DNA sequence analysis of a putative primary replicon from a cryptic plasmid pNM214 of a hydrocarbon utilizing marine <Emphasis Type=Italic>Bacillus</Emphasis> strain NM21

Marine Bacillus strain NM21 isolated from hydrocarbon-contaminated site at Naval Harbour, Mumbai grows on high-speed diesel as a source of carbon and energy. This bacterium harbours four plasmids in it. The smallest plasmid, pNM214 was digested with EcoRI enzyme and cloned in pUC19 vector. The clone Om4 containing largest insert of >3.5 kb was sequenced by primer walking. DNA sequence analysis showed this fragment to be homologous to replication initiation protein (rep) gene and dso (double strand origin) of different plasmids from Bacillus subtilis and Bacillus pumilus species. The putative rep gene sequence of pNM214 showed 74.3–91.6% DNA identity to B. subtilis plasmids (pTA1015, pTA1060 and pTA1040) and 86.3% to 88.9% DNA identity to B. pumilus plasmids (pPL7065, pPL10 and pSH1452). The translated amino acid sequence of rep shows that it contains all the three conserved motifs present in the Rep protein of pC194 family of plasmids. DNA sequence comparison of putative dso of pNM214 with other bacillus plasmids belonging to pC194 group shows that it contains highly conserved nick site sequence 5′-TCTTTTCTTATCTTGATA-3′ and surrounding inverted repeats. Thus, it indicates that pNM214 to be a rolling circle replicating plasmid belonging to the pC194 group. The presence of rep and dso like sequences in the sequenced EcoRI fragment indicate that the cloned fragment contain putative primary replicon of pNM214.     

Enzymatic release of ferulic acid fromIpomoea batatas L. (sweet potato) stem

Ferulic acid is a phenolic compound that serves as a major biosynthetic precursor of vanillin in higher plants. We investigated the ability of the 3 commercial enzymes—Ultraflo L, Viscozyme L, and α-Amylase—to induce the release ferulic acid from theIpomoea batatas L. (sweet potato) stem. The rate of release for ferulic acid was optimal when Ultraflo L (1.0%) was used compared with the other enzymes, whereas Viscozyme L was most effective for the release of vanillic acid and vanillin. Thus, these enzymes may be useful for the large-scale production of ferulic acid and other phenolic compounds from sweet potato stem.     

Antibacterial and immuno-modulatory activity of ethanol extracts from Lespedeza sp. during Helicobacter pylori infections

Chemical therapeutics targeted against H. pylori may lead to host toxicity and pathogen eradication failures. In this study, ethanolic extracts from five Lespedeza sp. plants were shown to inhibit the gastric-pathogen H. pylori and to modulate cytokine production. Disc agar diffusion assays showed that Lespedeza sp. ethanol extracts possess potent anti-H. pylori activity. Among the five plant extracts, the extracts from L. cyrtobotrya demonstrated the highest anti-H. pylori effect. The growth inhibitory effect against H. pylori was initiated after six h of treatment with plant extracts and the effect remained for a continuous period of 48 h. Incubation of the gastric cells infected with H. pylori with 1.25 to 50 mg/mL of sp. plant extracts resulted in a reduction of the production of cytokine IL-8. The plant ethanol extracts generally had little influence on AGS cell viability, indicating their safety for the treatment of bacterial infections. Three active fractions of L. cyrtobotrya also demonstrated similar anti-H. pylori and immuno-modulatory effects. Taken together, these results provide evidence that Lespedeza sp. plant extracts might be potential sources of new host friendly anti-H. pylori agents.     

Multiphase hemodynamic simulation of pulsatile flow in a coronary artery

A multiphase transient non-Newtonian three-dimensional (3-D) computational fluid dynamics (CFD) simulation has been performed for pulsatile hemodynamics in an idealized curved section of a human coronary artery. We present the first prediction, to the authors' knowledge, of particulate buildup on the inside curvature using the multiphase theory of dense suspension hemodynamics. In this study, the particulates are red blood cells (RBCs). The location of RBC buildup on the inside curvature correlates with lower wall shear stress (WSS) relative to the outside curvature. These predictions provide insight into how blood-borne particulates interact with artery walls and hence, have relevance for understanding atherogenesis since clinical observations show that atherosclerotic plaques generally form on the inside curvatures of arteries. The buildup of RBCs on the inside curvature is driven by the secondary flow and higher residence times. The higher viscosity in the central portion of the curved vessel tends to block their flow, causing them to migrate preferentially through the boundary layer. The reason for this is the nearly neutrally buoyant nature of the dense two-phase hemodynamic flow. The two-phase non-Newtonian viscosity model predicts greater shear thinning than the single-phase non-Newtonian model. Consequently, the secondary flow induced in the curvature is weaker. The waveforms for computed hemodynamic parameters, such as hematocrit, WSS, and viscosity, follow the prescribed inlet velocity waveforms. The lower oscillatory WSS produced on the inside curvature has implications for understanding thickening of the intimal layer.     

Physical Characteristics of Spores of Food-Associated Isolates of the Bacillus cereus Group

All 47 food-borne isolates of Bacillus cereus sensu stricto, as well as 10 of 12 food-borne, enterotoxigenic isolates of Bacillus thuringiensis, possessed appendages. Spores were moderately to highly hydrophobic, and each had a net negative charge. These characteristics indicate that spores of food-associated B. thuringiensis and not only B. cereus sensu stricto have high potential to adhere to inert surfaces.Bacillus cereus is a worldwide food-borne pathogen causing diarrheal or emetic-type illnesses. The presumptive toxins have been identified in each case (2, 9, 11). We recently reported that the diarrheal type was the more common toxigenic type in U.S. retail rice and seafood (5, 24). Spores of B. thuringiensis and B. mycoides were also isolated from rice. Best known for the insecticidal activity of its parasporal crystals, Bacillus thuringiensis has been associated with gastroenteritis and isolated in rare cases from outbreaks of food-borne illness (17). Isolates of B. thuringiensis, including those isolated from commercial insecticides, have been shown to produce one or both of the enterotoxins HBL and NHE (6, 14, 18, 22, 23, 28).Bacterial spores can adhere to inert surfaces of food processing equipment due to their surface properties and structures (8, 29). Spores of certain Clostridium and Bacillus species possess appendage-like structures (1, 12, 15, 19) which may contribute to biofilm formation (3, 30). Previous studies of the physical properties of spores of the B. cereus group have focused primarily on environmental isolates. Here the spore morphology, hydrophobic characteristics, and net negative charge of food-borne and food poisoning-associated isolates of the B. cereus group were investigated including potentially enterotoxigenic B. thuringiensis.The diarrheal-type B. cereus strains 85 and 133, B. thuringiensis strains 105 and 129, and B. mycoides strain 157 isolated from rice were examined in detail in this study. These B. cereus and B. thuringiensis strains were selected on the basis of the presence of the nhe or hbl gene along with the ability to produce the corresponding gene product at elevated titers as previously described (5). Bacillus subtilis (ATCC 6633) was used as a comparative reference in the surface charge and hydrophobicity studies. In addition to the above isolates, the presence of appendages on spores of the following was also examined by negative staining: 32 isolates of diarrheal B. cereus isolated from seafood (24), an NHE-positive control strain (ATCC 1230/88), 12 emetic-type B. cereus isolates from food poisoning outbreaks (20), and 10 food-borne isolates of B. thuringiensis (in addition to the above two) (5). Spores were produced as previously described (4).The spores of B. thuringiensis were separated from the inclusion bodies (IB) by centrifuging in step gradients of 0.6, 1.0, 1.4, and 1.8 g/ml sucrose. Cleaned spore suspensions in sodium-potassium phosphate buffer were diluted to an A₆₀₀ of 0.4. Two to three ml of the diluted suspension was layered on top of the gradient. The gradients were centrifuged for 2.5 h in a swinging bucket rotor (Dupont-Sorvall) at 450 × g at 10°C. A visible white layer of spores was collected from the bottom with a Pasteur pipette. The layer was washed with 0.85% saline (at least twice) and stored in the same at 4°C. Following these procedures, spore suspensions had <20% inclusions (relative to spores) as observed by phase-contrast microscopy.Spore hydrophobicity was measured using the bacterial adhesion to hydrocarbon (BATH) assay (26) modified from the observations reported elsewhere (16). The mean and the standard error were calculated from a minimum of seven measurements. ζ potentials of the spores were measured in a Malvern Ζetasizer model nano 2S (Malvern Instruments, Westborough, MA) using the Smoluchowski equation (26). The spores were suspended in saline (0.15 M) at a pH of 6 to 7. The ζ potentials were determined from a mean value of five measurements.Spores of two diarrheal B. cereus strains, two B. thuringiensis strains, and one B. mycoides strain were examined by transmission electron microscopy (TEM). Appendages were observed on B. cereus (Fig. 1a, b, and c) and B. thuringiensis (Fig. 1d and e) but not B. mycoides (Fig. ​(Fig.1f).1f). In contrast, exosporia were seen in all the isolates examined (Fig. ​(Fig.1).1). Similar observations for environmental and clinical samples of these species have been reported by others (13, 16, 21, 29). The number of appendages observed here varied among strains. In the case of B. cereus these ranged from three to four (isolate 133) to four to nine (isolate 85). On the other hand, B. thuringiensis 129 had a higher number (12 to 18) of appendages per spore. The appendage length for B. cereus varied from 0.45 to 3.8 μm. Appendages appeared tube-like in appearance, with an average diameter of 13.6 nm (Fig. ​(Fig.1b)1b) as determined by Image J software (http://rsb.info.nih.gov/ij). For each species, examined appendages were often lophotrichous (Fig. ​(Fig.1e)1e) though peritrichous appendages were more common. All 35 food-borne B. cereus isolates examined in this study and one B. cereus NHE control strain possessed appendages, as did 12 of 12 food poisoning-associated, emetic-type B. cereus isolates. Appendages are a common but not universal feature of the B. cereus group. Whether the number and length of spore appendages of the B. cereus group are species associated or due to their fragility and loss during the preparation procedures (16, 30) remains a possibility. That all 47 B. cereus sensu stricto isolates examined here possessed spore appendages suggests that these structures are characteristic of this species. There is some controversy as to their role in adhesion (27).Open in a separate windowFIG. 1.Electron micrographs of Bacillus cereus group spores. (a) Shadowed image of B. cereus 85 showing appendages and exosporium; (b) appendage alone; (c) B. cereus 133 showing appendage and exosporium; (d) B. thuringiensis 129 showing appendage, exosporium, and inclusion; (e) negative stain of B. thuringiensis 129 showing lophotrichous appendages; (f) shadowed image of B. mycoides 157 showing exosporium and lack of appendages.Large differences in the relative hydrophobicity of five food-borne isolates representing three Bacillus species of the B. cereus group were not observed among the species examined (Table ​(Table1).1). The hydrophobicity values for the isolates tested were in a narrow range of 42.4 to 55.6%. Similar values for spores of B. cereus sensu stricto have been reported by others (16). B. subtilis ATCC 6633 was included for comparative purposes. Its relative hydrophobicity (Table ​(Table1)1) was the lowest among spores examined and similar to that reported by Husmark and Ronner (16) for this strain. Using the same procedures, Doyle et al. (7) reported adherence values of 38.3% and 61.4% for two isolates of B. thuringiensis, compared to 42.4% and 55.6% observed here for B. thuringiensis (Table ​(Table1).1). From the values obtained here, our isolates can be classified to be moderately to highly hydrophobic. Exosporia have been proposed to be responsible for surface hydrophobicity of spores (16, 25). As mentioned above, exosporia were observed here in all isolates examined.

TABLE 1.

Relative hydrophobicity and charge of spores of food-borne isolates of the B. cereus group

Adipokinetic hormone enhances nodule formation and phenoloxidase activation in adult locusts injected with bacterial lipopolysaccharide

Interactions between the locust endocrine and immune systems have been studied in vivo in relation to nodule formation and activation of the prophenoloxidase cascade in the haemolymph. Injection of bacterial lipopolysaccharide (LPS) extracted from Escherichia coli induces nodule formation in larval and adult locusts but does not increase phenoloxidase activity in the haemolymph. Nodule formation starts rapidly after injection of LPS and is virtually complete within 8 h, nodules occurring mainly associated with the dorsal diaphragm on either side of the heart, but sometimes with smaller numbers associated with the ventral diaphragm on either side of the nerve cord. Co-injection of adipokinetic hormone-I (Lom-AKH-I) with LPS stimulates greater numbers of nodules to be formed in larval and adult locusts, and activates phenoloxidase in the haemolymph of mature adults but not of nymphs. The effect of co-injection of Lom-AKH-I with LPS on nodule formation is seen at low doses of hormone; only 0.4 pmol of Lom-AKH-I per adult locust is needed to produce a 50% increase in the number of nodules formed. When different components of LPS from the E. coli Rd mutant are tested, the mono- and the diphosphoryl Lipid A components have similar effects to the intact LPS. Remarkably, detoxified LPS activates phenoloxidase in the absence of Lom-AKH-I, although co-injection with hormone does enhance this response. Both diphosphoryl Lipid A and detoxified LPS induce a level of nodule formation that is enhanced by co-injection of Lom-AKH-I, but monophosphoryl Lipid A does not initiate nodule formation even when injected with hormone. Co-injection of a water-soluble inhibitor of eicosanoid synthesis, diclofenac (2-[(2, 6-dichlorophenyl)amino] benzeneacetic acid), reduces nodule formation in response to injections of LPS (both in the absence and presence of hormone) in a dose-dependent manner, but does not prevent activation of phenoloxidase in adult locusts. It is shown that nodule formation and activation of the prophenoloxidase in locust haemolymph can both be enhanced by Lom-AKH-I, but it is argued that these processes involve distinct mechanisms in which eicosanoid synthesis is important for nodule formation, but not for the increased phenoloxidase activity.