Large plasmids in ruminal strains of Selenomonas ruminantium

The plasmid content of six different isolates of Selenomonas ruminantium from the rumen of sheep, cows or goats was examined by electron microscopy. In addition to small plasmids (< 12 kb) studied previously, all six strains contained at least one plasmid larger than 20 kb. Plasmid sizes of 1·4, 2·1, 2·4, 5·0, 6·2, 20·4, 20·8, 22·7, 23·3, 29·3, 30·7, 34·4 and 42·6 kb were estimated from contour length measurements. DNA-DNA hybridization experiments revealed homology among the large plasmids from five strains, while the 20·8 kb plasmid from a sixth isolate showed no apparent relationship with the plasmids of the other strains.     

New plasmidovars of Yersinia pestis isolated in Mongolia]

The plasmid spectres of 122 strains of Yersinia pestis isolated in Mongolia from patients, wild mammals and arthropods were studied. The populations of three plasmidovars of Yersinia pestis were found to be circulating in the natural foci of plague in Mongolia. The first plasmidovar harbours three plasmids with mol masses 6, 47, 65 Md. The second and third plasmidovars contain the plasmids with mol masses 6, 16, 47, 65 Md and 8, 47, 75-80 Md.     

Genetic evidence for plasmid-encoded lactococcin production inLactococcus lactis subsp.lactis 484

Lactococcus lactis subsp.lactis 484 produced a proteinaceous antibacterial substance designated as lactococcin capable of inhibiting members of theLactococcus group,Bacillus cereus, Staphylococcus aureus, andSalmonella typhi. Growth of this culture in the presence of 2–30 g/ml of ethidium bromide or acriflavin or novobiocin, and at elevated temperature (39° and 41°C), could not produce any lactococcin-negative (Lap^–) variants. However, protoplast-induced curing with lysozyme was successful in developing Lap^– derivatives. Two types of cured derivatives, namely Lac^– Lap⁺ and Lac^– Lap^–, were obtained. Lap^– variants were also lacking sucrose-fermenting ability (Suc⁺) and lactococcin resistance (Lap^r). The lactose-negative (Lac^–) variants and Lap⁺ were clearly lacking the largest (65 Md) plasmid. However, Lap^– Suc^– Lap^s variants lost a 2 Md plasmid.L. lactis subsp.lactis 484 transferred lactose-fermenting ability as well as Lap⁺ Suc⁺ Lap^r phenotypes simultaneously toL. lactis subsp.lactis LM 2306 and LM 0230 by surface mating at a frequency of 10^–4 and 10^–1 per donor respectively. However, cured Lac^– Lap^– transconjugants could not transfer Lac⁺ Lap⁺ Suc⁺ Lap^r phenotypes to any of these recipient strains. Our results indicate that Lac⁺ and Lap⁺ Suc⁺ Lap^r phenotypes are associated with 65 Md and 2 Md plasmids respectively. Conjugal transfer of 2 Md plasmid is possible only in the presence of a conjugative 65 Md plasmid.     

Acid resistance of attached organisms and its implications for the pathogenicity of plasmid-bearing Escherichia coli

Organisms of Escherichia coli attached to glass beads in a model attachment system were more resistant to acid than were unattached organisms and this applied to cultures exposed to either pH 2·5 or 3·5. Attached organisms of both Col^- and Col V, I-K94⁺ strains showed the effect but with prolonged exposure to pH 2·5, the attached Col V⁺ organisms appeared more resistant than the attached Col^- ones, possibly because they formed a thicker surface layer. It is proposed that the increased resistance of attached organisms to pH 2·5 might allow the survival, in gastric acid, of organisms attached to food particles. This would be more significant for the Col V⁺ strains because the plasmid enhances enterobacterial attachment properties. The increased resistance of attached organisms to pH 3·5 might be significant for survival of particle- or surface-bound organisms in the acidic environment of the phagocyte especially since lactate (which occurs in phagocytes) enhanced the effect of exposure to pH 3·5.     

Plasmids of Staphylococcus aureus associated with live and processed poultry

A series of 23 strains of Staphylococcus aureus originally isolated from processed poultry was screened for the presence of plasmids. Plasmids were more common in strains of Staph. aureus characteristically associated with live poultry than with strains endemic in poultry plants and strains of human origin. Two plasmids with sizes of 1·65 and 18·2 kilobase pairs (kBp) were present in three strains considered typical of Staph. aureus forma specialis 'altilis' and two plasmids with sizes of 1·65 and 17 kBp were present in three of four strains of Staph. aureus var. gallinae. A 1·65 kBp plasmid was present in all seven strains of these poultry biotypes and in three of 14 'endemic' strains. All the 1·65 kBp plasmids were shown by blot hybridization to share sequence homology. There was also some sequence homology between the 18·2 kBp and 17 kBp plasmids. These results were supported by restriction enzyme digest analyses. A study of cured derivatives of strain PS221 f.sp. 'altilis' suggested that the 18·2 kBp plasmid encoded the genetic determinant(s) responsible for caseolysis. Both the 1·65 and the 18·2 kBp plasmids also exerted an effect on the production of acid from lactose. In no other characteristic did cured strains resemble the plasmid-free 'endemic' strains. This was therefore consistent with the notion that the genetic determinants associated with the cultural characteristics of endemic strains are chromosomally located.     

Transposon mutagenesis, elimination and mobilization of plasmids in nitrogen-fixating bacterium Azospirillum brasilense Sp245

The expressed difference in the plasmid profile of A. brasilense Sp245 is registered as a result of Tn5-Mob-mutability. Integration of the vector pSUP5011 into one of the A. brasilense Sp245 plasmid and using of the Tn5-Mob transposon to mobilize the 85Md cryptic plasmid are reported. The properties of A. brasilense Sp245 with the mutant plasmids composition (surface structure, acetylene and nitrate reduction, ability to a number of carbohydrates utilization, formation of melanin, antibiotics resistance specter) have been analyzed. The transposon Tn5-Mob insertion into the 85Md plasmid resulted in isolation of a mutant excreting a melanin-like pigment into the medium. The results suppose 85Md plasmid participation in melaninogenesis.     

Detection of plasmids in strains of Bacteroides ureolyticus isolated from the genital tract

Plasmids were detected in 17 of 24 clinical isolates of Bacteroides ureolyticus. All 17 strains harboured a single large plasmid of ≥24·5 MD but two also had smaller plasmids of 2·3 and 3·9 MD, respectively. The function of the plasmids is unknown.     

Habituation to acid in Escherichia coli: conditions for habituation and its effects on plasmid transfer

Induction of acid resistance (habituation) in Escherichia coli at pH 5·0 took ca 5 min in broth at 37°C and 30–60 min in minimal medium. Induction occurred at a range of pH values from 4·0 to 6·0; it was dependent on continuing protein and RNA synthesis but substantial acid resistance appeared in the presence of nalidixic acid. Acid resistance was long-lasting; organisms grown at pH 5·0 retained most of their resistance after 2 h growth at pH 7·0. Organisms grown at pH 5·0 showed increased synthesis of a number of cytoplasmic proteins compared with the level in cells grown at pH 7·0. DNA repair-deficient strains carrying recA, uvrA or polAl mutations were more acid-sensitive than the repair-proficient parents but were able to habituate at pH 5·0. Organisms grown at pH 5·0 transferred the ColV plasmid much more effectively at acid pH than did those grown at pH 7·0 and habituated recipients appeared better able to repair incoming acid-damaged plasmid DNA than did those that were non-habituated. Induction of acid resistance at pH 5·0 may be significant for the survival of organisms exposed to periodic discharges of acid effluent in the aquatic environment and habituation may also allow plasmid transfer and repair of acid-damaged plasmid DNA during or after such exposure.     

Purification and characterization of galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum

Galacto-oligosaccharide-producing β-galactosidase from Sirobasidium magnum CBS6803 was purified to homogeneity with a yield of 60% by DEAE–toyopearl, butyl–toyopearl, p -aminobenzyl 1-thio-β- d -galactopyranoside–agarose and concanavalin A–agarose columns, from a solubilized cell wall preparation. The isoelectric point (pI) of purified β-galactosidase was 3·8, and the relative molecular mass was 67 000 as estimated by SDS gel electrophoresis, and 135 000 as estimated by gel filtration. Optimal β-galactosidase activity was observed at a temperature and pH of 65°C and pH 4·5–5·5, respectively. The K _m values for o -nitrophenyl-β- d -galactopyranoside and lactose were 14·3 and 5·5 mmol l⁻¹, respectively, and the V _max values for these substrates were 33·4 and 94·5 μmol min⁻¹ mg of protein⁻¹, respectively. In addition this enzyme possessed a high level of transgalactosylation activity, and 72 mg ml⁻¹ galacto-oligosaccharide was produced from 200 mg ml⁻¹ lactose.     

An efficient transformation system for Bifidobacterium spp.

This study describes a broad host transformation protocol that enables the uptake of plasmid DNA into 10 different species of Bifidobacterium , some of which have never been transformed before. The vector pNC7 (4·9 kb) was used to optimize the electroporation protocol. Transformation efficiencies ranged from 3·6×10⁻¹ to 1·2×10⁵ transformations per μg DNA. The impact of growth medium composition and electric field strength on transformation efficiency were independently optimized. Electrocompetent cells were grown in Iwata medium broth enriched with Actilight^RP 16%, harvested during the early exponential growth phase, and pulsed at 12·5 kV cm⁻¹, 100 Ω and 25 μF.     

Mathematical modelling of the heat resistance of Listeria monocytogenes

The heat resistance of Listeria monocytogenes phagovar 2389/2425/3274/2671/47/108/340 (1992 French outbreak strain) in broth was studied at 55, 60 and 65 °C. Experiments were carried out on bacterial cultures in three different physiological states: cultures at the end of the log phase, cultures heat-shocked at 42 °C for 1 h, and subcultures of cells resistant to prolonged heating. Survivor curves were better fitted using a sigmoidal equation than the classical log-linear model. This approach was justified by the existence of heat resistance distributions within the bacterial populations. Peaks (log₁₀ of heating time) of heat resistance distributions of untreated, heat-shocked, and selected cultures at 55, 60 and 65 °C were 0·34, −0·90 and −1·84 min, 0·74, −0·51 and −1·24 min, and 0·17, −0·94 and−1·45 min, respectively. The widths of the distributions are proportional to 0·29, 0·36and 0·41 min^0·5, 0·26, 0·36 and 0·41 min^0·5, and 0·34, 0·44 and 0·41 min^0·5. An increase in thethermal tolerance could then be induced by sublethal heat shock or by selection of heatresistant cells.     

Chitinolytic activity of an endophytic strain of Bacillus cereus

Bacillus cereus strain 65, previously isolated as an endophyte of Sinapis , wasshown to produce and excrete a chitinase with an apparent molecular mass of 36 kDa. Theenzyme was classified as a chitobiosidase because it was able to cleave diacetylchitobiose(GlcNAc)₂ from the non-reducing end of trimeric chitin derivatives. The chitinaseexhibited activity over the pH range 4·5–7 5 and was stable between pH 4·0 and8·5. The enzyme had an isoelectric point of 6·4.Application of B. cereus 65directly to soil significantly protected cotton seedlings from root rot disease caused by Rhizoctonia solani .     

Plasmid profiles of Neisseria gonorrhoeae in Peninsular Malaysia

The plasmid profiles of 160 strains of Neisseria gonorrhoeae isolated in Peninsular Malaysia, comprising 80 penicillinase-producing (PPNG) and 80 non-penicillinase-producing (non-PPNG) isolates, were determined. The 80 PPNG isolates were divided into two plasmid groups. All of them harbored two common plasmid species, a 4.4 megadalton (Md) R plasmid previously associated with beta-lactamase production in PPNG strains from the Far East and a 2.6 Md multicopy plasmid of unknown function. In addition to these two plasmids, 60 (75%) PPNG isolates also carried a large 24.5 Md conjugative plasmid. In contrast, the 80 non-PPNG strains were divided into three plasmid groups. All of them possessed the 2.6 Md cryptic plasmid, and 35 (44%) isolates also harbored the 24.5 Md transfer plasmid. Besides these two plasmids, one non-PPNG isolate carried an additional 7.8 Md cryptic plasmid.     

Differential survival of juvenile sockeye and coho salmon exposed to low dissolved oxygen during winter

Juvenile sockeye salmon (43–78 mm) survived 100% for 24 h in cages in ice–covered Black Lake, Alaska at oxygen saturations >65% (9 mg l^–1), but only 45% at 24% saturation (3·0–3·3 mg l^–1) and none at <17% saturation (2·3 mg l^–1). All juvenile coho (50–120 mm) survived 100% for 24 h down to 21% oxygen saturation (3·1 mg l^–1), and all 50 coho survived 4–5 days at 23–24% saturation (3·2–3·3 mg l^–1).     

Partial purification, characterization and regulation of cellulolytic enzymes from Trichoderma longibrachiatum

A net purification of 9·46-, 18·6- and 16·7-fold for filter paper (FP) hydrolytic activity, carboxymethyl (CM) cellulase and β-glucosidase, respectively was achieved through ion exchange and gel chromatographies. The purified enzyme preparation showed an optimal pH of 5·0 for CM cellulase and 5·5 for the other two components. The enzyme activities increased up to 60°–65°C for the three enzyme components and they were stable at 30° or 40°C and pH 4·5 to 5·0 after 20–30 min treatment. The four enzyme components, that is, two FP activities (unadsorbed and adsorbed), a CM cellulase and a β-glucosidase, had K_m values of 47·6 mg, 33·3 mg, 4·0 mg and 0·18 mmol/l with V _max of 4, 1·28, 66·5 and 1·28 units per mg protein. The molecular weights as determined with SDS-PAGE were found to be 44000, 38000, 55000 and 63000 for the above four enzyme components in the same sequence. A distinct type of synergistic action was observed between these components by their action on dewaxed cotton. Glycerol at 1% strongly repressed the formation of all the cellulolytic enzymes. The role of proteolytic enzymes in in vitro inactivation of cellulases was not apparent.     

Plasmids and transposons and their stability and mutability in bacteria isolated during an outbreak of hospital infection.

In an outbreak of hospital infection caused by Klebsiella aerogenes type K-16 isolates over a 3-month period carried, apparently unaltered, a cryptic 90-Megadalton (Md) plasmid (unclassified) and a multiple-resistance 65-Md plasmid of IncM. The IncM plasmid, identified in environmentally related strains of Citrobacter koseri and Escherichia coli, showed minor variations from that in the klebsiella vector. The IncM plasmids, as well as all wild host strains cured of the IncM plasmids, carried a transposable DNA sequence, encoding trimethoprim and, in every case but one, streptomycin resistance. This transposon appeared identical with Tn7, previously identified in unrelated plasmids in bacteria from different environments.     

Study of the dependence of gene expression of plague pathogen plasmid 65MD on the host bacterium genome

The conjugative cointegrate containing Yersinia pestis 65 Md plasmid coding for the production of traction I antigen and mouse toxin has been transferred into Yersinia pseudotuberculosis, Yersinia enterocolitica and Escherichia coli cells. Analysis of the transconjugants obtained has confirmed the connection of the genetical determinants for the mentioned bacterial products with the 65 Md plasmid. The similar level of fra and tox-genes expression has been found in Yersinia cells while fraction I was not produced in Escherichia coli CA cells. The data on the increased synthesis of fraction I at 40 degrees C as compared with the one at 37 degrees C has been obtained. In some cases the production remained at the same level characteristic of the 37 degrees C. The possibility of formation of different Yersinia Fra+ recombinants in the mixed natural epizootic foci and their role in epizootic infections are discussed.     

Conjugative R-plasmid of facultative methylotrophic bacteria Pseudomonas sp. M

50 Md conjugative plasmid, designated pM3, has been found in the cells from natural isolates of Pseudomonas sp M. The plasmid determines the resistance to tetracycline and streptomycin and is capable of conjugative transfer between the cells of Pseudomonas and Escherichia coli. The conjugative derivatives of pM3 deleted for 14 Md of molecular mass were isolated after acridine dyes treatment of cells harbouring plasmid pM3. The discovered plasmid was not shown to belong to IncP1 incompatibility group.     

Disulfide assignment of the C-terminal cysteine knot of agouti-related protein (AGRP) by direct sequencing analysis.

We have assigned the disulfide structure of Md-65 agouti-related protein (Md65-AGRP) using differential reduction and alkylation followed by direct sequencing analysis. The mature human AGRP is a single polypeptide chain of 112 amino acid residues, consisting of an N-terminal acidic region and a unique C-terminal cysteine-rich domain. The C-terminal domain, a 48 amino acid peptide named Md65-AGRP, was expressed in Escherichia coil cells and refolded under different conditions from the mature recombinant protein. The disulfide bonds in the cystine knot structure of Md65-AGRP were partially reduced using tris(2-carboxyethyl) phosphine (TCEP) under acidic conditions, followed by alkylation with N-ethylmaleimide (NEM). The procedure generated several isoforms with varying degrees of NEM alkylation. The multiple forms of Md65-AGRP generated by partial reduction and NEM modification were then completely reduced and carboxymethylated to identify unreactive disulfide bonds. Differentially labeled Md65-AGRP were directly sequenced and analyzed by MALDI mass spectrometry. The results confirmed that Md65-AGRP contained the same disulfide structure as that of Md5-AGRP reported previously [Bures, E. J., Hui, J. O., Young, Y. et al. (1998) Biochemistry 37, 12172-12177].     

Morphology and fine structure of the heart of the burbot, a cold stenothermal fish

The ventricle of the burbot Lota lota heart comprised 0·148 ± 0·006% of the body mass which is nearly two-fold heavier than the relative ventricular mass ( M _V) of other similarly sized teleosts. The shape of the ventricle is pyramidal and the wall is exclusively composed of spongious muscle without a distinct compact layer. The atrium forms 0·017 ± 0·002% of the body mass. Length, width, sarcolemmal surface area and volume of enzymatically isolated myocytes from burbot ventricle were 147·2 ± 10·2 μm, 6·3 ± 0·4 μm, 2440·8 · 251·5 μm² and 2356·8 ± 316·6 μm³, respectively. The myofibrils were peripherally located and their volume density was remarkably high: 65 ± 2 and 68 ± 3% in ventricle and atrium, respectively ( P >0·05). Although not particularly conspicuous, some nonjunctional and junctional sarcoplasmic reticulum (SR) was present in both atrial and ventricular myocytes. The SR formed peripheral couplings with the sarcolemma and the junctional clefts were frequently occupied by foot processes. These findings suggest that cold-adaptation is achieved by cardiac enlargement, high volume density of myofibrils and well-developed peripheral couplings in the SR in the heart of stenothermal burbot.