Tributyltin-resistant bacteria from estuarine and freshwater sediments.

Resistance to tributyltin (TBT) was examined in populations from TBT-polluted sediments and nonpolluted sediments from an estuary and from fresh water as well as in pure cultures isolated from those sediments. The 50% effective concentrations (EC50s) for populations were higher at a TBT-polluted freshwater site than at a site without TBT, suggesting that TBT selected for a TBT-resistant population. In contrast, EC50s were significantly lower for populations from a TBT-contaminated estuarine site than for those from a site without TBT, suggesting that other factors in addition to TBT determine whether populations become resistant. EC50s for populations from TBT-contaminated freshwater sediments were nearly 30 times higher than those for populations from TBT-contaminated estuarine sediments. We defined a TBT-resistant bacterium as one which grows on trypticase soy agar containing 8.4 microM TBT, a concentration which prevented the growth of 90% of the culturable bacteria from these sediments. The toxicity of TBT in laboratory media was influenced markedly by the composition of the medium and whether it was liquid or solid. Ten TBT-resistant isolates from estuarine sediments and 19 from freshwater sediments were identified to the genus level. Two isolates, each a Bacillus sp., may be the first gram-positive bacteria isolated from fresh water in the presence of a high concentration of TBT. There was a high incidence of resistance to heavy metals: metal resistance indices were 0.76 for estuarine isolates and 0.68 for freshwater isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperexpression of a Bacillus thuringiensis delta-endotoxin-encoding gene in Escherichia coli: properties of the product.

Conditions for hyperexpression, in Escherichia coli, of the Bacillus thuringiensis var, kurstaki gene, cryIA9(c)73, encoding an insecticidal crystal protein, CryIA(c)73, were investigated by varying the promoter type, host cell, plasmid copy number, the second codon and number of terminators. The cryIA(c)73 gene was cloned into three E. coli expression vectors, pKK223-3 (Ptac promoter), pET-3a (P phi 10 promoter), and pUC19 (Ptac promoter). The level of cryIA(c)73 expression was measured by ELISA and compared to total cellular protein over growth periods of 24 and 48 h. Maximum expression levels of 284 microgram CryIA(C)73/ml (48% of cellular protein) were obtained in shake flasks with the Ptac promoter in E. coli JM103. Optimal conditions were found to be low-copy-number plasmid (pBR322 ori), 48 h of growth, in lon+ cells. A change of the gene's second codon to AAA can improve expression by two to three fold but is undetectable in the presence of a strong E. coli promoter. The cryIA(c)73 gene product, in E. coli, formed crystals with the same lattice structure as the native crystals formed in B. thuringiensis (as visualized by electron microscopy). Bioassay results (insect toxicity and specificity) of the crystal produced in E. coli were similar to that produced in B. thuringiensis.

Scanning electron microscopy of Malassezia furfur attachment to Broviac catheters.

Malassezia furfur has been increasingly associated with Broviac-catheter-related sepsis in infants receiving fat emulsions for parenteral alimentation. We examined by scanning electron microscopy the appearance of M. furfur attached to Broviac catheter segments mock-infected in vitro and to Broviac catheters removed from two infants with catheter-related sepsis. In vitro attachment occurred equally on external and internal surfaces of the catheters. Although some organisms were attached next to surface defects in the catheters, we could not determine if such defects were preferential sites of attachment. In the patient catheters, a dense coating of yeast cells was found adhering to the luminal surface, most abundantly near the tip. No organisms were seen on the external surface of the catheters. These findings show the need to examine the mechanisms of intraluminal catheter colonization in order to understand better the pathogenesis of M. furfur infections.

Cadmium transport by a Cd2+-sensitive and a Cd2+-resistant strain of Bacillus subtilis.

Cadmium uptake by a Cd2+-sensitive (1A1) and a Cd2+-resistant mutant (1A1r) strain of Bacillus subtilis was investigated. Uptake of 109Cd2+ was determined for cells of both strains grown in tryptone broth and in broth containing tryptone, yeast extract, and glucose (TYG). The extent of 109Cd2+ uptake by cells of 1A1r was less than by cells of 1A1 under both growth conditions. In both growth media, 109Cd2+ uptake by 1A1 cells demonstrated saturation kinetics and was energy dependent. In both TYG and tryptone broth, 109Cd2+ uptake by 1A1 cells was inhibited by the addition of unlabeled Mn2+. Although lower in magnitude, the kinetics of 109Cd2+ uptake by 1A1r cells were similar to those of 1A1 cells when grown in tryptone broth. However, no obvious saturation kinetics, energy dependence, temperature sensitivity, or inhibition of 109Cd2+ uptake by the addition of unlabeled Mn2+ was observed in 1A1r cells grown in TYG. Differential Mn2+ accumulation by 1A1r cells in TYG and tryptone broth correlated with differential 109Cd2+ uptake by 1A1r cells in these media.

Hexavalent chromium-resistant bacteria isolated from river sediments.

Hexavalent chromium [Cr(VI)] is a known carcinogen and mutagen; however, the actual mechanisms of Cr toxicity are unknown. Two approaches were used to isolate Cr(VI)-resistant bacteria from metal-contaminated river sediments. Diluted sediments were plated directly onto a peptone-yeast extract (PYE) medium containing 0 to 100 micrograms of Cr(VI) ml-1. Approximately 8.4 x 10(5) CFU g-1 were recovered on 0 microgram of Cr(VI) ml-1, whereas 4.0 x 10(2) CFU g-1 were recovered on PYE plus 100 micrograms of Cr(VI) ml-1. Alternatively, continuous culture enrichment techniques were employed using PYE and 100 micrograms Cr(VI) ml-1 input at dilution rates of 0.02 and 0.10 h-1. After six residence periods, 10(9) CFU were recovered on PYE agar containing 0 microgram of Cr(VI) ml-1 and 10(7) CFU on PYE agar plus 100 micrograms of Cr(VI) ml-1. Of 89 isolates obtained by direct plating onto PYE, 47% were resistant to 100 micrograms of Cr(VI) ml-1, and 29% were resistant to 250 micrograms of Cr(VI) ml-1. When the same isolates were plated onto PYE containing Cr(III), 88% were resistant to 100 micrograms ml-1 but only 2% were resistant to 250 micrograms ml-1. Cr, Co, Sb, and Zn were found in significantly higher concentrations at an industry-related contaminated site than at a site 11 km downstream. Total Cr in the sediments at the contaminated site averaged 586 micrograms (dry weight) g-1, and the downstream site averaged 71 micrograms (dry weight) g-1. The Cr recovered from acid-digested Ottawa River sediment samples was predominantly hexavalent. Five acid digestion procedures followed by atomic absorption spectroscopy were compared and found to be 30 to 70% efficient for recovery of Cr relative to neutron activation analysis. A population of aerobic, heterotrophic bacteria was recovered from sediments containing elevated levels of Cr.(ABSTRACT TRUNCATED AT 250 WORDS)

Freeze-fracture confirmation of the presence of a core in the specialized tip structure of Mycoplasma pneumoniae.

The presence of a specialized terminal region in Mycoplasma pneumoniae was seen in thin sections viewed in an electron microscope. Actively growing cells were examined by the freeze-fracture technique in the absence of fixation to further establish the core as a significant structural entity. Cross fractures revealed a cytoplasmic matrix surrounding a central core structure of about 54 nm. This structure disappeared rapidly in aging cells. The convex protoplastic faces of the membrane around the core had characteristic 5- to 10-nm intramembrane particles evenly distributed across the cell surface, with no apparent difference in the region of the specialized tip. A periodicity previously noted in negatively stained preparations was clearly defined here in thin sections. Attachment of actively growing cells to sheep erythrocytes was seen primarily as a side attachment rather than attachment at the tip alone. This association between the mycoplasma and the sheep erythrocytes seriously deformed the sheep erythrocytes, but no membrane fusion could be detected.

Exospore formation in Methylosinus trichosporium.

Formation of exospores in Methylosinus trichosporium was examined by electron microscopy; serial sectioning was used to visualize the shape and location of the developing exospore in relation to the vegetative cell. The initial stage was the formation of a budlike enlargement on one end of the vegetative cell. The enlargement was surrounded by the exospore capsule, and the cell wall was continuous around both the cell and the developing exospore. A constriction occurred in the area where the budlike structure was attached to the vegetative cell, and the constriction continued to form until the immature exospore was detached from the vegetative cell. The cup-shaped immature exospore was surrounded by the exospore capsule, which appeared to hold the exospore close to the vegetative cell. After separation from the vegetative cell, the immature exospore developed further by forming the exospore wall and by becoming spherical.

Intracellular structures of Mycoplasma pneumoniae revealed after membrane removal.

Mycoplasma pneumoniae was grown on Formvar- and carbon-coated electron microscope grids and treated with the nonionic detergent Triton X-100 to gently remove the membrane and cytoplasm. The detergent mixture was composed of 0.5% Triton X-100 in SSR-2 broth base. After this treatment, the grids were rinsed in a mixture of 0.1 M KCl, 5 mM MgCl2, and 6 mM potassium phosphate buffer (pH 7.05) and negatively stained with uranyl acetate. The Triton X-100-resistant remains of M. pneumoniae after gentle removal of the membrane and cytoplasm consisted of fibrous structures oriented similarly to the undisrupted cells. The thin fibers displayed a negative staining quality and diameter analogous to that of rabbit muscle F-actin. The fibrous moieties ended in rodlike condensations which appeared striated in negatively stained and shadowed preparations. These striations were regular, and the majority of rod structures had lengths of 220 to 300 nm and widths of 50 to 80 nm. Specific antibody to rabbit muscle actin, produced in guinea pigs, was used in indirect immunofluorescence of the M. pneumoniae colonies. Fluorescence was detected, with concentrations at the colony center and at the tips of filamentous cells.

Structure of Methylosinus trichosporium exospores.

Methylosinus trichosporium exospores did not display a well-defined cortex or an exosporium. A thick, electron-dense exospore wall was characteristic of the exospores. Located on the exterior of the exospore wall was a cell wall to which a well-defined capsule was attached. An extensive lamellar intracytoplasmic membrane system characteristic of the kind in vegetative cells of this bacterium was present along the interior periphery of the exospore wall. Upon germination of M. trichosporium exospores, the thick exospore wall gradually disappeared and a germ tube formed. The intracytoplasmic membranes of the exospores extended into the germ tube which did not possess the extensive fibrillar capsule observed on the dormant exospore. Cup-shaped exospores which have an ultrastructure similar to that of mature exospores except that they are invaginated also germinated upon exposure to methane.

Characteristics of Streptomyces coelicolor A3(2) aerial spore rodlet mosaic.

Cytochemical analysis of Streptomyces coelicolor (A3(2) indicated that the aerial growth rodlet mosaic is a polysaccharide. Statistical analysis of frequency distributions of individual rodlet lengths from control and ether-reoriented spore mosaics indicated that the rodlet fibrillar image is the result of individual particulates, rather than evaginations in a continuous sheet of material. A model of the mature sport envelope was developed from freeze-etch-replicated, thin-sectioned, and critical point dried S. coelicolor A3(2) mature spores. The rodlet mosaic was situated between the outer spore wall and an external granuloma matrix. Mixture spore envelope layers from the inner surface to the external surface are plasma membrane, inner spore wall, outer spore wall, rodlet mosaic, an undefined granular matrix, and the sheath. The granular matrix had an uneven thickness and much of the matrix was frequently absent from the interspore spaces of mature spore chains. Streptomyces coelicolor A3(2) mosaic rodlets were isolated by acetic acid refluxing, then ethanol precipitation. Complete acid hydrolysis of rodlets released on sugar which cochromatographed with D-glucosamine-HCl and released acetic acid at 139% of the expected level. Cell associated rodlet mosaics and isolated mosaic rodlets were hydrolyzed with chitinase. Infrared spectra of isolated rodlets were similar to crab chitin spectra.

Ultrastructure of the capsule of Bacillus megaterium ATCC 19213.

The ultrastructure of the firmly adherent capsule produced by Bacillus megaterium cultured on fructose mineral salts medium was examined using thin sectioning, freeze-etching, and critical point drying by transmission and scanning electron microscopy. The capsule material was shown to be fibrillar, with most fibrils containing bulbous protrusions. Two types of fibres were resolved. These were termed primary and cross-linking fibres. Primary fibres originated at the cell wall and had a diameter of 34-50 nm. They also contained bulbous protrusions and enlarged areas where branching occurred. Cross-linking fibres connected the primary fibres. The cross-linking fibres were much smaller, usually 15 micro m in diameter, and contained few enlarged areas. The primary fibres originated at sites on the cell wall approximately equidistant and 0.26 micro m apart.

Electron microscopy of Mycoplasma pneumoniae microcolonies grown on solid surfaces.

Mycoplasma pneumoniae sprain CL-8 was studied by using various surfaces for adherence and growth. Cells grown on Epon 812, Formvar, carbon, and glass were of similar morphology. Thin Epon pieces were good material for culturing the organisms and examining thin-sectioned microcolonies by transmission electron microscopy.

Liquid nitrogen cryo-impacting: a new concept for cell disruption.

High-efficiency disruption of bacteria can be accomplished in 2 or more min by the new procedure of liquid nitrogen cryo-impacting. Release of the dipicolinic acid-Ca2+ chelate paralleled the breakage of Bacillus megaterium endospores. Lactate dehydrogenase activity was much better in supernates from liquid nitrogen cryo-impacting-broken Escherichia coli cells than in those from sonically treated and broken E. coli cells.

Babesia sp.: the relationship of stage of development to structure of intra- and extracellular parasites.

The relationship of stage of development to structure in Babesia rodhaini and B. microti was studied by freeze-etching, carbon replication, and thin sectioning. The trophozoites of these Babesia are surrounded by a single membrane over most of their surfaces, but in some regions of the cytoplasm, membranous structures may parallel the plasmalemma, providing a double membrane. Merozoites are produced from trophozoites by budding. An early external indication that a bud will form is the organization of a pellicle complex at the site of the bud. The trophozoite nucleus divides before bud formation starts. The pellicle forms by development of a thick subplasmalemmal layer just under the plasmalemma in a bulging area that contains one of the daughter nuclei. The subplasmalemma layer of the pellicle of babesial merozoites is similar to that of plasmodial merozoites as it is divided into a series of roughly hexagonal plates by a reticular network which resembles coarse chicken wire. Thus, not only are the merozoites of these two species of Babesia budded in a fashion similar to that by which plasmodial merozoites are budded, but they also have similar pellicles.

A mixed bacterial population in a continuous culture with and without kaolinite.

Chromobacterium lividum and a Pseudomonas sp. were grown in pure and mixed continuous culture with and without the clay-mineral, kaolinite. Irrespective of the growth conditions, C. lividum adhered to the wall of the culture vessel whereas the Pseudomonas sp. showed no such tendency, at least visually. During mixed culture studies, the organism which was initially established in the culture dominated. The ratio between C. lividum and the Pseudomonas sp. was about 20:1 when C. lividum was first established and 1:2 when the Pseudomonas sp. was first grown. The indirect fluorescent antibody technique provided a rapid method for differentiating the mixed cultures when the bacterial concentration was sufficient for microscopic analysis. During both pure and mixed continuous culture studies, the addition of kaolinite reduced the C. lividum but not the Pseudomonas sp. population.