Photolyase confers resistance to UV light but does not contribute to the symbiotic benefit of bioluminescence in Vibrio fischeri ES114

Recent reports suggest that the selective advantage of bioluminescence for bacteria is mediated by light-dependent stimulation of photolyase to repair DNA lesions. Despite evidence for this model, photolyase mutants have not been characterized in a naturally bioluminescent bacterium, nor has this hypothesis been tested in bioluminescent bacteria under natural conditions. We have now characterized the photolyase encoded by phr in the bioluminescent bacterium Vibrio fischeri ES114. Consistent with Phr possessing photolyase activity, phr conferred light-dependent resistance to UV light. However, upon comparing ES114 to a phr mutant and a dark Delta luxCDABEG mutant, we found that bioluminescence did not detectably affect photolyase-mediated resistance to UV light. Addition of the light-stimulating autoinducer N-3-oxo-hexanoyl homoserine lactone appeared to increase UV resistance, but this was independent of photolyase or bioluminescence. Moreover, although bioluminescence confers an advantage for V. fischeri during colonization of its natural host, Euprymna scolopes, the phr mutant colonized this host to the same level as the wild type. Taken together, our results indicate that at least in V. fischeri strain ES114, the benefits of bioluminescence during symbiotic colonization are not mediated by photolyase, and although some UV resistance mechanism may be coregulated with bioluminescence, we found no evidence that light production benefits cells by stimulating photolyase in this strain.     

The regulation of tissue plasminogen activator activity by human fibroblasts

We have found that live and ethanol-fixed fibroblasts, when covered with conditioned medium containing tissue plasminogen activator, associate with the enzyme and remove it from the medium. Binding of tissue plasminogen activator to fixed cells showed equilibrium kinetics with maximal uptake corresponding to 2.4 units of enzyme per 10(6) fixed cells. Enzyme bound to fixed cells could activate plasminogen and produce plaques of caseinolysis in casein-plasminogen-agar overlays. Electrophoretic analysis showed it covalently attached to a fibroblast component with a molecular weight of 40,000-50,000. Sequestration of tissue plasminogen activator by live fibroblasts showed nonsaturable first order kinetics with a rate constant of 0.465/hr. We conclude that active enzyme is bound to a surface receptor, then internalized and degraded. Fibroblasts did not release the binding molecule into the medium; binding of tissue plasminogen activator from the medium was unaffected by heparin or thrombin. This phenomenon differs from that described by Baker et al. and ascribed to "proteasenexin."     

Evolutionary perspectives in a mutualism of sepiolid squid and bioluminescent bacteria: combined usage of microbial experimental evolution and temporal population genetics

The symbiosis between marine bioluminescent Vibrio bacteria and the sepiolid squid Euprymna is a model for studying animal-bacterial Interactions. Vibrio symbionts native to particular Euprymna species are competitively dominant, capable of outcompeting foreign Vibrio strains from other Euprymna host species. Despite competitive dominance, secondary colonization events by invading nonnative Vibrio fischeri have occurred. Competitive dominance can be offset through superior nonnative numbers and advantage of early start host colonization by nonnatives, granting nonnative vibrios an opportunity to establish beachheads in foreign Euprymna hosts. Here, we show that nonnative V. fischeri are capable of rapid adaptation to novel sepiolid squid hosts by serially passaging V. fischeri JRM200 (native to Hawaiian Euprymna scolopes) lines through the novel Australian squid host E. tasmanica for 500 generations. These experiments were complemented by a temporal population genetics survey of V. fischeri, collected from E. tasmanica over a decade, which provided a perspective from the natural history of V. fischeri evolution over 15,000-20,000 generations in E. tasmanica. No symbiont anagenic evolution within squids was observed, as competitive dominance does not purge V. fischeri genetic diversity through time. Instead, abiotic factors affecting abundance of V. fischeri variants in the planktonic phase sustain temporal symbiont diversity, a property itself of ecological constraints imposed by V. fischeri host adaptation.     

An inhibitor-like binding mode of a carbonic anhydrase activator within the active site of isoform II

The 2,4,6-trimethylpyridinium derivative of histamine is an effective activator of the zinc enzyme carbonic anhydrase (CA, EC 4.2.1.1). However, unlike other CA activators, which bind at the entrance of the active site cavity, an X-ray crystal structure of hCA II in complex with the 1-[2-(1H-imidazol-4-yl)-ethyl]-2,4,6-trimethylpyridinium salt evidenced a binding mode never observed before either for activators or inhibitors of this enzyme, with the 2,4,6-trimethylpyridinium ring pointing towards the metal ion deep within the enzyme cavity, and several strong hydrophobic interactions stabilizing the adduct. Indeed, incubation of the activator with the enzyme for several days leads to potent inhibitory effects. This is the first example of a CA activator which after a longer contact with the enzyme behaves as an inhibitor.     

Localisation of luciferase in luminous marine bacteria by gold immunocytochemical labelling

Abstract Bioluminescent bacteria are able to produce light by an oxidative reaction involving the enzyme luciferase. The biochemistry of the reaction has been well documented. It has been postulated from the reaction pathway that the enzyme luciferase is membrane bound. Two species of luminous bacteria, Vibrio fischeri and V. harveyi , were isolated from sea water, fixed and embedded in resin for transmission electron microscopy. A commercially available V. fischeri bacterial luciferase was used to immunise rats for production of an antibody against luciferase. This antibody was coupled with a protein A gold probe and used to immunolabel the sections of bacteria. It was shown that the luciferase was associated with the inner membrane, and to a lesser extent present in the cytoplasm. Our results confirm that the luciferase is predominantly membrane bound.     

Oxygen metabolism of catalase-negative and catalase-positive strains of Lactobacillus plantarum.

Two catalase-negative strains of Lactobacillus plantarum and a strain producing the atypical, nonheme catalase were studied to determine if the ability to produce the atypical catalase conferred any growth advantage upon the producing strain. Both catalase-negative strains grew more rapidly than the catalase-positive strain under aerobic or anaerobic conditions in a glucose-containing, complex medium. Upon exhaustion of glucose from the medium, all three strains continued growth under aerobic but not under anaerobic conditions. The continued aerobic growth was accompanied by production of acetic acid in addition to the lactic acid produced during growth on glucose. Oxygen was taken up by exponential phase-cell suspensions grown on glucose when glucose or glycerol were used as substrates. Cells harvested from glucose-exhausted medium oxidized glucose, glycerol, and pyruvate. Oxygen utilization by a catalase-negative strain increased as did the specific activity of reduced nicotinamide adenine dinucleotide peroxidase during late growth in the glucose-exhausted medium. The catalase-positive strain and the catalase-negative strain tested both possessed low but readily detectable levels of superoxide dismutase throughout growth. The growth responses are discussed in terms of the presence of enzymes which would allow the cells to remove potentially damaging reduction products of O2.     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.     

Novel properties of human monocyte plasminogen activator

Human peripheral monocytes stimulated by either muramyl dipeptide [N-acetyl-muramoyl-L-alanyl-D-isoglutamine], bacterial lipopolysaccharide or lymphokine-containing supernatants of human lymphocytes, could be shown to produce and secrete appreciable activities of a 52 000-Mr plasminogen activator. This enzyme was suppressed in control and stimulated cultures by dexamethasone (0.1 microM). Monocyte plasminogen activator could only be assayed under conditions of low ionic strength and had no detectable activity at 0.15 M NaCl. Intracellular enzyme was present as a proenzyme, requiring activation by preincubation with plasminogen containing traces of plasmin, before its activity could be seen on sodium dodecyl sulphate/polyacrylamide gel electrophoresis by a fibrin overlay method. Secreted enzyme was in the active form. Further incubation of lysate or supernatant plasminogen activator with plasminogen did not produce any active enzyme species of Mr 36 000, unlike incubations of urokinase with plasminogen. Moreover, comparisons with other plasminogen activators of Mr 52 000 from transformed cell lines showed that the monocyte activator was unique in its resistance to monocyte minactivin, a specific inactivator of urokinase-type plasminogen activators, and in its sensitivity to human alpha 2-macroglobulin. It was therefore concluded that human monocyte plasminogen activator, although sharing an Mr of 52 000 in common with other such activators, is not identical to the high Mr form of urokinase or the plasminogen activators of transformed cells. On present evidence it is the least likely of these enzymes to be active extracellularly under normal physiological conditions.     

Construction and detection of bioluminescent strains of Bacillus subtilis

Bioluminescence ( lux ) genes from Vibrio fischeri and V. harveyi were introduced into Bacillus subtilis on a plasmid vector and by chromosomal integration. The plasmid-bearing strain was highly luminescent and stable under antibiotic selection, but luminescence was lost in the absence of selection and following sporulation and germination. The chromosomally marked strains emitted less light but were found to be stable without the requirement for antibiotic selection and following sporulation and germination. Individual luminescing colonies of both B. subtilis strains could be detected against a high background of non-bioluminescent indigenous soil microbial colonies on agar plates using a charge-coupled device camera. These bioluminescent Gram-positive strains could be of value in studies concerning the survival and spread of genetically-modified micro-organisms in soil environments.     

Bioluminescence of Vibrio fischeri in continuous culture: optimal conditions for stability and intensity of photoemission

Continuous cultivation of the bioluminescent bacterium, Vibrio fischeri NRRL-B-11177, in a fermenter provided a constant supply of bacteria in exponential growth phase. These bacteria may be used as stable bioindicators to measure perturbation of the metabolic state by physical and chemical agents. However, optimization of light emission necessitates careful choice of growth medium and culture operating conditions. Optimized conditions that supported long-term cultivation of V. fischeri NRRL-B-11177 with stable intense bioluminescence were: a dilution rate of 0.08-0.09h(-1), air supply of 600mlmin(-1), stirring of 300-350rpm at a constant incubation temperature within the range of 20 to 26 degrees C and pH7.8. These conditions were as successful in a 500-ml as in a 10-ml fermenter. This system provided a reliable long-term (more than 1 month) continuous culture facility for the reproducible measurement of perturbation of V. fischeri by monitoring changes in luminescence.     

Classification of ethylene-producing bacteria in terms of biosynthetic pathways to ethylene

Two hundred and twenty-nine ethylene-producing strains of bacteria were identified among 757 bacterial strains which included 13 strains of chemolithotrophs. The ethylene-producing bacetria were classified into three groups, namely, l-methionine-dependent, 2-ketoglutarate-dependent and meat extract-dependent, with reference to their respective biosynthetic pathways to ethylene. Two hundred and twenty-five l-methionine-dependent strains were obtained, while the only 2-ketoglutarate-dependent strain was Pseudomonas syringae pv. phaseolicola PK2. Three strains of chemolithotrophs had ethylene-forming capacity, and Thiobacillus novellus IFO 12443 had a novel ethylene-forming system which was dependent upon the addition of meat extract into the culture medium. The ethylene-forming systems of two of the strains of Thiobacillus ferrooxidans have not yet been characterized. Several strains of non-ethylene-producing bacteria failed to produce ethylene, even when l-methionine was added to the culture medium. We examined the causes of their lack of ethylene-producing ability and judged that these strains are either NADH:Fe(III)EDTA oxidoreductase-less or methionine-uptake activity-less.     

Growth and siderophore production inBradyrhizobium (lupin) strains under iron limitation

SixBradyrhizobium (lupin) strains were evaluated for their ability to produce siderophores using four chemical assays. Two strains gave positive reactions with chrome azurol S assay (CAS) and produced hydroxamate-type siderophores. The other four strains gave negative results for siderophore production using the four assays. Generation time, growth yield and hydroxamate production of one strain (WPBS 3201 D) were affected by the iron concentration of the culture medium and the previous culture history of the cells. Resuspension of washed cells grown previously in media supplemented with 0 and 20 μmol/L Fe into differing iron regimes (0, 0.5, 1, 2, 4, 8, 10, 15 and 20 μmol/L Fe) suggest that the extent of hydroxamate production depended on the growth history of the cells. Cells pregrown in 20 μmol/L Fe produced a high amount of hydroxamates compared with cells pregrown in iron-free medium when resuspended in medium containing up to 4 μmol/L Fe. Cells pregrown in 20 μmol/L Fe were more sensitive to iron repression than those pregrown in 0.5 μmol/L Fe. Mannitol was the best carbon source for siderophore production. Siderophore synthesis was inhibited by 4-chloromercuribenzenesulfonic acid, 2,4-dinitrophenol, sodium azide and MgCl₂ suggesting that an energized membrane and a mercapto group are essential and required for hydroxamate synthesis in strain WPB5 3201 D.     

Isolation of the major serine protease inhibitor from the 5-day serum-free conditioned medium of human embryonic lung cells and demonstration that it is fetuin

Human embryonic lung (HuEL) cells in culture produce large amounts of the enzyme, plasminogen activator, and thus generate substantial amounts of active plasmin. Despite the presence of plasmin, however, HuEL cells grow in ordered, flattened, adherent sheets. It seemed of interest to characterize protease inhibitors that might be present in HuEL cultures and which might account for this apparent contradiction. This paper reports the isolation and purification of the major serine protease inhibitor in 5-day serum-free conditioned medium (CM) from HuEL cells, and the purification of an identical molecule from fetal bovine serum (FBS). Both the CM-derived inhibitor and the FBS-derived inhibitor are identical with fetuin, the major glycoprotein of FBS. The CM-derived inhibitor is apparently derived from the FBS used to supplement the growth medium of HuEL cells between serum-free CM collection periods. It is not labeled metabolically with 3H-leucine. Its electrophoretic behavior is indistinguishable from that of standard fetuin in SDS-PAGE, non-SDS basic pH,PAGE, and isoelectric focusing. The CM-derived inhibitor and standard fetuin inhibit trypsin and plasmin with similar efficiencies, but neither inhibits chymotrypsin, pancreatic elastase, or plasminogen activator. They are immunologically indistinguishable. The suggestion is made that fetuin, and possibly other protease inhibitors present in HuEL cell cultures, may be concentrated locally by HuEL cells and gradually released back into the medium in the absence of serum. These molecules may serve to protect HuEL cells against proteases they generate.     

Plasminogen activator in the rat ovary. Production and gonadotropin regulation of the enzyme in granulosa and thecal cells

The production of plasminogen activator by ovarian granulosa cells has been previously reported to be temporally correlated with ovulation in the rat and to be under hormonal control of gonadotropins. We have examined the type of plasminogen activator produced by granulosa cells and also investigated other ovarian cell types for synthesis of this enzyme. Using antibodies specific for tissue-type or urokinase-type plasminogen activator, we have found that granulosa cells produce exclusively the tissue-type enzyme. However, in cultures of whole follicles isolated from the ovary, there is primarily synthesis of urokinase-type plasminogen activator. Examination of other isolated ovarian cell types has demonstrated that thecal cells secrete the urokinase-type plasminogen activator and that the production of this enzyme is also regulated by gonadotropins and temporally correlated with ovulation. These results suggest that ovulation requires both types of plasminogen activator and that the neighboring granulosa and thecal cells cooperate to ensure rupture of the follicle wall and unimpeded passage of the ovum into the oviduct.     

ATP:AMP phosphotransferase from baker''s yeast. Purification and properties

Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14-h/10-h light/dark regime, contain a peak of ribonucleoside-diphosphate reductase activity and maximum deoxyribonucleoside 5'-triphosphate concentrations at the 12th hour of the cell cycle, coinciding with DNA synthesis and preceding the formation of eight daughter cells. The intracellular dTTP pool reaches 4.5 pmol and the other pools 2-3 pmol/10(6) cells. Algal reductase activity is sensitive to cycloheximide, but not to lincomycin. These correlations demonstrate the functioning of the NDP leads to dNDP leads to dNTP pathway of DNA precursor biosynthesis in plant cells. In the presence of 20 micrograms 5-fluorodeoxyuridine/ml, an inhibitor of thymidylate synthesis, the dTTP pool is rapidly depleted and DNA synthesis ceases. 5-Fluorouracil and methotrexate produce similar effects. At the same time the ribonucleotide reductase activity and also the dATP pool are greatly increased, especially when fluorodeoxyuridine treatment is combined with continued illumination of the algae. In contrast, arabinosylcytosine, an inhibitor of DNA replication, has no effect on ribonucleotide reduction. The control of de novo enzyme synthesis in the eucaryotic algae therefore appears to depend on the presence of dTTP (or a related nucleotide), but not directly coupled to DNA synthesis. This interdependence resembles the situation observed in HeLa cells, while it may differ in detail from control mechanisms of ribonucleotide reductase studied in bacteria.