摘要:

BAL-31 deletion products of the DNA fragment containing thevnfHpromoter and upstream region, when cloned in a transcriptional fusion vector and analyzed forvnfHexpression inAzotobacter vinelandii, revealed that the upstream activator sequence of thevnfHpromoter lies about 140 nucleotides upstream of the promoter. Subsequent substitution and deletion analysis by oligonucleotide-directed mutagenesis in the upstream region of thevnfHpromoter showed that sequences 5'-GTACCATGCGGAAC-3' and 5'-GTACCTGCGGGTAC-3', located 170 and 140 nucleotides upstream of thevnfHpromoter, respectively, are both required forvnfHexpression. Addition of four nucleotides in the intervening sequence between thevnfHpromoter and the putative VnfA (analog of NifA of the conventional molybdenum-dependent nitrogen-fixation pathway) binding site resulted in a drastic reduction of expression from thevnfHpromoter inAzotobacter vinelandii, where as addition of 10 nucleotides in the intervening sequence did not affect the expression. Therefore, the face of the helix-dependent contact appeared to be important. DNA bending seemed to play a crucial role in expression fromvnfHpromoter. The intervening sequence exhibited characteristics of sequence-dependent intrinsically curved DNA, as shown by anomalous low gel mobility with polyacrylamide gel electrophoresis, electron microscopy, and computer simulated curvature analysis. Distamycin at very low concentrations significantly reduced the anomaly in electrophoretic mobility of the intervening DNA sequence.Key words:Azotobacter vinelandii,vnfA,vnfH, promoter-lacZfusion, DNA bending.

ISSN:0008-4166    

DOI:10.1139/w98-011

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The present work was designed to study the effect of saline stress on the attachment ofAzospirillum brasilenseto maize and wheat roots. We demonstrate that both attachment steps (adsorption and anchoring) are altered. A 100-kDa protein disappeared under these experimental conditions. Coincidently, a 100-kDa flagellum protein has been identified as the agent responsible for adsorption. However, the adhesive properties of bacteria appear to involve other factors, such as the ionic strength of the medium. The impairment of anchoring ability was correlated with alterations in exopolysaccharide, glucan, and lipopolysaccharide contents.Key words:Azospirillum brasilense, saline stress,Azospirillum-root interaction.

ISSN:0008-4166    

DOI:10.1139/w98-024

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

Polysaccharide lyases that can degrade glycosaminoglycans (GAGs) were identified in an anaerobic strain living in the human intestine. The strain was isolated from the stool of a healthy male and identified asBacteroidessp. strain HJ-15. A detailed taxonomical study indicated the species is a strain ofBacteroides stercoris. The isolate was cultured and the polysaccharide lyase activity was partially purified. This enzyme preparation could act on GAGs containing either glucosamine or galactosamine suggesting the presence of both heparinases and chondroitinases. Various GAGs were incubated with the partially purified enzyme and the products formed were analyzed by strong anion-exchange high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy. These studies demonstrated the presence of at least two types of polysaccharide lyases: heparin lyase and chondroitin sulfate lyase. The eliminative mechanism of these lyase enzymes was confirmed through the isolation of unsaturated disaccharide products. The heparin lyase acted on both heparin and acharan sulfate, a GAG recently isolated fromAchatina fulica. TheBacteroideschondroitin lyase, acted on chondroitin sulfates A, B (dermatan sulfate), and C, resembling chondroitin lyase ABC. The presence of a GAG-degrading organism in human intestine may pose problems for the effective oral administration of GAG drugs.Key words:Bacteroides stercoris, glycosaminoglycan, nuclear magnetic resonance spectroscopy, polysaccharide lyase, heparinase, chondroitinase.

ISSN:0008-4166    

DOI:10.1139/w98-027

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

HeLa cells were established as a model system to study the invasiveness and biology ofLegionella pneumophila. In this model, invasion could be distinguished from adherence; virulent strains ofL. pneumophilawere adherent and invasive, whereas nonvirulent strains were adherent but poorly invasive. Invasion was rapid and did not require de novo bacterial protein synthesis, suggesting that the invasion factor is constitutively expressed by virulent strains. Entry into HeLa cells required actin polymerization and an intact microtubule cytoskeleton and was only moderately inhibited by the presence of 100 mM glucose or galactose. Intracellular replication of virulentL. pneumophilatook place in ribosome-studded complex endosomes and led to the formation of free bacteria-laden vesicles presumably released from lysed HeLa cells, These free vesicles (referred to as mature vesicles) were isolated in continuous density gradients of Percoll. The bacteria contained in the isolated mature vesicles had a unique envelope structure and were highly adherent to HeLa cells, characteristics that correlated with a bright red appearance after the Giménez stain (Giménez positive). Plate-grown legionellae and replicating legionellae, harboured in complex endosomes, displayed a typical Gram-negative envelope and stained green after the Giménez stain (Giménez negative). Chronically infected cultures of HeLa cells were also established that may be a useful tool for studying long-term interactions between virulentL. pneumophilaand mammalian cells. HeLa cells constitute a valuable model system that offers unique opportunities to study parasite-directed endocytosis, as well as stage specific host-parasite interactions.Key words:Legionella pneumophila, HeLa cells, invasion mechanisms, intracellular pathogens.

ISSN:0008-4166    

DOI:10.1139/w98-023

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The ability of various electroplated coatings (cobalt, zinc, copper, and cobalt-containing alloys of nickel, zinc, chromium, etc.) to inhibit the growth of pathogenic bacteria (Gram-positive bacteriaEnterococcus faecalisand methicillin-resistantStaphylococcus aureusand Gram-negative bacteriaEscherichia coli,Pseudomonas aeruginosa, andKlebsiella pneumoniae) was determined by a drop-method antibacterial experiment. The amounts of H2O2produced and metal ions dissolved from the surfaces of various electroplated coatings were measured and it was found that the inhibitory ability of coatings corresponded to the amounts of H2O2produced. The more significant the inhibition of the coating to bacterial growth, the greater the amount of H2O2production. In addition, the bacterial survival rates on the surfaces of coatings were almost zero when H2O2was produced in amounts greater than 10-6mmol/cm2. However, the dominant concentrations of metal ions dissolved from coatings were outside of the bacterial lethal range.Key words: electroplated coatings, pathogenic bacteria, inhibitory ability, hydrogen peroxide.

ISSN:0008-4166    

DOI:10.1139/w98-030

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The adherence ofStreptococcus pneumoniaeto epithelial (A549) lung cells was studied and the bacterial binding distribution was found to be nonrandom (non-Gaussian). Analysis of the dependency of bacterial binding on the cell cycle of A549 cells revealed that approximately 1.8 times more bacteria bind to G2cells than to G0-G1phase cells. Furthermore, bacterial binding curves exhibited a plateau of binding to G2cells at a normalized bacteria to cell ratio approximately 1.8 times larger than that at which the plateau of binding to G0-G1cell was observed. Since G2cells are on average 1.4-1.8 times larger than G0-G1cells,the results indicate that bacterial binding is proportional to cell size and not to the preferential binding (higher affinity) of bacteria to A549 cells in the G2phase. Finally, the non-Gaussian distribution of bacterial binding could be mathematically modeled by a linear combination of three Gaussian distributions each representing bacterial binding to cells in a particular phase of the cell cycle (G0-G1, S, and G2-M). Because the Gaussian function contains a term that takes into account the relative number of cells in each of the phases, this last result implies that the overall (non-Gaussian) binding distribution (and hence the median of bacterial binding) can be highly sensitive to the relative proportion of cells in the various phases of the cell cycle.Key words: bacterial adherence,Streptococcus pneumoniae, flow cytometry, cell cycle.

ISSN:0008-4166    

DOI:10.1139/w98-025

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The neurotoxin domoic acid is produced in quantity by the diatomPseudo-nitzschia multiseriesand is released to the environment directly and indirectly via food chains. Presumably there is a mechanism for the biodegradation and disposal of domoic acid and as bacteria are logical candidates for such an activity, a search for bacteria competent to carry out biodegradation of domoic acid was initiated. Extensive trials with a wide variety of bacteria isolated mainly from muds and waters taken from the marine environment showed that the ability to grow on or degrade domoic acid was rare; in fact, domoic acid was inhibitory to resting cells or growing cultures of most of these bacteria. In contrast, using enrichment techniques, it was possible to isolate from molluscan species that eliminate domoic acid readily, i.e., blue mussels (Mytilus edulis) and soft-shell clams (Mya arenaria), bacteria that exhibited growth with and biodegradation of domoic acid when supplemented with low concentrations of growth factors. The species that retain domoic acid for lengthy periods, such as sea scallops (Placopecten magellanicus)and red mussels(Modiolus modiolus), only occasionally yielded bacteria with this capability. The differences may be a result of the mechanisms used by the different shellfish in dealing with domoic acid, i.e., freely available in the blue mussels and soft shell clams but likely sequestered in the digestive glands of sea scallops and red mussels and thus, largely unavailable for bacterial utilization. The results show thatMytilus edulisandMya arenaria,almost uniquely, are prime and reliable sources of domoic acid utilizing bacteria. These findings suggest a strong possibility that autochthonous bacteria may be significant factors in the elimination of the neurotoxin in these two species of shellfish.Key words: bacteria, neurotoxin, domoic acid, elimination, bivalve molluscs.

ISSN:0008-4166    

DOI:10.1139/w98-028

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The organic hydroperoxidest-butyl hydroperoxide, cumene hydroperoxide, and peracetic acid were found to act similarly to hydrogen peroxide in causing inactivation of enzymes within intact spores ofBacillus megateriumATCC 19213 concomitant with mortality. Spores treated with lethal levels of the agents were germinated and permeabilized for enzyme assays. The hierarchy of sensitivities among enolase, glucose-6-phosphate dehydrogenase (G6Pdh), and pyruvate kinase to inactivation varied somewhat with the specific hydroperoxide used, possibly because of differences in the types of radicals generated. However, each agent inactivated each of the enzymes, albeit at different rates. Comparative assessments of enzyme inactivation by lethal levels of H2O2or by moist heat showed that some enzymes, such as G6Pdh, are highly sensitive to inactivation, while others, such as ATPases, are much more resistant. The enzymes G6Pdh and aldolase were highly sensitive to hydroperoxide inactivation and also to moist heat, while pyruvate kinase was much more sensitive to hydroperoxides than to moist heat. Our overall interpretation of the findings is that hydroperoxides and moist heat can produce cumulative damage to sensitive enzymes within spores, which progressively diminishes the capacities of the cells to undergo the outgrowth required for return to vegetative life.Key words: bacterial spores, hydroperoxides,Bacillus megaterium, enzyme inactivation.

ISSN:0008-4166    

DOI:10.1139/w98-026

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

Pichia anomalainhibits growth ofPenicillium roquefortiin high-moisture winter wheat, barley, and oats in cases where a malfunctioning airtight feed-storage system allows air to leak in. To imitate air leakage to such a storage system, grain was inoculated, packed in glass tubes with a restricted air supply, and incubated at 25°C. Yeast and mold colony-forming units (CFU) were counted on selective media after 14 days.Pichia anomalareached a density of about 5 x 107CFU/g on all tested cereals except in spring wheat (cv. Dragon), where a density of 109CFU/g was reached. In winter wheat (cv. Kosack),Penicillium roquefortireached a density of 106CFU/g in grain that had not been inoculated with yeast and 105CFU/g in co-culture with 5 x 103CFU/g ofPichia anomala. At 5 x 104Pichia anomala, growth ofPenicillum roquefortiwas totally inhibited. Similar results were obtained with spring wheat (cv. Dragon), barley (cv. Golf), and oats (cv. Svea). However, spring wheat cv. Dragon was generally much less conducive to growth ofPenicillium roqueforti. On rye (cv. Motto),Penicillium roquefortidid not grow in monoculture or when co-cultured withPichia anomala. No differences in antagonistic activity ofPichia anomalaor sensitivity ofPenicillium roqueforti, respectively, were found between the three isolates tested of each species.Key words: biological control, postharvest, mold, cereal grain, storage.

ISSN:0008-4166    

DOI:10.1139/w98-018

出版商:NRC Research Press    

年代:1998

数据来源: NRC

摘要:

The lipopolysaccharides LPS I and LPS II, isolated from the hypovirulent EV40 strain ofYersinia pestis, are composed only of type R lipopolysaccharides. This type consists of two forms, a and b, depending on their solubility pattern in a solvent mixture containing varying proportions of chloroform, methanol, hexane, and hydrochloric acid. LPS I consists of one subtype, RIb, while LPS II consists of two subtypes, RIIa and RIIb. Analysis by gel electrophoresis shows that the mass of these lipopolysaccharide forms are in the vicinity of 2000-3000 Da. The RIb and RIIb subtypes, which are found in the majority of lipopolysaccharide I and II fractions, are composed of ketoses and amines that are similar to those occurring in LPS I and LPS II. In contrast, the two subtypes RIIa and RIIb are different both in terms of the composition of lipid A and the extent of its substitution. Certain fractions of RIIa contain only lipid A and 3-deoxy-D-manno-octulosonic acid (KDO), while other fractions of RIIb possess a lipid A, which is not substituted by arabinose. The whole set of these R-type lipopolysaccharide forms are excellent models for the study of the role of the primary structure of the polysaccharide region, and for the effect of lipid A substitution on the biological activity of bacterial lipopolysaccharides.Key words:Yersinia pestis, hypovirulence, lipopolysaccharides, R type.[Journal translation]

ISSN:0008-4166    

DOI:10.1139/w98-029

出版商:NRC Research Press    

年代:1998

数据来源: NRC