Biomedical subjects
Publications and source records attributed to S E Stevens.
The oxidation of heterocyclic sulfur compounds reported to be part of the macrostructure of coal and petroleum was investigated. The oxidation of thianthrene solubilized in 10% dimethylformamide to thianthrene monosulfoxide in the presence of hydrogen peroxide was catalyzed by the ligninase from Phanerochaete chrysosporium.
Loops and braids in filaments of the cyanobacterium Mastigocladus laminosus were observed. Braided filaments were generally in the form of a right-handed helix (87%) but were occasionally observed as left-handed helices (13%). It was demonstrated by time-lapse photomicrography that braids continued to grow as braids and that loops coiled into braids as growth proceeded. Measurements of the distance between grooves in 74 braids yielded an average distance of 13 +/- 3 micron, a result which suggests that braid formation is not random. We propose that the braids arise as a consequence of the helical growth of cells that make up the filaments of M. laminosus.
A blend of nylon fiber and silver-coated nylon fiber (the latter known as X-static) was used in these experiments. This fiber was bactericidal when bacteria were exposed to it directly or to an extract derived from its prior incubation in salt solution. At ambient temperatures, a rapid exponential decrease of survival occurred, usually after a delay of approximately 1 h. The rate of killing (decrease of survival) increased with an increase in X-static percentage of the fiber blend, temperature of fiber extraction, concentration of Tris buffer present during extraction, and temperature at which bacteria were exposed to the extract. When bacteria were exposed to the extract at 37 degrees C as opposed to ambient temperature, there was no delay in onset of killing. Escherichia coli was generally the indicator organism tested, but comparable results were also found for Pseudomonas, Klebsiella, Staphylococcus, and Streptococcus species. The rate of killing increased with increasing silver ion concentration of the fiber extract, as determined through atomic absorption spectrophotometry. The rate of killing was greater and the onset was earlier with an extract containing silver ions from fiber than with a salt solution containing the same concentration of silver ions from silver nitrate. Studies of the kinetics of ion release suggested that X-static may be an effective, sustained-release antibacterial agent.
The cyanobacterium Mastigocladus laminosus produces motile hormogonia which move by gliding motility. These hormogonia were characterized in terms of their morphology, state of differentiation of the cells, optimal temperature for production and motility, minimal nutritional requirements to sustain motility, liberation of the hormogonium from its parental trichome, average surface velocity, and maximal concentration of agar through which the hormogonium may move. We found that an average hormogonium consisted of 13.6 cells of only the narrow-cell-type morphology. Gliding motility and the production of hormogonia were maximal at 45 degrees C. Agarose plus 0.20 mM Ca2+ was sufficient to sustain gliding motility. Hormogonia were liberated from the parental trichome by formation and lysis of a necridium. The average surface velocity of a hormogonium was 1.7 micron/s with a maximal velocity of 3 micron/s. Hormogonia were motile through 7% agar. Motile hormogonia leave a record of their passage in the form of easily visible tracks on the surface of solid media. Three types of tracks were observed: straight, sinusoidal, and circular. Normal, forward-directed motion involves screwlike rotation that describes a right-handed helix. However, observations are presented which suggest that rotational motion is not a prerequisite for gliding motility in this cyanobacterium.
In Cyanophora paradoxa, the allophycocyanin apoprotein subunits, alpha and beta, are encoded in the cyanelle (plastid) genome. These genes were transferred to the cyanobacterium Synechococcus sp. PCC 7002 on a plasmid replicon. Phycobilisomes isolated from transformed cyanobacteria were found to contain C. paradoxa allophycocyanin subunits. Thus, these plastid genes are expressed in the cyanobacterium as polypeptides which become linked to a chromophore and are incorporated into the light-harvesting apparatus.
The isolation of several biosynthetic genes from a cyanobacterium, Agmenellum quadruplicatum, by complementation of auxotrophic mutations in Escherichia coli, and their partial characterization, is described. Although our search for such genes has not been exhaustive, it appears that complementation of E. coli mutations may be of limited utility for the identification and/or isolation of cyanobacterial genes. Despite some overlap in the complementation abilities of these isolated cyanobacterial DNA fragments, the genes that we have studied in some detail are not located in operons. We have used mutagenized versions of these cyanobacterial DNA fragments to produce mutant phenotypes in the cyanobacterium, but clean auxotrophs were not obtained. Complementation of these mutant phenotypes can be obtained when the appropriate wild-type DNA fragment is introduced into the cyanobacterium on a shuttle vector. Recombination between two copies of a cyanobacterial gene occurs at high frequency in the cyanobacterium.
The unicellular cyanobacterium Agmenellum quadruplicatum PR-6 grows in the presence of light on agar containing 10 microM 3-(3,4 dichlorophenyl)-1,1-dimethylurea and 1 to 30 mM glycerol. A derivative strain, PR-6G2, was tolerant of 100 mM glycerol. Photoheterotrophic growth conditions had little effect on transformation competence but did decrease the viability of single cells plated onto agar, particularly cells of the parent strain.
Heterospecific transformation occurred between cyanobacteria currently classified in either the genus Synechococcus or Synechocystis. Cyanobacterial strains 73109 and 6906 were capable of physiological transformation.
A biphasic plasmid vector was used to introduce the Escherichia coli K-12 lac operon into the unicellular cyanobacterium Agmenellum quadruplicatum PR-6. The PR-6 transformants expressed beta-galactosidase at nearly as high a level as did Escherichia coli transformants. In order to accomplish this, it was necessary to obtain PR-6 mutants that could be transformed by plasmids with unmodified recognition sites for the endogenous PR-6 restriction endonuclease Aqu I. These mutants were generated by a variation of the ectopic mutagenesis techniques that have been used in other naturally transforming bacteria. The ability to assay the expression of lacZ in PR-6 paves the way for the construction of gene fusions with various PR-6 promoters and quantitation of their expression under specific in vivo conditions.
The genes for the alpha- and beta-subunit apoproteins of allophycocyanin (AP) were isolated from the cyanelle genome of Cyanophora paradoxa and subjected to nucleotide sequence analysis. The AP beta-subunit apoprotein gene was localized to a 7.8-kilobase-pair Pst I restriction fragment from cyanelle DNA by hybridization with a tetradecameric oligonucleotide probe. Sequence analysis using that oligonucleotide and its complement as primers for the dideoxy chain-termination sequencing method confirmed the presence of both AP alpha- and beta-subunit genes on this restriction fragment. Additional oligonucleotide primers were synthesized as sequencing progressed and were used to determine rapidly the nucleotide sequence of a 1336-base-pair region of this cloned fragment. This strategy allowed the sequencing to be completed without a detailed restriction map and without extensive and time-consuming subcloning. The sequenced region contains two open reading frames whose deduced amino acid sequences are 81-85% homologous to cyanobacterial and red algal AP subunits whose amino acid sequences have been determined. The two open reading frames are in the same orientation and are separated by 39 base pairs. AP alpha is 5' to AP beta and both coding sequences are preceded by a polypurine, Shine-Dalgarno-type sequence. Sequences upstream from AP alpha closely resemble the Escherichia coli consensus promoter sequences and also show considerable homology to promoter sequences for several chloroplast-encoded psbA genes. A 56-base-pair palindromic sequence downstream from the AP beta gene could play a role in the termination of transcription or translation. The allophycocyanin apoprotein subunit genes are located on the large single-copy region of the cyanelle genome.
The effects of nitrogen starvation on the morphology and ultrastructure of the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The internal ultrastructural characteristics of vegetative cells changed markedly during nitrogen starvation. Carboxysomes were degraded, while polyphosphate bodies and lipid bodies accumulated. The ultrastructure of mature heterocysts was also affected by nitrogen starvation; their intracytoplasmic membranes vesiculated to form vacuolelike structures and, eventually, large empty regions in the cytoplasm. Nitrogen starvation stimulated extensive heterocyst differentiation in M. laminosus, producing heterocyst frequencies of 17.5% in narrow filaments and 28.3% in wide filaments within 44 h after transfer to N-free conditions. Cells in wide filaments differentiated so extensively that only 16.8% of them failed to initiate the differentiation process within 44 h.
The genes for the following proteins were localized by hybridization analysis on the cyanelle genome of Cyanophora paradoxa: the alpha and beta subunits of phycocyanin (cpcA and cpcB); the alpha and beta subunits of allophycocyanin (apcA and apcB); the large and small subunits of ribulose-1,5-bisphosphate carboxylase (rbcL and rbcS); the two putative chlorophyll alpha-binding apoproteins of the photosystem I-P700 complex (psaA and psaB); four apoproteins believed to be components of the photosystem II core complex (psbA, psbB, psbC, and psbD); the two apoprotein subunits of cytochrome b-559 which is also found in the core complex of photosystem II (psbE and psbF); three subunits of the ATP synthase complex (atpA and atpBE); and the cytochrome f apoprotein (petA). Eighty-five percent of the genome was cloned as BamHI, BglII, or PstI fragments. These cloned fragments were used to construct a physical map of the cyanelle genome and to localize more precisely some of the genes listed above. The genes for phycocyanin and allophycocyanin were not clustered and were separated by about 25 kilobases. Although the rbcL gene was adjacent to the atpBE genes and the psbC and psbD genes were adjacent, the arrangement of other genes encoding various polypeptide subunits of protein complexes involved in photosynthetic functions was dissimilar to that observed for known chloroplast genomes. These results are consistent with the independent development of this cyanelle from a cyanobacterial endosymbiont.
Phycocyanin (PC) is a light-harvesting protein common to blue-green and red algae. We have isolated the genes for the two apoprotein subunits, alpha and beta, of PC from the blue-green alga Agmenellum quadruplicatum PR-6. We synthesized eight sense-strand tetradecameric oligonucleotide probes that could encode a particular pentapeptide in PC alpha from A. quadruplicatum. Only one probe hybridized with total RNA from this organism. This oligonucleotide showed homology to a unique restriction fragment when used to probe Southern blots of A. quadruplicatum DNA. The probe-homologous 3.1-kilobase pair HindIII fragment was cloned. The nucleotide sequence of a 1.7-kilobase pair segment of this clone was determined. Two open reading frames are contained in this region, which correspond in deduced amino acid sequence to PC alpha and PC beta subunits. Both coding sequences are in the same orientation, separated by 105 base pairs, with the PC beta gene 5' to the PC alpha gene. Each gene has a Shine-Dalgarno-type sequence near the initiation codon. Codon frequencies in the two genes may be correlated with the abundance of their products. The deduced amino acid sequences of the gene products show considerable homology with the alpha and beta PC subunits from other species.
The morphological and ultrastructural aspects of heterocyst differentiation in the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The earliest differentiation stages involved cytoplasmic changes, including (i) rapid degradation of carboxysomes, (ii) degradation of polysaccharide granules, and (iii) accumulation of electron-dense ribosomal or protein material (or both). Intermediate differentiation stages involved synthesis of a homogeneous extra wall layer, development of necks leading to adjacent cells, and elaboration of a complex system of intracytoplasmic membranes. Late differentiation stages included further development of necks and continued elaboration of membranes. Mature heterocysts possessed a uniformly electron-dense cytoplasm that contained large numbers of closely packed membranes, some of which were arranged in lamellar stacks. Mature heterocysts lacked all of the inclusion bodies present in undifferentiated vegetative cells, but contained a number of unusual spherical inclusions of variable electron density. Cells in both narrow and wide filaments were capable of differentiating. No regular heterocyst spacing pattern was observed in the narrow filaments; the number of vegetative cells between consecutive heterocysts of any given filament varied by a factor of 10. Heterocysts developed at a variety of locations in the wide, branching filaments, although the majority of them were situated adjacent to branch points. M. laminosus displayed a marked tendency to produce sets of adjacent heterocysts or proheterocysts (or both) that were not separated from each other by vegetative cells. Groups of four or more adjacent heterocysts or proheterocysts occurred frequently in wide filaments, and in some of these filaments virtually all of the cells appeared to be capable of differentiating into heterocysts.
A new light microscopic method for identifying heterocysts and proheterocysts in morphologically complex cyanobacteria was evaluated for reliability and usefulness. Mature heterocysts and proheterocysts could be distinguished readily from vegetative cells in 0.25 micron sections of fixed and embedded material after staining with toluidine blue. Examination by light and electron microscopy of the same specimens indicated that the staining reactions which served to differentiate these cell types were both reproducible and accurate. Light microscopic analysis of serial sections stained with toluidine blue greatly facilitated localization of heterocysts and proheterocysts in the complex, branching cyanobacterium, Mastigocladus laminosus, even when its filaments of cells were intertwined in thick mats.
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A computer-aided reconstruction system was evaluated in regard to its usefulness for illustrating the three-dimensional ultrastructure of cyanobacterial (blue-green algal) cells. The system readily depicted the intracellular locations of specialized inclusion bodies. The photosynthetic thylakoid membrane system was more difficult to depict because of its intricate substructure, yet could be illustrated effectively by using procedures designed to minimize multiple overlapping of thylakoid components. By eliminating unwanted or unimportant cell structures, by examining selected portions of cells, and by rotating images to obtain maximum clarity, it was possible to produce reconstructions that effectively showed the overall three-dimensional arrangement of ultrastructural features within the cell.
The first complete three-dimensional ultrastructural reconstruction of a cyanobacterium was accomplished with high-voltage electron microscopy and computer-aided assembly of serial sections. The precise arrangement of subcellular features within the cell body was very consistent from one cell to another. Specialized inclusion bodies always occupied specific intracellular locations. The photosynthetic thylakoid membranes entirely surrounded the central portion of the cytoplasm, thereby compartmentalizing it from the rest of the cell. The thylakoid membranes formed an interconnecting network of concentric shells, merging only at the inner surface of the cytoplasmic membrane. The thylakoids were in contact with the cytoplasmic membrane at several locations, apparently to maintain the overall configuration of the thylakoid system. These results clarified several unresolved issues regarding structure-function relationships in cyanobacteria.