated LPS of a few serologically related Proteus strains and O-antisera against P. mirabilis TG 276-90 and CCUG 4669 (O34) were used. Serological characterization of P. mirabilis TG 276-90 O-specific polysaccharide was done using enzyme immunosorbent assay, passive immunohemolysis test (PIH), inhibition of these tests, SDS/PAGE and Western blot techniques, absorption of rabbit polyclonal O-antisera, and repeated PIH test. RESULTS: Structural and serological investigations showed that the O-polysaccharides of P. mirabilis TG 276-90 and P. vulgaris O34 are identical and that their LPSs differ only in epitopes in the core part. Therefore these two strains could be classified into the same Proteus O34 serogroup. CONCLUSIONS: The serological data showed that the beta-D-GalpNAc-(1--> 4)-alpha-D-GalpNAc disaccharide is an important epitope of the P. mirabilis TG 276-90 and P. vulgaris O34 LPSs, shared by the P. mirabilis O16 and P. vulgaris TG 251 LPSs. It is responsible for cross-reactions with P. mirabilis TG 276-90 and P. vulgaris O34 O-antisera.

Structure of the O-polysaccharide of Proteus serogroup O34 containing 2-acetamido-2-deoxy-alpha-D-galactosyl phosphate.

On mild acid degradation of the lipopolysaccharide of Proteus vulgaris O34, strain CCUG 4669, the O-polysaccharide was cleaved at a glycosyl-phosphate linkage that is present in the main chain. The resultant phosphorylated oligosaccharides and an alkali-treated lipopolysaccharide were studied by sugar and methylation analyses along with 1H and 13C NMR spectroscopy, and the following structure of the branched tetrasaccharide phosphate repeating unit of the O-polysaccharide was established: [carbohydrate structure: see text]The O-polysaccharide of Proteus mirabilis strain TG 276 was found to have the same structure and, based on the structural and serological data, this strain was proposed to be classified into the same Proteus serogroup O34.

Structure of the N-acetyl-L-rhamnosamine-containing O-polysaccharide of Proteus vulgaris TG 155 from a new Proteus serogroup, O55.

The O-polysaccharide of the lipopolysaccharide (LPS) of Proteus vulgaris TG 155 was found to contain 2-acetamido-2,6-dideoxy-L-mannose (N-acetyl-L-rhamnosamine, L-RhaNAc), a monosaccharide that occurs rarely in Nature. The following structure of the O-polysaccharide was established by NMR spectroscopy, including 2D COSY, TOCSY, ROESY and 1H,13C HSQC experiments, along with chemical methods: [carbohydrate structure in text] Rabbit polyclonal O-antiserum against P. vulgaris TG 155 reacted with both core and O-polysaccharide moieties of the homologous LPS but showed no cross-reactivity with other LPS from the complete set of serologically different Proteus strains. Based on the unique O-polysaccharide structure and the serological data, we propose classifying P. vulgaris TG 155 into a new, separate Proteus O-serogroup, O55.

Structure of the O-polysaccharide of Providencia alcalifaciens O21 containing 3-formamido-3,6-dideoxy-D-galactose.

The O-polysaccharide (O-antigen) of Providencia alcalifaciens O21 was obtained by mild acid degradation of the lipopolysaccharide and studied by chemical methods and NMR spectroscopy. It was found that the polysaccharide is built up of branched pentasaccharide repeating units with a terminal residue of 3-formamido-3,6-dideoxy-D-galactose (D-Fuc3NFo) and has the following structure: [structure: see text]. Anti-P. alcalifaciens O21 serum cross-reacted with the O-antigen of Proteus vulgaris O47, which contains a GalNAc trisaccharide similar to that present in the P. alcalifaciens O21 O-polysaccharide.

Structure of the O-polysaccharide of Proteus vulgaris O44: a new O-antigen that contains an amide of D-glucuronic acid with L-alanine.

The O-polysaccharide of Proteus vulgaris O44, strain PrK 67/57 was studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, ROESY, H-detected 1H, 13C HMQC, HMQC-TOCSY and HMBC experiments. The polysaccharide was found to contain an amide of D-glucuronic acid with L-alanine [D-GlcA6(L-Ala)], and the following structure of the linear pentasaccharide repeating unit was established: [structure: see text]. The structural data of the O-polysaccharide and the results of serological studies with P. vulgaris O44 O-antiserum showed that the strain studied is unique among Proteus bacteria, which is in agreement with its classification in a separate Proteus serogroup, O44.

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids.

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: [Figure: see text], where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N(epsilon)-[(R)-1-carboxyethyl]-L-lysine ("alaninolysine"), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.