Nucleotide sequence of the ethidium efflux gene from Escherichia coli

The nucleotide sequence of the gene specifying the ethidium efflux system of Escherichia coli has been determined. The translated open reading frame has identified a membrane-bound polypeptide of 110 amino acids (11,960 Da) which shares 42% identity with a staphylococcal protein specifying resistance to ethidium.     

Tail-associated structural protein gp61 of Staphylococcus aureus phage φMR11 has bifunctional lytic activity

A tailed bacteriophage, φMR11 (siphovirus), was selected as a candidate therapeutic phage against Staphylococcus aureus infections. Gene 61, one of the 67 ORFs identified, is located in the morphogenic module. The gene product (gp61) has lytic domains homologous to CHAP (corresponding to an amidase function) at its N-terminus and lysozyme subfamily 2 (LYZ2) at its C-terminus. Each domain of gp61 was purified as a recombinant protein. Both the amidase [amino acids (aa) 1–150] and the lysozyme (aa 401–624) domains but not the linker domain (aa 151–400) caused efficient lysis of S . aureus . Immunoelectron microscopy localized gp61 to the tail tip of the φMR11 phage. These data strongly suggest that gp61 is a tail-associated lytic factor involved in local cell-wall degradation, allowing the subsequent injection of φMR11 DNA into the host cytoplasm. Staphylococcus aureus lysogenized with φMR11 was also lysed by both proteins. Staphylococcus aureus strains on which φMR11 phage can only produce spots but not plaques were also lysed by each protein, indicating that gp61 may be involved in 'lysis from without'. This is the first report of the presence of a tail-associated virion protein that acts as a lysin, in an S. aureus phage.     

Land surface phenology as an indicator of biodiversity patterns

With the rapid decline in biodiversity worldwide it is imperative to develop procedures for assessing changes in biodiversity across space. The synoptic view provided by imaging remote sensors constitutes a suitable approach for analyzing biodiversity from local to regional scales. A procedure based on the close relationship between floristic similarity and the similarity in land surface phenology was recently developed and successfully applied to assess diversity patterns using time series imagery acquired by the Moderate Resolution Imaging Spectro-radiometer (MODIS). However, as it depends on high temporal resolution remotely sensed data (e.g., MODIS), the procedure is constrained by the coarse spatial resolution characterizing these high temporal resolution data. Using an optimized technique for image fusion, we combined high temporal resolution data acquired by the MODIS sensor system with moderate spatial resolution data acquired by the Landsat TM/ETM+ sensor systems. Our results show that the MODIS/Landsat data fusion allows the characterization of land surface phenology at higher spatial resolutions, which better corresponded with information acquired within vegetation survey plots established in temperate montane forests located in Wolong Nature Reserve, Sichuan Province, China. As such, the procedure is useful for capturing changes in biodiversity induced by disturbances operating at large spatial scales and constitutes a suitable tool for monitoring and managing biodiversity.     

Isolation and characterization of Zymomonas mobilis mutants resistant to octadecyltrimethylammonium chloride,a detergent acting on hopanoid-producing bacteria

Growth of the hopanoid-producing bacterium Zymomonas mobilis was inhibited at low concentrations of the cationic detergent octadecyltrimethylammoniumchloride (OTAC). A relationship between sensitivity of Zymomonas mobilis to OTAC, presence of hopanoids and ethanol tolerance was postulated. Mutants resistant to OTAC were isolated from strains ZM1 and ZM4. They did not present any alteration of the hopanoid content and their squalene cyclases showed the same sensitity to OTAC as the parent enzymes. Resistance to OTAC paralleled pleiotropic effects including, enhanced accessibility of the membrane-bound alkaline phosphatase, important release of proteins from cells by Tris/HCl treatment, increased resistance to antibiotics and increased sensitivity to ethanol. In addition, OTAC^R mutants were also characterized by the synthesis or the overproduction of an outer membrane protein (F53) not detected on 2D-PAGE maps of parent strains and by a normal heat shock response. The role of hopanoids, heat shock proteins, protein F53 and membrane organization in ethanol tolerance is discussed.Abbreviations OTAC octadecyltrimethylammoniumchloride - SLS sodium lauryl sarcosinate     

Biological characterization of the lytic cycle of actinophage φA7 in Streptomyces antibioticus

Some basic parameters of the lytic development of phage phi A7 in Streptomyces antibioticus are described. One-step growth experiments demonstrated that at 28 degrees C phi A7 has a latent period of about 60 min and an exponential growth period of about 35 min. The average burst size ranged from 70-100 plaque forming units per infected cell. At the same temperature 50% of the virions were adsorbed to germ tubes of S. antibioticus in about 10 min. This corresponds to an adsorption constant of 6.5 x 10(-10) ml/min. The phage was unable to adsorb the host at other stages of the life cycle (spores or mycelium). Divalent cations are not required for phi A7 stability but Ca2+ proved to be essential for adsorption and also for a later stage of the vegetative development of the phage.     

Turnover of cell surface-bound capsular polysaccharide in Staphylococcus aureus

The capsular polysaccharide (CPS) of Staphylococcus aureus strain Smith was labelled by growth of bacteria in the presence of radioactive N-acetylglucosamine and was separated from labelled cell wall components by affinity chromatography on wheat germ agglutinin following dissolution of the cells by lysostaphin. The products were partially characterised chemically and immunochemically. Similar labelled components were found in the culture fluid during growth. In a pulse-chase experiment, cell-bound CPS was released continuously into the culture fluid at the same rate as cell wall turnover and there was no evidence of direct excretion of CPS.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Vitronectin and type-I collagen binding by Staphylococcus aureus and coagulase-negative staphylococci

Abstract Binding of ¹²⁵I-labelled type-I collagen and ¹²⁵I-labelled vitronectin (human serum spreading factor or S-protein) was studied using Staphylococcus aureus and coagulase-negative staphylococci of different species. Binding of collagen and vitronectin was time dependent for S. aureus ISP 546, and S. haemolyticus E 2498/86. Co-operative binding of vitronectin and collagen by staphylococcal cells was demonstrated. Binding to S. haemolyticus E 2498/86 was more rapid and was enhanced in vitronectin/collagen mixtures than for either protein separately. Furthermore, pre-incubation of staphylococcal cells with unlabelled collagen enhanced vitronectin binding. When cells of S. haemolyticus E 2498/86 were treated with pronase E, proteinase K, subtilopeptidase A or trypsin, vitronectin-binding was decreased by 50% or more, whereas collagen-binding was protease resistant. For the strains of S. aureus tested, both vitronectin and collagen binding were found to be protease sensitive. Type-I collagen peptides inhibited collagen-binding to S. haemolyticus E 2498/86, whereas vitronectin-binding was not affected perhaps indicating different receptors for these proteins. The binding of both collagen and vitronectin was shown to be reversible, since bound ¹²⁵I-collagen and ¹²⁵I-vitronectin were displaced after adding excess of the homologous protein.     

Inhibition ofCandida albicans andStaphylococcus aureus by phenolic compounds from the terrestrial cyanobacteriumNostoc muscorum

Phenolic compounds were determined in methanolic extract from the algal mass of aNostoc muscorum culture. Bioassays with two human pathogens,Candida albicans andStaphylococcus aureus indicated that algal phenolic compounds evoked significant growth inhibition for both species (89.1% and 88.2%, respectively). It is suggested that this strong inhibitory effect is of potential medicinal value.