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PubMed2006

Lytic activity of recombinant bacteriophage phi11 and phi12 endolysins on whole cells and biofilms of Staphylococcus aureus.

The recombinant phi11 endolysin hydrolyzed heat-killed staphylococci as well as staphylococcal biofilms. Cell wall targeting appeared to be a prerequisite for lysis of whole cells, and the combined action of the endopeptidase and amidase domains was necessary for maximum activity. In contrast, the phi12 endolysin was inactive and caused aggregation of the cells.

Amidohydrolases↗

PubMed1986

Degradation of Pyridine by Micrococcus luteus Isolated from Soil.

An organism capable of growth on pyridine was isolated from soil by enrichment culture techniques and identified as Micrococcus luteus. The organism oxidized pyridine for energy and released N contained in the pyridine ring as ammonium. The organism could not grow on mono- or disubstituted pyridinecarboxylic acids or hydroxy-, chloro-, amino-, or methylpyridines. Cell extracts of M. luteus could not degrade pyridine, 2-, 3-, or 4-hydroxypyridines or 2,3-dihydroxypyridine, regardless of added cofactors or cell particulate fraction. The organism had a NAD-linked succinate-semialdehyde dehydrogenase which was induced by pyridine. Cell extracts of M. luteus had constitutive amidase activity, and washed cells degraded formate and formamide without a lag. These data are consistent with a previously reported pathway for pyridine metabolism by species of Bacillus, Brevibacterium, and Corynebacterium. Cells of M. luteus were permeable to pyridinecarboxylic acids, monohydroxypyridines, 2,3-dihydroxypyridine, and monoamino- and methylpyridines. The results provide new evidence that the metabolism of pyridine by microorganisms does not require initial hydroxylation of the ring and that permeability barriers do not account for the extremely limited range of substrate isomers used by pyridine degraders.

Journal Article↗

PubMed1988

Nitrile Hydratase-Catalyzed Production of Nicotinamide from 3-Cyanopyridine in Rhodococcus rhodochrous J1.

Nitrile hydratase, which occurs abundantly in cells of Rhodococcus rhodochrous J1 isolated from soil samples, catalyzes the hydration of 3-cyanopyridine to nicotinamide. By using resting cells, the reaction conditions for nicotinamide production were optimized. Under the optimum conditions, 100% of the added 12 M 3-cyanopyridine was converted to nicotinamide without the formation of nicotinic acid, and the highest yield achieved was 1,465 g of nicotinamide per liter of reaction mixture containing resting cells (1.48 g as dry cell weight) in 9 h. The nicotinamide produced was crystallized and then identified physicochemically. The further conversion of the nicotinamide to nicotinic acid was due to the low activity of nicotinamide as a substrate for the amidase(s) present in this organism.

Journal Article↗

PubMed1991

Production of R-(-)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750.

R-(-)-Mandelic acid was produced from racemic mandelonitrile by Alcaligenes faecalis ATCC 8750. Ammonium acetate or L-glutamic acid as the carbon source and n-butyronitrile as the inducer in the culture medium were effective for bacterial growth and the induction of R-(-)-mandelic acid-producing activity. The R-(-)-mandelic acid formed from mandelonitrile by resting cells was present in a 100% enantiomeric excess. A. faecalis ATCC 8750 has an R-enantioselective nitrilase for mandelonitrile and an amidase for mandelamide. As R-(-)-mandelic acid was produced from racemic mandelonitrile in a yield of 91%, whereas no S-mandelonitrile was left, the S-mandelonitrile remaining in the reaction is spontaneously racemized because of the chemical equilibrium and is used as the substrate. Consequently, almost all the mandelonitrile is consumed and converted to R-(-)-mandelic acid. R-(-)-Mandelic acid was also produced when benzaldehyde plus HCN was used as the substrate.

Acetonitriles↗

PubMed1992

Cloning and expression of a conjugated bile acid hydrolase gene from Lactobacillus plantarum by using a direct plate assay.

The conjugated bile acid hydrolase gene from the silage isolate Lactobacillus plantarum 80 was cloned and expressed in Escherichia coli MC1061. For the screening of this hydrolase gene within the gene bank, a direct plate assay developed by Dashkevicz and Feighner (M. P. Dashkevicz and S. D. Feighner, Appl. Environ. Microbiol. 53:331-336, 1989) was adapted to the growth requirements of E. coli. Because of hydrolysis and medium acidification, hydrolase-active colonies were surrounded with big halos of precipitated, free bile acids. This phenomenon was also obtained when the gene was cloned into a multicopy shuttle vector and subsequently reintroduced into the parental Lactobacillus strain. The cbh gene and surrounding regions were characterized by nucleotide sequence analysis. The deduced amino acid sequence was shown to have 52% similarity with a penicillin V amidase from Bacillus sphaericus. Preliminary characterization of the gene product showed that it is a cholylglycine hydrolase (EC 3.5.1.24) with only slight activity against taurine conjugates. The optimum pH was between 4.7 and 5.5. Optimum temperature ranged from 30 to 45 degrees C. Southern blot analysis indicated that the cloned gene has similarity with genomic DNA of bile acid hydrolase-active Lactobacillus spp. of intestinal origin.

Amidohydrolases↗

PubMed1995

Autolysis of lactococci: detection of lytic enzymes by polyacrylamide gel electrophoresis and characterization in buffer systems.

Lactococcal strains were screened for bacteriolytic activity against Micrococcus luteus cells, lactococcal cells, and cell walls. Thirty strains were screened for bacteriolytic activity against cells and cell walls incorporated into agar medium. Enzymes from all strains hydrolyzed the substrates; however, the activity against Micrococcus cells was much higher than against Lactococcus cells or cell walls. Electrophoretic profiles of bacteriolytic activities of culture supernatants, sodium dodecyl sulfate-treated cell extracts, cell wall fractions, and cell extracts were analyzed in sodium dodecyl sulfate-polyacrylamide gels containing M. luteus cells or lactococcal cell walls as the substrate. The 22 strains tested contained two to five lytic bands in the culture supernatant, ranging in size between 32 and 53 kDa. The cell extracts, the sodium dodecyl sulfate-treated cell extracts, and the cell wall fractions revealed two lytic bands of 47 and 53 kDa. Effects of external factors on autolysis of some strains were determined in buffer systems. Optimal autolysis was observed in the exponential growth phase at pH 6.0 to 7.5 and at a temperature of 30(deg)C. Two of three strains tested seemed to contain a glycosidase, and all three strains contained an N-acetylmuramyl-l-alanine amidase or an endopeptidase.

Journal Article↗

PubMed2000

Gene cloning and expression and secretion of Listeria monocytogenes bacteriophage-lytic enzymes in Lactococcus lactis.

Bacteriophage lysins (Ply), or endolysins, are phage-encoded cell wall lytic enzymes which are synthesized late during virus multiplication and mediate the release of progeny virions. Bacteriophages of the pathogen Listeria monocytogenes encode endolysin enzymes which specifically hydrolyze the cross-linking peptide bridges in Listeria peptidoglycan. Ply118 is a 30.8-kDa L-alanoyl-D-glutamate peptidase and Ply511 (36.5 kDa) acts as N-acetylmuramoyl-L-alanine amidase. In order to establish dairy starter cultures with biopreservation properties against L. monocytogenes contaminations, we have introduced ply118 and ply511 into Lactococcus lactis MG1363 by using a pTRKH2 backbone. The genes were expressed under control of the lactococcal promoter P32, which proved superior to other promoters (P21 and P59) tested in this study. High levels of active enzymes were produced and accumulated in the cytoplasmic cell fractions but were not released from the cells at significant levels. Therefore, ply511 was genetically fused with the (SP)slpA nucleotide sequence encoding the Lactobacillus brevis S-layer protein signal peptide. Expression of (SP)slpA-ply511 from pSL-PL511 resulted in secretion of functional Ply511 enzyme from L. lactis cells. One clone expressed an unusually strong lytic activity, which was found to be due to a 115-bp deletion that occurred within the 3'-end coding sequence of (SP)slpA-ply511, which caused a frameshift mutation and generated a stop codon. Surprisingly, the resulting carboxy-terminal deletion of 80 amino acids in the truncated Ply511 Delta(S262-K341) mutant polypeptide strongly increased its lytic activity. Proteolytic processing of the secretion competent (SP)SlpA-Ply511 propeptide following membrane translocation had no influence on enzyme activity. Immunoblotting experiments using both cytoplasmic and supernatant fractions indicated that the enzyme was quantitatively exported from the cells and secreted into the surrounding medium, where it caused rapid lysis of L. monocytogenes cells. Moreover, transformation of pSL-PL511 delta C into L. lactis Bu2-129, a lactose-utilizing strain that can be employed for fermentation of milk, also resulted in secretion of functional enzyme and showed that the vector is compatible with the native lactococcal plasmids.

Bacteriolysis↗

PubMed2003

Prospecting for novel biocatalysts in a soil metagenome.

The metagenomes of complex microbial communities are rich sources of novel biocatalysts. We exploited the metagenome of a mixed microbial population for isolation of more than 15 different genes encoding novel biocatalysts by using a combined cultivation and direct cloning strategy. A 16S rRNA sequence analysis revealed the presence of hitherto uncultured microbes closely related to the genera Pseudomonas, Agrobacterium, Xanthomonas, Microbulbifer, and Janthinobacterium. Total genomic DNA from this bacterial community was used to construct cosmid DNA libraries, which were functionally searched for novel enzymes of biotechnological value. Our searches in combination with cosmid sequencing resulted in identification of four clones encoding 12 putative agarase genes, most of which were organized in clusters consisting of two or three genes. Interestingly, nine of these agarase genes probably originated from gene duplications. Furthermore, we identified by DNA sequencing several other biocatalyst-encoding genes, including genes encoding a putative stereoselective amidase (amiA), two cellulases (gnuB and uvs080), an alpha-amylase (amyA), a 1,4-alpha-glucan branching enzyme (amyB), and two pectate lyases (pelA and uvs119). Also, a conserved cluster of two lipase genes was identified, which was linked to genes encoding a type I secretion system. The novel gene aguB was overexpressed in Escherichia coli, and the enzyme activities were determined. Finally, we describe more than 162 kb of DNA sequence that provides a strong platform for further characterization of this microbial consortium.

Bacteria↗

PubMed2003

On the origins of cyanuric acid hydrolase: purification, substrates, and prevalence of AtzD from Pseudomonas sp. strain ADP.

Cyanuric acid hydrolase (AtzD) from Pseudomonas sp. strain ADP was purified to homogeneity. Of 22 cyclic amides and triazine compounds tested, only cyanuric acid and N-methylisocyanuric acid were substrates. Other cyclic amidases were found not to hydrolyze cyanuric acid. Ten bacteria that use cyanuric acid as a sole nitrogen source for growth were found to contain either atzD or trzD, but not both genes.

Amidohydrolases↗

PubMed2004

Cloning and characterization of the bile salt hydrolase genes (bsh) from Bifidobacterium bifidum strains.

Biochemical characterization of the purified bile salt hydrolase (BSH) from Bifidobacterium bifidum ATCC 11863 revealed some distinct characteristics not observed in other species of Bifidobacterium. The bsh gene was cloned from B. bifidum, and the DNA flanking the bsh gene was sequenced. Comparison of the deduced amino acid sequence of the cloned gene with previously known sequences revealed high homology with BSH enzymes from several microorganisms and penicillin V amidase (PVA) of Bacillus sphaericus. The proposed active sites of PVA were highly conserved, including that of the Cys-1 residue. The importance of the SH group in the N-terminal cysteine was confirmed by substitution of Cys with chemically and structurally similar residues, Ser or Thr, both of which resulted in an inactive enzyme. The transcriptional start point of the bsh gene has been determined by primer extension analysis. Unlike Bifidobacterium longum bsh, B. bifidum bsh was transcribed as a monocistronic unit, which was confirmed by Northern blot analysis. PCR amplification with the type-specific primer set revealed the high level of sequence homology in their bsh genes within the species of B. bifidum.

Amidohydrolases↗

PubMed2006

Purification and characterization of TrzF: biuret hydrolysis by allophanate hydrolase supports growth.

TrzF, the allophanate hydrolase from Enterobacter cloacae strain 99, was cloned, overexpressed in the presence of a chaperone protein, and purified to homogeneity. Native TrzF had a subunit molecular weight of 65,401 and a subunit stoichiometry of alpha(2) and did not contain significant levels of metals. TrzF showed time-dependent inhibition by phenyl phosphorodiamidate and is a member of the amidase signature protein family. TrzF was highly active in the hydrolysis of allophanate but was not active with urea, despite having been previously considered a urea amidolyase. TrzF showed lower activity with malonamate, malonamide, and biuret. The allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, was also shown to hydrolyze biuret slowly. Since biuret and allophanate are consecutive metabolites in cyanuric acid metabolism, the low level of biuret hydrolase activity can have physiological significance. A recombinant Escherichia coli strain containing atzD, encoding cyanuric acid hydrolase that produces biuret, and atzF grew slowly on cyanuric acid as a source of nitrogen. The amount of growth produced was consistent with the liberation of 3 mol of ammonia from cyanuric acid. In vitro, TrzF was shown to hydrolyze biuret to liberate 3 mol of ammonia. The biuret hydrolyzing activity of TrzF might also be physiologically relevant in native strains. E. cloacae strain 99 grows on cyanuric acid with a significant accumulation of biuret.

Allophanate Hydrolase↗

PubMed2005

Recognition of Staphylococcus aureus by the innate immune system.

The gram-positive bacterium Staphylococcus aureus is a major pathogen responsible for a variety of diseases ranging from minor skin infections to life-threatening conditions such as sepsis. Cell wall-associated and secreted proteins (e.g., protein A, hemolysins, and phenol-soluble modulin) and cell wall components (e.g., peptidoglycan and alanylated lipoteichoic acid) have been shown to be inflammatory, and these staphylococcal components may contribute to sepsis. On the host side, many host factors have been implicated in the innate detection of staphylococcal components. One class of pattern recognition molecules, Toll-like receptor 2, has been shown to function as the transmembrane component involved in the detection of staphylococcal lipoteichoic acid and phenol-soluble modulin and is involved in the synthesis of inflammatory cytokines by monocytes/macrophages in response to these components. Nod2 (nucleotide-binding oligomerization domain 2) is the intracellular sensor for muramyl dipeptide, the minimal bioactive structure of peptidoglycan, and it may contribute to the innate immune defense against S. aureus. The staphylococcal virulence factor protein A was recently shown to interact directly with tumor necrosis factor receptor 1 in airway epithelium and to reproduce the effects of tumor necrosis factor alpha. Finally, peptidoglycan recognition protein L is an amidase that inactivates the proinflammatory activities of peptidoglycan. However, peptidoglycan recognition protein L probably plays a minor role in the innate immune response to S. aureus. Thus, several innate immunity receptors may be implicated in host defense against S. aureus.

Humans↗

PubMed1982

Soluble peptidoglycan-polysaccharide fragments of the bacterial cell wall induce acute inflammation.

Peptidoglycan (PG)-polysaccharide (PS) polymers derived from group A streptococcal cell walls were solubilized by M-1 mutanolysin (endo-N-acetylmuramidase) and phage-associated lysin (N-acetylmuramyl-l-alanine amidase). Fragments were isolated by ultrafiltration and a series of gel filtrations and were injected intravenously into Sprague-Dawley rats. No fragments with a molecular weight of less than 5 x 10(6) were able to induce arthritis by systemic injection. However, the enzyme-derived fragments displayed a new biological activity. High-molecular-weight PG-PS fragments ( congruent with500,000) derived from mutanolysin digests induced a severe edematous reaction in the front and hind limbs. The response started 5 to 10 min postinjection, reached maximum intensity in approximately 30 min, and disappeared by 10 h. The smallest dose capable of eliciting the response was 0.31 mug/g of body weight. Low-molecular-weight PG-PS ( congruent with30,000) derived from the mutanolysin digests and the PG-PS fragments isolated from phage-associated lysin digests also induced edema; however, a higher dose was required to elicit the same response as that produced by high-molecular-weight PG-PS fragments. The active fragments contained rhamnose, glucosamine, muramic acid, alanine, glutamic acid, and lysine in various molar ratios. PG-PS fragments obtained by sonic degradation of cell walls (molecular weight >/=5.3 x 10(6)), as well as enzyme-treated PG preparations and muramyl dipeptide, failed to elicit the response. These findings indicate that PG-PS fragments of sizes too small to be arthritogenic can affect the vascular endothelium to induce a rapidly developing edema. Fragments with this biological property could have a key role in the pathogenesis of experimental arthritis by influencing the tissue distribution of arthritogenic PG-PS.

Animals↗

PubMed1983

Resistance of O-acetylated gonococcal peptidoglycan to human peptidoglycan-degrading enzymes.

Two naturally occurring forms of gonococcal peptidoglycan (PG) were tested for their susceptibility to human PG hydrolases. Purified 3H-labeled PG substituted extensively with O-acetyl derivatives (O-PG; from Neisseria gonorrhoeae FA19) and 14C-labeled O-acetyl-deficient PG (non-O-PG; from N. gonorrhoeae RD5) were mixed together and treated with either normal human sera (NHS) or with lysozyme purified from human polymorphonuclear leukocytes (PMN-LZ). The initial rate of hydrolysis of O-PG by NHS or by PMN-LZ was two- to fourfold less than that of its non-O-PG counterpart in the same tube. When the reactions were allowed to go to completion. NHS solubilized both PGs completely, whereas PMN-LZ solubilized all of the non-O-PG and left ca. 60% of the O-PG insoluble. The PMN-LZ-soluble fraction of O-PG consisted largely of glycosidically linked fragments with molecular weights greater than ca. 10(4), whereas the corresponding non-O-PG was degraded to lower-molecular-weight fragments, exclusively. At completion, NHS hydrolyzed both PGs to fragments whose size was equal to or smaller than that of the free disaccharide unit of PG, suggesting that human sera contain a peptide-splitting (amidase) activity and a glycosidase activity, in addition to that of the well-known muramidase. NHS also promoted the release of high-molecular-weight PG fragments from intact gonococci. The persistence of human hydrolase-resistant PG in the form of soluble macromolecular fragments may potentiate the biological effects of gonococcal PG in vivo.

Acetylation↗

PubMed2005

Gene silencing and overexpression of porcine peptidoglycan recognition protein long isoforms: involvement in beta-defensin-1 expression.

Peptidoglycan recognition proteins (PGRPs) are a group of newly identified proteins with emerging functions in mammalian innate immunity. Here we report the identification and characterization of two long isoforms of porcine PGRP. Their complete cDNA sequences encode predicted peptides of 252 and 598 residues and are named pPGRP-L1 and pPGRP-L2, respectively. These porcine isoforms share identical PGRP domains at their C terminus, which are highly conserved with human and mouse orthologs. pPGRP-L1 is expressed constitutively in several tissues, including bone marrow, intestine, liver, spleen, kidney, and skin. pPGRP-L2 is highly expressed in the duodenum and liver, and expression in intestinal tissues is increased by Salmonella infection. In intestinal cells, expression of both pPGRP-L1 and pPGRP-L2 is increased by bacterial infection. Recombinant pPGRP-L1 and pPGRP-L2 have N-acetylmuramoyl-L-alanine amidase activity. Loss-of-function and gain-of-function experiments indicate that these two pPGRPs are involved in expression of the antimicrobial peptide beta-defensin-1. Silencing of pPGRP-L2 in intestinal cells challenged with Listeria monocytogenes results in downregulation of beta-defensin-1. Conversely, overexpression of pPGRP-L1 or pPGRP-L2 dramatically upregulates expression of beta-defensin-1. Collectively, these findings suggest that porcine PGRPs are involved in antimicrobial peptide expression.

Amino Acid Sequence↗

PubMed1969

Specificity of a bacteriolytic enzyme from Pseudomonas aeruginosa.

A bacteriolytic enzyme isolated from shake-flask cultures of Pseudomonas aeruginosa and capable of lysing cells of Staphylococcus aureus was purified approximately 500-fold by passage through diethylaminoethyl cellulose and chromatography on carboxymethyl-cellulose. The purified enzyme was shown to act as an endopeptidase, cleaving the pentaglycine cross-bridges of the cell wall peptidoglycan at d-alanyl-glycine and glycyl-glycine linkages with the release of di-, tri-, and tetraglycine fragments. Release of NH(2)-alanine indicated weak N-acetylmuramyl-l-alanine amidase activity, but most of the residual peptide remained attached to the glycan. No hydrolysis of the glycan occurred. The lytic spectrum of the enzyme toward a variety of other cell walls of known peptidoglycan composition indicated relatively high specificity for peptidoglycans with polyglycine bridges.

Aminohydrolases↗

PubMed1970

Extracellular cell wall lytic enzyme from Staphylococcus aureus: purification and partial characterization.

An autolysin obtained from culture fluid of Staphylococcus aureus strain 8507 was purified 3,000-fold. One milligram of this preparation (S-5DL) will solubilize 12 mg of cell wall in 1 hr. The major activity is N-acetylmuramyl-l-alanine amidase. Recovery of lytic activity in the purified preparation was repeatably only 20% of the starting level. This suggests that other cell wall lytic enzymes may be present in the starting material. The S-5DL enzyme has been compared to freeze-thaw extracted enzyme (AFZ). Both enzymes precipitate in 0.01 m KPO(4) (pH 6.0) and dissolve in 0.1 to 0.7 m NaCl. Fifty per cent of the AFZ activity and 66% of the S-5DL activity bind rapidly to cell walls of S. aureus at 0 C in the presence of magnesium ion. None of the AFZ activity and 66% of the S-5DL activity bind to cell walls at 0 C in the absence of magnesium ion. The cell walls of nine different strains of S. aureus were compared for level of native autolysin activity. These same walls after inactivation of the native autolysin were tested for susceptibility to the S-5DL enzyme.

Amidohydrolases↗

PubMed1972

New centrifugation technique for isolating enzymes from large cell structures: isolation and characterization of two Bacillus subtilis autolysins.

Bacillus subtilis cell walls can be centrifuged through a linear gradient of 0 to 2 m LiCl and 10 to 25% sucrose so that different autolysins are removed by different salt concentrations and banded in separate positions as the walls pass through the gradient. Using this technique we have found that B. subtilis cell walls are isolated with two autolytic enzymes attached. One autolysin, a glycosidase, can be eluted from walls with 0.5 m LiCl, has a pH optimum between 5 and 8, is relatively heat-sensitive, and has a molecular weight of 60,000. The other autolysin, an alanine amidase, can be eluted from walls with 1.5 m LiCl, has a pH optimum around 8, is relatively heat-stable, has a molecular weight of 35,000, and is present in quantities ten times greater than the glycosidase.

Alanine↗