Purification and properties of a lytic enzyme from the cell wall of Chlorella ellipsoidea C-87

Cell wall lytic activity was detected in the culture medium and cell wall of 1AM Chlorella ellipsoidea C-87. The enzymes of both fractions had their highest activity at pH 5. The lytic activity bound to the cell wall consisted of a polysaccharide releasing enzyme, an exo-type enzyme releasing disaccharide, and glucosidase; but only the polysaccharide releasing enzyme was solubilized by lithium chloride. A polysaccharide releasing enzyme with a molecular weight around 40 kDa was isolated from the culture medium. Hemicellulose is degraded by the polysaccharide releasing enzyme, and the rigid wall by the exo-type enzyme.     

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.     

Application of subproteomics in the characterization of Gram-positive bacteria

Gram-positive bacteria cause a series of diseases in human, animals and plants. There has been increasing interest in efforts to investigate pathogenesis of bacteria using multiple "omic" strategies including proteomics. Proteins in different cell fractions of bacteria may play different vital roles in various physiological processes, such as adhesion, invasion, internalization, sensing, respiration, oxidative stress protection and pathogenicity. Subproteomics specifically focuses on the pre-fractionated cellular proteins and thus may be able to characterize more low-abundance molecules that are usually overlooked by the traditional whole-cell proteomics, providing comprehensive information for further investigations. This review intends to outline the current progress, challenges and future development of subproteomics in the characterization of Gram-positive bacteria. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Methods of cell lysis and effect of detergents for the recovery of nitrile metabolizing enzyme from Amycolatopsis sp. IITR215

Nitrile metabolizing enzymes are of great industrial interest for the selective bio-transformation of nitriles and surface modification of synthetic polymers under mild reaction conditions. In the present work, isolated strain Amycolatopsis sp. IITR215 was cultivated in the bench top bioreactor for the recovery of maximum biomass of whole cell catalyst. Effect of different lyoprotectants was studied on nitrile metabolizing enzyme from Amycolatopsis sp. IITR215 in which sorbitol proved to be an efficient lyoprotectant. In physical and mechanical methods, only 30% activity was recovered while 85% activity was achieved in the enzymatic method using 2 g/l lysozyme. Very less activity was recovered during stationary phase when cells were grown in mineral base media containing 1 g/l yeast. Therefore, recovery of intracellular enzymes was enhanced by using different concentrations of sodium cholate and deoxycholate.     

重庆地区儿童2000年至2004年首位革兰阴性细菌和阳性细菌的耐药性监测

目的了解重庆地区儿童感染的分离至临床标本的首位革兰阴性细菌和阳性细菌对常用抗生素的耐药趋势,指导临床合理使用抗生素。方法常规方法分离、培养细菌,应用美国德灵公司WalkAway-40细菌鉴定仪对2000年至2004年我院细菌室分离至临床标本的首位革兰阴性细菌和阳性细菌共2854株进行细菌鉴定及药敏试验。结果2000年至2004年检出的首位革兰阴性细菌和阳性细菌分别为大肠埃希菌和金黄色葡萄球菌。2000年至2004年前5位革兰阴性菌5777株,革兰阳性菌1565株,其中大肠埃希菌2090株,金黄色葡萄球菌764株,分别占36.2%和48.8%;5年间大肠埃希菌对氨苄西林、头孢吡肟、头孢西丁、庆大霉素、亚胺培南、环丙沙星、头孢噻肟、头孢他啶的总耐药率分别为80.9%、37.5%、15.4%、54.0%、0.8%、34.0%、46.6%、46.2%;金黄色葡萄球菌对青霉素、红霉素、复方新诺明、万古霉素、阿莫西林/克拉维酸的总耐药率分别为95.6%、63.4%、5.8%、0%、11.0%。结论通过细菌耐药监测发现：大肠埃希菌对常用抗生素的总耐药率变化不大,金黄色葡萄球菌对常用抗生素的总耐药率有下降趋势,应引起临床医生重视。     

Expression of bacteriophage PhiX174 lysis gene E in Staphylococcus carnosus TM300

Abstract Expression of the cloned PhiX174 gene E causes lysis of the Gram-negative bacterium Escherichia coli , which led to the proposal that a two-membrane system is necessary for the protein E lysis function. Gene E was cloned in an E. coli/Bacillus subtilis shuttle vector and expressed in the Gram-positive bacterium Staphylococcus carnosus TM300. Regulated gene E expression had a lethal effect on S. carnosus ; however, no lysis was detected, lending support to the hypothesis.     

Fungus fruit body lytic enzyme from a myxomycete,Badhamia utricularis

Chitinase,β-1,3-glucanase, cellulase, xylanase and protease activity were detected in a crude enzyme preparation obtained from a slime mold (Badhamia utricularis) which was grown on autoclaved mycelia ofPholiota nameko in a petri dish. The optimal pH of the enzyme preparation for lytic activity against fruit bodies ofLentinus edodes was 4.0, and those ofβ-1,3-glucanase and cellulase were the same. On the other hand, chitinase and protease showed optimal activity at pH 5.0 and 8.0, respectively. The lytic activity was stable below 40°C but completely inactivated at 70°C, and was most stable at pH 5.0. The studies of the optimal pH, thermal stability, and pH stability, and isoelectric focusing analysis of the enzyme preparation suggest that chitinase,β-1,3-glucanase and cellulase activities may be responsible for lysis of fruit bodies of some mushrooms. The crude enzyme preparation from the slime mold lysed fruit bodies of several mushrooms more efficiently than did commercial lytic enzymes preparations (Driselase and Usukizyme).     

Production and properties of a cellobiose-oxidizing enzyme from a newly isolatedcellulolytic bacterium Cytophaga sp. LX-7

A cell-bound cellobiose-oxidizing enzyme was produced by cellulolytic Cytophaga sp. LX-7. It was found that both the cellulosic substrates and the soluble carbohydrate substrates tested promoted the production of the cellobiose-oxidizing enzyme, and the highest specific activities were obtained with cellulose powder MN300, carboxy- methylcellulose CM22, maltose and cellobiose. Among the nitrogen sources examined, peptone gave the best cellobiose-oxidizing enzyme production, whereas inorganic nitrogen sources gave very poor growth. The medium buffered with Tris/HCl, pH 7.1, yielded the highest levels of cellobiose-oxidizing enzyme activity and the temperature optimum for crude enzyme activity was 40°C.     

An X-ray study of the cytoplasmic membranes of two Gram-positive bacteria

X-ray diffraction diagrams of partially disordered one-dimensional lattices of isolated bacterial cytoplasmic membranes are described and they provide a basis for suggesting possible molecular structures of bacterial membranes.Biochemical and electron microscope evidence points towards a lipid bilayer with a high degree of fluidity. The protein molecules are in a disordered configuration in the membrane.     

Detection of heavy metal ion resistance genes in Gram-positive and Gram-negative bacteria isolated from a lead-contaminated site

Resistance to a range of heavy metal ions wasdetermined for lead-resistant and other bacteria whichhad been isolated from a battery-manufacturing sitecontaminated with high concentrations of lead. Several Gram-positive (belonging to the genera Arthrobacter and Corynebacterium) andGram-negative (Alcaligenes species) isolateswere resistant to lead, mercury, cadmium, cobalt,zinc and copper, although the levels of resistance tothe different metal ions were specific for eachisolate. Polymerase chain reaction, DNA-DNAhybridization and DNA sequencing were used to explorethe nature of genetic systems responsible for themetal resistance in eight of the isolates. SpecificDNA sequences could be amplified from the genomic DNAof all the isolates using primers for sections of themer (mercury resistance determinant on thetransposon Tn501) and pco (copperresistance determinant on the plasmid pRJ1004) geneticsystems. Positive hybridizations with mer andpco probes indicated that the amplified segmentswere highly homologous to these genes. Some of thePCR products were cloned and partially sequenced, andthe regions sequenced were highly homologous to theappropriate regions of the mer and pcodeterminants. These results demonstrate the widedistribution of mercury and copper resistance genes inboth Gram-positive and Gram-negative isolates obtainedfrom this lead-contaminated soil. In contrast, theczc (cobalt, zinc and cadmium resistance) andchr (chromate resistance) genes could not beamplified from DNAs of some isolates, indicating thelimited contribution, if any, of these genetic systemsto the metal ion resistance of these isolates.     

Importance of Gram-positive naphthalene-degrading bacteria in oil-contaminated tropical marine sediments

AIMS: The aim of this study was to isolate, characterize and evaluate the importance of naphthalene-degrading bacterial strains from oil-contaminated tropical marine sediments. METHODS AND RESULTS: Three Gram-positive naphthalene-degrading bacteria were isolated from oil-contaminated tropical intertidal marine sediments by direct isolation or enrichment using naphthalene as the sole source of carbon and energy. Bacillus naphthovorans strain MN-003 can also grow on benzene, toluene, xylene and diesel fuel while Micrococcus sp. str. MN-006 can also grow on benzene. Staphylococcus sp. str. MN-005 can only degrade naphthalene and was not able to use the other aromatic hydrocarbons tested. Strain MN-003 possessed the highest maximal specific growth rate with naphthalene as sole carbon source. An enrichment culture fed with naphthalene as sole carbon source exhibited a significant increase in the relative abundances of the three isolates after 21 days of incubation. The three isolates constituted greater than 69% of the culturable naphthalene-degrading microbial community. Strain MN-003 outcompeted and dominated the other two isolates in competition studies involving batch cultures inoculated with equal cell densities of the three isolates and incubated with between 1 and 10 mg l-1 of naphthalene. CONCLUSIONS: Three Gram-positive naphthalene-degrading bacteria were successfully isolated from oil-contaminated tropical marine sediments. Gram-positive bacteria might play an important role in naphthalene degradation in the highly variable environment of oil-contaminated tropical intertidal marine sediments. Among the three isolates, strain MN-003 has the highest maximal specific growth rate when grown on naphthalene, and outgrew the other two isolates in competition experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: This research will aid in the development of bioremediation schemes for oil-contaminated marine environments. Strain MN-003 could potentially be exploited in such schemes.     

Intrinsic nitric oxide-stimulatory activity of lipoteichoic acids from different Gram-positive bacteria

Lipoteichoic acid (LTA) is a structural component of the cell walls of Gram-positive bacteria. Similar to lipopolysaccharide (LPS) which is expressed in Gram-negative bacteria, LTA exhibits immunostimulatory properties. Frequently observed positive response of LTA in the Limulus amebocyte lysate (LAL) assay has been interpreted as a sign of LPS contamination, raising doubts about the intrinsic immune activities of LTA. Regarding many similarities in immunobiological and physicochemical properties of LTA and LPS, we hypothesized that similar to LPS, the LAL reactivity of LTA might be due to its ability to bind to LAL. Our data confirm the positivity of Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis and Streptococcus pyogenes LTAs in the LAL test. The estimates of suspected LPS content were 605, 10.3, 6.2 and 127 pg/μg LTA, respectively. The effectiveness of LTAs to induce the NO production in rat peritoneal cells was remarkably higher than that of equivalent concentrations of reference LPS (Escherichia coli). The LPS-induced NO was inhibited by polymyxin B (PMX), the IC₅₀ of PMX:LPS concentration ratio (pg:pg) being 1050:1. Many fold higher concentrations of PMX were needed to partially suppress the NO-augmenting effects of LTAs, applied at concentrations representing the equivalents of LPS. Transposed to the concentrations of LTAs per se, the IC₅₀s of the PMX:LTA ratios (μg:μg) ranged from 0.3:1 (S. aureus) to 7.5:1 (B. subtilis). It is concluded that LTA is not necessarily contaminated with LPS. The results prove the intrinsic immunostimulatory properties of LTAs of Gram-positive bacteria. The positive response of LTA in the LAL assay results from its capacity to bind to LAL. In addition, LTA binds with high affinity to PMX.     

Evolution of peptidoglycan biosynthesis under the selective pressure of antibiotics in Gram-positive bacteria

Acquisition of resistance to the two classes of antibiotics therapeutically used against Gram-positive bacteria, the glycopeptides and the beta-lactams, has revealed an unexpected flexibility in the peptidoglycan assembly pathway. Glycopeptides select for diversification of the fifth position of stem pentapeptides because replacement of D-Ala by D-lactate or D-Ser at this position prevents binding of the drugs to peptidoglycan precursors. The substitution is generally well tolerated by the classical D,D-transpeptidases belonging to the penicillin-binding protein family, except by low-affinity enzymes. Total elimination of the fifth residue by a D,D-carboxypeptidase requires a novel cross-linking enzyme able to process the resulting tetrapeptide stems. This enzyme, an L,D-transpeptidase, confers cross-resistance to beta-lactams and glycopeptides. Diversification of the side chain of the precursors, presumably in response to the selective pressure of peptidoglycan endopeptidases, is controlled by aminoacyl transferases of the Fem family that redirect specific aminoacyl-tRNAs from translation to peptidoglycan synthesis. Diversification of the side chains has been accompanied by a parallel divergent evolution of the substrate specificity of the L,D-transpeptidases, in contrast to the D,D-transpeptidases, which display an unexpected broad specificity. This review focuses on the role of antibiotics in selecting or counter-selecting diversification of the structure of peptidoglycan precursors and their mode of polymerization.     

Characterization of Superoxide dismutase genes from Gram-positive bacteria by polymerase chain reaction using degenerate primers

Abstract An internal fragment representing approximately 85% of sod genes from seven Gram-positive bacteria was amplified by using degenerate primers in a polymerase chain reaction assay. The DNA sequences of sod polymerase chain reaction products from Clostridium perfringens, Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae , and Streptococcus pyogenes were determined. Comparisons of their deduced amino acid sequences with those of the corresponding regions of the SOD proteins from Bacillus stearothermophilus, Listeria monocytogenes , and Streptococcus mutans revealed strong relatedness. Phylogenetic analysis of SOD peptides showed that members of the genera Streptococcus and those of the genera Enterococcus constitute two well-supported monophyletic groups. The method described in this study provides a means for easy recovery of sod genes and the construction of sod mutants of various Gram-positive pathogens.     

Quantitative assessment of total and Gram-positive aerobic bacteria in fresh and ambient-temperature-stored sub-tropical marine fish

The current study was undertaken to enumerate Gram-positive bacteria in fresh sub-tropical marine fish and determine the effect of ambient storage (25°C) on the Gram-positive bacterial count. Total and Gram-positive bacteria were enumerated in the muscles, gills and gut of fresh and stored Pseudocaranx dentex, Pagrus auratus and Mugil cephalus on tryptone soya agar (TSA) and TSA with 0.25% phenylethyl alcohol (PEA), respectively. Initial studies indicated that PEA significantly reduced total aerobic bacterial count (TABC) whereas control Gram-positive bacteria were not affected by 0.25% PEA. TABC significantly increased in all fish body parts, whereas Gram-positive aerobic bacterial count (GABC) significantly increased only in the muscles and gills during ambient storage for 15 h. The TABC of the fish species increased from 4.00, 6.13 and 4.58 log cfu g⁻¹, respectively in the muscles, gills, and gut to 6.31, 7.31 and 7.23 log cfu g⁻¹ by the end of storage. GABC increased from 2.00, 3.52 and 2.20 log cfu g⁻¹ to 4.70, 5.85 and 3.36 log cfu g⁻¹. Within each species, TABC were significantly higher in the gills compared to that of muscles and gut; however, no significant differences were found in GABC between muscles and gills. This study demonstrated the potential importance of Gram-positive bacteria in sub-tropical marine fish and their spoilage.     

Design and synthesis of biaryloxazolidinone derivatives containing a rhodanine or thiohydantoin moiety as novel antibacterial agents against Gram-positive bacteria

Novel biaryloxazolidinone derivatives containing a rhodanine or thiohydantoin moiety were designed, synthesized and evaluated for their antibacterial activity. The key compounds 7 and 9 were synthesized by the knoevenagel condensation of intermediate aldehyde 5 with rhodanine derivatives 6a?6b. The preliminary study showed that compounds 7, 9 and 10e exhibited potent antibacterial activity with MIC values of 0.125?µg/mL against S. aureus, MRSA, MSSA, LREF and VRE pathogens, using linezolid and radezolid as the positive controls. The most promising compound 10e exhibited potent antibacterial activity against tested clinical isolates of MRSA, MSSA, VRE and LREF with MIC values in the range of 0.125–0.5?µg/mL, and the potency of 10e against clinical isolates of LREF was 64-fold higher than that of linezolid. Moreover, compound 10e was non-cytotoxic with an IC₅₀ value of 91.04?μM against HepG2 cell. Together, compound 10e might serve as a novel antibacterial agent for further investigation.     

Species-specificity of the lytic activity of the cell wall in the genus Chlorella

Cell wall lytic activity was compared among strains IAM C-27, C-87, SAG 211-1c, -1d, -9a, -8b, -8c, -8l, -11f, -8k, -11g, and -11h/9 of the genus Chlorella . The optimal pH was alkaline in strains with glucosamine as the characteristic group of the rigid wall, and acidic in strains characterised by glucan groups. The lytic enzymes of strains in the former type of algae lyzed the cell wall mainly to soluble high molecular oligosaccharides. The lytic activity of the Chlorella cell wall thus appears species- and strain-speicific.     

裂解系统在细菌载体疫苗中的应用

重组细菌载体疫苗因其能够诱导机体产生粘膜免疫、体液免疫和细胞免疫的特点,已经被广泛用作递送保护性抗原和核酸疫苗的载体来预防某些传染病。但是重组到细菌载体疫苗中的保护性抗原和核酸难以穿越细菌细胞壁释放到宿主细胞内发挥作用,残留在动物或畜禽产品中的疫苗菌株还可能造成环境的污染和疫苗菌株的传播。而有效解决这些问题的方法是构建一种细菌自动裂解系统,使疫苗菌株能够在体外培养时正常生长而在体内环境中自动裂解死亡。目前主要应用的细菌裂解系统包括:基于调控延迟肽聚糖合成的裂解系统、基于噬菌体裂解蛋白调控的裂解系统、基于毒素-抗毒素系统(Toxin-antitoxin system)的裂解系统。此外,一种潜在的基于细菌Ⅵ型分泌系统(Type Ⅵ secretion system,T6SS)的裂解系统也有望成为构建自动裂解菌株的新方法。文中将着重对这几种裂解系统的调控机制进行阐述。     

Distribution of glutathione transferases in Gram-positive bacteria and Archaea

Glutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea. An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum. A comparison of the identified sequences with well-characterized GSTs belonging to both Gram-negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily. This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea.     

Immunological crossreactivity to the catabolite control protein CcpA from Bacillus megaterium is found in many Gram-positive bacteria

Abstract The catabolite control protein CcpA from Bacillus megaterium was overproduced as a fusion protein to a 6xhis affinity tag and purified to homogeneity. Polyclonal antibodies of high affinity and specificity were raised against the purified protein. The serum did not crossreact with purified Lac repressor despite the fact that CcpA and LacI belong to the same protein family. Using this antiserum we identified proteins that share antigenic determinants with CcpA in many Gram-positive bacteria, including bacilli, staphylococci, streptococci, lactic acid bacteria, and some actinomycetes.