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Volume 136,
Issue 5,
1990
Volume 136, Issue 5, 1990
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Siderophore-independent acquisition of transferrin-bound iron by Haemophilus influenzae type b
More LessThe specificity by which Haemophilus species acquired iron from transferrin (TF) was investigated. In a plate bioassay H. influenzae used iron bound to human, bovine and rabbit TFs but not mouse, rat, dog, horse, guinea-pig, pig or ovo- TFs or human and bovine lactoferrins. In contrast, H. pleuropneumoniae used iron only from pig TF whilst H. parainfluenza was unable to utilize iron bound to any of the human or animal TFs tested. The inhibition of growth imposed on H. influenzae type b strain Eagan by the addition of the synthetic iron chelator EDDA to the culture medium was reversed by 30% iron-saturated human TF added directly to the medium but not when the TF was contained inside a dialysis bag. Dot-blotting of whole cells revealed that human TF bound to the surface of bacteria cultured in iron-restricted but not in iron-plentiful media. Incubation of whole bacterial cells in the presence of the proteolytic enzyme trypsin also abolished TF-binding activity, suggesting that the TF receptor was a protein. In competition dot blotting experiments, human and bovine but not rabbit, dog, mouse or guinea-pig TFs blocked the binding of a horseradish peroxidase-human TF conjugate. SDS-PAGE and Western blotting of outer membranes revealed the presence of a TF-binding protein of approximately 72 kDa. These results suggest that the acquisition of TF-bound iron by H. influenzae type b probably involves a direct interaction with an outer-membrane protein which shows some TF-species specificity.
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Cobalamin-dependent 1,2-propanediol utilization by Salmonella typhimurium
More LessThe enteric bacterium Salmonella typhimurium utilizes 1,2-propanediol as a sole carbon and energy source during aerobic growth, but only when the cells are also provided with cobalamin as a nutritional supplement. This metabolism is mediated by the cobalamin-dependent propanediol dehydratase enzyme pathway. Thirty-three insertion mutants were isolated that lacked the ability to utilize propanediol, but retained the ability to degrade propionate. This phenotype is consistent with specific blocks in one or more steps of the propanediol dehydratase pathway. Enzyme assays confirmed that propanediol dehydratase activity was absent in some of the mutants. Thus, the affected genes were designated pdu (for defects in propanediol utilization). Seventeen mutants carried pdu:: lac operon fusions, and these fusions were induced by propanediol in the culture medium. All of the pdu mutations were located in a single region (41 map units) on the S. typhimurium chromosome between the his (histidine biosynthesis) and branch I cob (cobalamin biosynthesis) operons. They were shown to be P22-cotransducible with a branch I cob marker at a mean frequency of 12%. Mutants that carried deletions of the genetic material between his and cob also failed to utilize propanediol as a sole carbon source. Based upon the formation of duplications and deletions between different pairs of his:: Mu dA insertions, the pdu genes were transcribed in a clockwise direction relative to the S. typhimurium genetic map.
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Activation of Ca2+influx by metabolic substrates in Saccharomyces cerevisiae: role of membrane potential and cellular ATP levels
More LessInflux of Ca2+into cells of Saccharomyces cerevisiae was measured under non-steady-state conditions, which enable measurements of the initial rate of transport across plasma membranes without interference by the vacuolar Ca2+transport system. Removal of glucose from the incubation medium led to inactivation of Ca2+influx within 5 min. Readdition of glucose led to a transient increase in the rate of Ca2+transport, reaching a peak after 3–5 min. A second increase was observed 60–80 min later. To examine whether the first transient activation of Ca2+influx by glucose was mediated by membrane hyperpolarization, influx of 45Ca2+was measured in the presence and absence of metabolic substrates (glucose, glycerol, and glucose plus antimycin A) in cells hyperpolarized to different values of membrane potential (∆ψ). Logarithms of the rate of Ca2+influx were plotted against values of ∆ψ. Two different slopes were obtained, depending upon whether the metabolic substrate was present or absent. Ca2+influx in the presence of the metabolic substrates was always higher than expected by their effect on ∆ψ. Glycerol plus antimycin A did not affect Ca2+influx. It was concluded that metabolized substrates activate Ca2+influx not only by effects on ∆ψ but also by additional mechanism(s). Since no simple correlation between Ca2+influx and intracellular ATP levels was observed, it was concluded that ATP levels do not affect the initial rates of Ca2+transport across the plasma membrane of S. cerevisiae.
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Misregulation of maltose uptake in a glucose repression defective mutant of Saccharomyces cerevisiae leads to glucose poisoning
More LessIn hex2 mutants of Saccharomyces cerevisiae, which are defective in glucose repression of several enzymes, growth is inhibited if maltose is present in the medium. After adding [14C]maltose to cultures growing with ethanol, maltose metabolism was followed in both hex2 mutant and wild-type cells. The amount of radioactivity incorporated was much higher in hex2 than in wild-type cells. Most of the radioactivity in hex2 cells was located in the low molecular mass fraction. Pulse-chase experiments showed that 2 h after addition of maltose, hex2 cells hydrolysed maltose to glucose, which was partially excreted into the medium. 31P-NMR studies gave evidence that turnover of sugar phosphates was completely abolished in hex2 cells after 2 h incubation with maltose. 13C-NMR spectra confirmed these results: unlike those for the wild-type, no resonances corresponding to fermentation products (ethanol, glycerol) were found for hex2 cells, whereas there were resonances corresponding to glucose. Although maltose is taken up by proton symport, the internal pH in the hex2 mutant did not change markedly during the 5 h after adding maltose. The intracellular accumulation of glucose seems to explain the inhibition of growth by maltose, probably by means of osmotic damage and/or unspecific O-glycosylation of proteins. Neither maltose permease nor maltase was over-expressed, and so these enzymes were not the cause of glucose accumulation. Hence, the coordination of maltose uptake, hydrolysis to glucose and glycolysis of glucose is not regulated simply by the specific activity of the catabolic enzymes involved. The results indicate that there is an unknown regulatory mechanism, under control of HEX2, which coordinates glycolytic flux and maltose uptake. Furthermore, the excretion of accumulated glucose into the medium gives clear evidence that at least one glucose carrier in S. cerevisiae acts passively and transports glucose in both directions.
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Growth-rate-dependent synthesis of K99 fimbrial subunits is regulated at the level of transcription
Increase in the production of the fimbrial adhesin K99 by enterotoxigenic Escherichia coli in continuous cultures at specific growth rates above 0·25 h−1was shown to be independent of the nature of the growth-limiting nutrient. The correlation between specific growth rate and K99 production was also found to be independent of the copy number of the K99 operon. Introduction of additional copies of the K99 regulatory region did not affect growth-ratedependent K99 production in wild-type strains, indicating that no hypothetical regulatory host factor is titrated by the K99 regulatory region. Regulation at the transcriptional level was measured with galactokinase gene fusions. The transcription of the fimbrial subunit gene increased with an increase in specific growth rate. This growth-ratedependent transcription was found to originate from the strong promoter PA. Transcription originating from the weaker promoter PB was independent of growth rate. The results indicated that transcriptional regulation at PA is involved in the growth-rate-dependent regulation of K99 production.
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A grpE mutant of Escherichia coli is more resistant to heat than the wild-type
More LessThe grpE gene of Escherichia coli is essential for bacteriophage λ DNA replication and is also necessary for host RNA and DNA synthesis at high temperature. A grpE mutant of E. coli was found to be substantially more resistant to 50°C heat treatment than the wild-type. Upon receiving a 42°C heat shock for 15 min, both the wild-type and the grpE mutant became more resistant to heat (i.e. they became thermotolerant). A grpE + revertant behaved similarly to the wild-type in that it was more sensitive to heat than grpE cells. In addition, grpE cells had the same H2O2 and UV sensitivity as the wild-type. This implies that the conditions for which a grpE mutation is beneficial are unique to heat exposure and are not caused by H2O2 or UV exposure. Furthermore, synthesis of heat-shock proteins occurred sooner in the grpE mutant than in the wild-type, indicating that the grpE gene of E. coli may influence the regulation of the heat-shock response.
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Dictyostelium discoideum spore germination: increases in proteinase activity are not directly coupled to the emergence of myxamoebae
More LessThe accumulation of proteinase activity during the germination of Dictyostelium discoideum spores was examined under conditions in which the timing of germination events was varied. Spores had a single major proteinase, the aspartic proteinase ddAP58 (proteinase E), while the extracellular matrix which surrounds the spores in the fruiting body contained a number of lower-M r cysteine proteinases, the most active being ddCP18. Very little peptide-nitroanilide-hydrolysing activity was detectable in spores, although some was associated with the matrix. During spore germination there were large increases in activity towards N-carbobenzoxy-l-tyrosyl-l-lysyl-l-arginine 4-nitroanilide and N-benzoyl-l-prolyl-l-phenylalanyl-l-arginine 4-nitroanilide. The enzymes responsible for these activities (referred to as ZYKRase and BzPFRase respectively) were not identical as BzPFRase was much more sensitive to the cysteine proteinase inhibitor E-64 than was ZYKRase. When spores were heatactivated, the increases in activity coincided with the emergence of myxamoebae and the appearance of cysteine proteinases detected using electrophoresis in gelatin gels. For autoactivated spores, emergence was delayed by 0·5 to 1 h and proteinase accumulation lagged slightly behind emergence. If the spores were activated with DMSO, or if heat-activated spores were treated with sucrose after swelling, proteinase accumulation proceeded more rapidly than emergence. Thus temporal control of the accumulation of proteinases during germination varies according to the conditions used. In heat-activated spores, the timing of the increase was similar to that observed previously for β-glucosidase and trehalase, but the temporal controls were not the same as those for the latter enzymes when the other activation conditions were used. The results show that proteinase accumulation is not directly coupled to emergence but could be more closely linked to the late swelling stage which precedes emergence.
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Purification and characterization of d-2-haloacid dehalogenase from Pseudomonas putida strain AJ1/23
More LessA d-2-haloacid dehalogenase was isolated and purified to homogeneity from Pseudomonas putida strain AJ1/23. The enzyme catalysed the stereospecific dehalogenation of the d-isomer of 2-chloropropionate. Using a new ionchromatograph assay, the enzyme was found to catalyse the dehalogenation of short-chain 2-halocarboxylic acids. Maximum enzyme activity occurred at pH 9·5 and 50 °C and the enzyme was insensitive to most -SH reagents. The enzyme has an M r of about 135000 and appears to be composed of four subunits of identical M r.
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Adhesive proteins of haemagglutinating Staphylococcus aureus isolated from bovine mastitis
More LessTwo proteins derived from the cell wall of Staphylococcus aureus, exhibiting apparent molecular masses of 116 kDa and 145 kDa, were found to bind to human buccal and bovine lactiferous sinus epithelial cells. By using antibodies specific for fibronectin-binding protein of S. aureus of human origin, the 116 kDa protein, but not the 145 kDa protein, was identified as a fibronectin-binding protein. The 145 kDa protein bound to bovine fat globule membranes, human buccal epithelial cells, bovine lactiferous sinus epithelial cells and sheep erythrocytes. The properties of the 145 kDa protein suggest that it is an adhesin with a possible role in the early stages of the development of bovine mastitis.
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Germination of Dictyostelium discoideum spores: inhibition of the autoactivation pathway by vanadate
More LessAddition of vanadate to spores of Dictyostelium discoideum engaged in the process of autoactivation resulted in an inhibition of germination which was dependent on the time of addition of the compound. If vanadate was added directly after the washing of dormant spores, then few spores swelled or released myxamoebae. Autoactivator was not secreted from vanadate-inhibited spores. Addition of vanadate to spores that had begun to autoactivate in moist fruiting bodies did not completely inhibit germination. Spores blocked from autoactivation by vanadate responded to the presence of exogenous autoactivator by germinating, but the resulting myxamoebae contained prominent vesicles. Heat shock also overcame vanadate-induced inhibition. This inhibition of autoactivation by vanadate is similar to that imposed on spores by the natural autoinhibitor: thus (1) both compounds block the initiation of the autoactivation cascade and spores do not swell; (2) autoactivator does not transiently accumulate in the culture broths under these conditions; and (3) addition of exogenous autoactivator overrides the inhibition caused by these two compounds. It is hypothesized that vanadate blocks the synthesis/secretion of autoactivator by interfering with an early step in the membrane traffic involved in spore autophagy. Exogenous autoactivator appeared in the supernatants when autoactivating spores swelled but decreased just after the appearance of nascent myxamoebae. It was shown that the autoactivator is not cAMP or cGMP and that it is stable to treatment with beef heart cyclic nucleotide phosphodiesterase. Nevertheless, myxamoebae from autoactivating spores precociously began aggregation within 30 min of plating on non-nutrient agar.
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Chemotactic auto-aggregation in the water mould Achlya
More LessFree-swimming zoospores of several isolates of the oomycetous water mould Achlya demonstrate mutual attraction (adelphotaxis) which may result in auto-aggregation. In the apparent absence of exogenous chemotactic signals macroscopic globular aggregates or microscopic plaques of cysts are formed. Auto-aggregation was favoured by high concentrations of zoospores in suspension; the growth rate of spontaneous aggregates was estimated at 4–10 × 103spores min−1; growth of an aggregate was limited to about 7 min. By means of chemotactic assays, attractant activity for zoospores was detected in the vicinity of aggregates, and in zoospore or cyst supernatants. The attractant activity extracted from spore aggregates was heat-stable. In the absence of exogenous nutrients, the macroscopic aggregates of an homothallic isolate produced sporangia and oogonia. Aggregation was readily initiated as a result of zoospore chemotaxis towards capillary tubes containing exogenous attractant in an agar medium. Aggregating zoospores frequently assumed a slender swarm configuration moving directly towards the aggregate at the capillary-tube tip. Analysis of video recordings showed that many zoospores joining the swarm were attracted directly by the swarm rather than by the exogenous attractant. Such aggregation was a time-limited process, the duration of which was extended by the presence of amino acids in the zoospore suspension. Under natural conditions zoospore aggregation may be a dual-component chemotactic process triggered by exogenous attractants and amplified by chemical signals from the aggregating spores. The possible consequences of adelphotaxis and auto-aggregate formation, namely enhanced inoculum potential, syngamy and spore production, are discussed.
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Antigens in culture supernatant of Mycobacterium tuberculosis: epitopes defined by monoclonal and human antibodies
More LessAntigens of Mycobacterium tuberculosi found in the supernatant of heat-treated cultures were characterized in order to explore whether antigens from this source could be used for the development of a serological test. Culture supernatants and sonicates of 12, 25 and 39 d cultures were analysed by SDS-PAGE. In culture supernatant, major protein bands of 65, 24, and 12 kDa were visible after Coomassie brilliant blue staining. Using murine monoclonal antibodies in Western blots, a pattern of protein bands distinct from that of the corresponding M. tuberculosis sonicates was found in all the culture supernatants. Gel permeation chromatography, in the presence of SDS, was used to separate the major protein bands in the culture supernatant. In ELISA, sera from 20 of 26 patients with tuberculosis reacted with fractions containing mainly 24 kDa or 12 kDa proteins, whereas none of the control sera reacted. In Western blots, each patient serum had its own characteristic banding pattern with culture supernatant, but all the sera from tuberculosis patients and control subjects reacted with protein bands of 65, 61, 58, 30 and 24 kDa. The 12 kDa protein was recognized only by sera from patients with tuberculosis in both Western blots and ELISA. This suggests that different kinds of epitopes on proteins of M. tuberculosis are detected by human antibodies in Western blots and ELISA. We assume that epitopes recognized in Western blots by patients with tuberculosis and control subjects are ubiquitous and are also present on normal commensal bacteria. Epitopes recognized by only some patients with tuberculosis in Western blots may be linear and M. tuberculosis specific. Epitopes recognized by tuberculosis patients but by none of the control subjects in ELISA may be conformation related and M. tuberculosis specific. The major protein bands found in supernatants of heat-treated cultures, 24 and 12 kDa, possess epitopes that may be M. tuberculosis specific and are potentially valuable for the development of a serological test.
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Comparison of two glucoamylases from Hormoconis resinae
More LessTwo extracellular glucoamylases (EC 3.2.1.3), glucoamylase P and glucoamylase S, were purified to homogeneity from the culture medium of Hormoconis resinae (ATCC 20495; formerly Cladosporium resinae) by a new method. Their apparent molecular masses (71 kDa glucoamylase P; 78 kDa glucoamylase S) and catalytic properties agreed well with those previously reported in the literature. Heat inactivation studies suggested that the high debranching (1,6-glycosidic) activity of glucoamylase P preparations (measured with pullulan) may reside in the same protein molecule as its 1,4-glycosidic activity (measured with soluble starch). Although glucoamylase S had virtually no debranching activity, it cross-reacted with polyclonal antibodies raised against glucoamylase P, and the two enzymes had very similar amino acid compositions. However, peptide mapping and amino-terminal sequencing studies of the peptides showed that the two enzymes have different sequences and must be encoded by different genes.
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Cloning and expression of an α-amylase gene from Streptomyces thermoviolaceus CUB74 in Escherichia coli JM107 and S. lividans TK24
More LessA gene coding for a thermostable extracellular α-amylase, carried by a 5·7 kb BamHI chromosomal DNA fragment isolated from Streptomyces thermoviolaceus strain CUB74, was cloned into Escherichia coli JM107 using, as a cloning vector, the high-copy-number plasmid pUC8. E. coli containing a recombinant plasmid pQR300 expressed the amylase gene and exported the enzyme into the periplasmic space and the culture medium. The amylase protein expressed by E. coli had the same molecular mass (50 kDa) as that expressed by the Streptomyces parent strain, which suggests that the enzyme is processed similarly by both strains. The amylase gene was also cloned into Streptomyces lividans TK24 using pIJ702 as vector. The enzyme was stable at 70°C when CaCl2 was present.
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Immunolabelling of SR protein and serotype polysaccharide in Streptococcus mutatans
More LessImmunoelectron microscopy was used to determine the accessibility of gold-labelled monoclonal antibodies, specific for the SR protein and serotype polysaccharide, to the surface of viable cells of Streptococcus mutatis. The results indicate that both antigens are simultaneously accessible to their respective antibodies.
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Characterization of the Renibacterium salmoninarum haemagglutinin
More LessWater-extracted proteins from nine geographically diverse strains of Renibacterium salmoninarum, all of which agglutinated rabbit erythrocytes and rainbow trout spermatozoa, were compared by SDS-PAGE. Extracts from eight strains, including the type strain, ATCC 33209, were similar, containing a major protein of 57 kDa and a minor protein of 58 kDa. The SDS-PAGE protein profile of the Char strain did not contain the 58 kDa protein. A non-agglutinating strain, MT-239, which was also non-hydrophobic, did not produce any water-extractable protein. Immunoblot reactions with rabbit antiserum prepared against whole cells of the type strain demonstrated that the water-extracted haemagglutinins from the various strains were antigenically related. When purified by polyacrylamide gel zone electrophoresis, the haemagglutinin from R. salmoninarum ATCC 33209 formed a doublet band with molecular masses of 57 and 58 kDa, similar to the previously described F antigen. The water-extracted haemagglutinin agglutinated salmonid spermatozoa, was degraded by protease K and trypsin, and was shown to self-assemble onto the cell surface.
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Differential secretion of Dictyostelium discoideum proteinases
More LessProteinases secreted by Dictyostelium discoideum myxamoebae during growth and starvation were analysed using three arginine-containing peptide nitroanilides as substrates. The enzymes were secreted differentially, both during growth and after resuspension of the myxamoebae in phosphate buffer. Secretion was stimulated by the addition of sucrose to starvation buffer, but this did not affect the type of proteinase released. Activity towards N-benzoyl-l-prolyl-l-phenylalanyl-l-arginine 4-nitroanilide (BzPFRase activity) was released in large quantities, and over 80% of the total activity in starved suspensions was extracellular within 6 h of transfer to buffer containing sucrose. A smaller fraction of the total activity towards N-carbobenzoxy-l-arginyl-l-arginine 4-nitroanilide (ZRRase activity) was secreted. Extracellular activity towards N-carbobenzoxy-l-tyrosyl-l-lysyl-l-arginine 4-nitroanilide (ZYKRase) was either too low to be detected or present in quantities which were small enough to be accounted for by cell lysis. BzPFRase and ZRRase activities were both inactivated by E-64 and two peptidyldiazomethanes, specific inhibitors of cysteine proteinases. ZYKRase activity was also inhibited by peptidyldiazomethanes but E-64 was effective only at high concentrations. When electrophoresis in polyacrylamide gels containing gelatin (gelatin-SDS-PAGE) was used to analyse the proteinases involved, one cysteine proteinase, ddCP42, correlated exactly with the secreted BzPFRase activity under all conditions, although contributions to this activity from other proteinases are also likely. Overall, the results demonstrate significant differences between individual proteinases with respect to secretion which suggests that their physiological roles may differ.
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Characteristics of immunosuppressive macrophages induced in spleen cells by Mycobacterium avium complex infections in mice
More LessThe profile of generation and characteristics of splenic macrophages (MΦs) which suppress the concanavalin A (Con A) mitogenic response of splenic T cells (designated as ‘immunosuppressive MΦ’) in host CBA/JN mice during the course of Mycobacterium avium complex (MAC) infection were investigated. In MAC-infected mice, reductions in some cellular functions of host splenic T cells, such as the Con A mitogenic response and mixed leucocyte reaction, were seen around 2 weeks after challenge of organisms, and this was accompanied by appearance of immunosuppressive MΦs in spleen cells. In this case, increase in immunosuppressive MΦ activity was seen in terms of both activity per spleen and activity per individual MΦ. In this phase of the infection, MAC-induced splenic MΦs showed a markedly increased ability to produce reactive oxygen radicals in response to phorbol myristate acetate. Thus, the expression of suppressor activity of MAC-induced MΦs seems to be closely linked to their activated state. A large proportion of the immunosuppressive MΦs exhibited suppressor activity dependent on prostaglandins and membrane functions related to microfilaments. It was also found that the generation of IL-2-reactive T cell populations in response to Con A was markedly inhibited by MAC-induced splenic MΦs, whereas they caused no significant reduction in the IL-2-producing ability of normal spleen cells.
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Conjugative plasmid transfer from Escherichia coli to Clostridium acetobutylicum
More LessThe conjugation mechanism of IncP plasmids may be employed to mobilize small non-conjugative plasmids from Escherichia coli to a wide range of different organisms. This strategy has been adapted for use with the Grampositive anaerobe, Clostridium acetobutylicum NCIB 8052. Several shuttle vectors containing replicons from pAMβ1 (Enterococcus faecalis), pCB101 (Clostridium butyricum) or pWV01 (Streptococcus cremoris), together with the cis-acting oriT region of RK2, have been constructed, and transferred to and established in this organism. One of the vectors apparently contains a hot-spot for insertion of IS1. Conjugative mobilization of plasmids from E. coli will provide a useful alternative to electroporation for effecting gene transfer to this industrially important anaerobe.
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Use of a novel cassette to label phenotypically a cryptic plasmid of Bacillus subtilis and map loci involved in its stable maintenance
More LessIn order to facilitate studies on the maintenance of cryptic plasmids from Gram-positive bacteria we have constructed a novel cassette cAPG1000 (5·0kb) which carries both a selectable marker (chloramphenicol resistance from Staphylococcus aureus plasmid pC194) and a screenable marker (the xylE gene from the TOL plasmid of Pseudomonas putida expressed from a cloned promoter of Bacillus phage SPO2) and which is flanked by terminators to prevent transcription from the cassette activating or inhibiting loci adjacent to the site of insertion. To demonstrate the usefulness of this cassette we have mapped loci required for stable maintenance of an 8·6 kb cryptic plasmid endogenous to Bacillus subtilis (pPOD2000) from the properties of cAPG1000 insertion and insertion/deletion derivatives. We have identified the replication region as well as separate regions required for segregational and structural stability. The segregational mechanism is very efficient since it allows no detectable loss despite the fact that bacteria carrying the plasmid have a greatly increased mean generation time.
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