The nucleotide sequence of the puf operon from the purple photosynthetic bacterium, Rhodospirillum molischianum: Comparative analyses of light-harvesting proteins and the cytochrome subunits associated with the reaction centers

The nucleotide sequence of the puf operon of the purple bacterium, Rhodospirillum molischianum, was determined. The operon includes genes coding for the and subunits of the light-harvesting 1 (LH1) complex and the L, M, and cytochrome subunits of the reaction center complex. As in other purple bacteria, the genes are arranged within the operon in this order. As in Rubrivivax gelatinosus, the deduced amino acid sequence of the cytochrome subunit in Rsp. molischianum contains significant deletions at the attachment site to the M subunit compared with that of Rhodopseudomonas viridis. This suggests that the interaction between the cytochrome subunit and the LM core in Rsp. molischianum and Rvi. gelatinosus is different from that in Rps. viridis. Phylogenetic analysis of the light-harvesting proteins indicated that the LH1 and subunits of Rsp. molischianum are included in the lineage of LH1 polypeptides of the purple bacteria, while the LH2 and subunits are positioned apart from LH2 polypeptides of the other purple bacteria together with those of Chromatium vinosum. Based on these phylogenetic analyses, the classification of the light-harvesting proteins in purple bacteria is discussed.     

Defining patch contribution in source-sink metapopulations: the importance of including dispersal and its relevance to marine systems

In metapopulations, individual patch contribution (source or sink) is typically calculated as a patch growth rate (the intrinsic lambda, _I) dependent only upon local demographics. We demonstrate that when dispersal is explicitly included in the model, the growth rates for all patches calculated in an analogous manner (the observed lambda, _O) equilibrate to the overall metapopulation growth rate and thus no longer serve as a useful reflection of the demographic and dispersive characteristics of a given patch. In these situations we suggest an alternative method of estimating patch contribution (the contribution lambda, _C) in which a patch is decremented for losses that occur within it and credited for gains that occur anywhere in the metapopulation because of it. We compare values of _I, _O, and _C for individual patches in discrete-time density-independent metapopulation models of two organisms with very different life histories, mayflies with adult dispersal, and reef fish with larval dispersal. Results confirm that when dispersal is included only _C clearly indicates the contribution of a particular patch. _I–_C comparisons indicate that inclusion of dispersal in the mayfly model was only important if connectivity patterns were random or directional. In the reef fish model, however, results were very different when dispersal was included and there were many cases of patches being misidentified (e.g., as a source when it was really a sink) depending upon the metric used (_I or _C). Our results demonstrate the importance of including dispersal in metapopulation models when considering the contribution of individual patches.     

Wide hybridization in okra

Summary Crosses were made between members of the two West African okra types Soudanien and Guineen. All crosses succeeded in both directions and the F₁ plants which showed hybrid vigour for plant stature were partially sterile. Cytological observations of the F₁ plants revealed abnormal meiosis which resulted in the production of microspores of variable sizes. The frequency of viable pollen (as indicated by acetocarmine staining) was low in the hybrids: 35.80% (U.I.92× U.I.313) and 39.41% (1bk-1×U.I.215). The number of seeds produced per fruit was low in the hybrids and only a few of these seeds are viable. The possibility of gene transfer between the two okra types was discussed.     

Control of molecular weight of poly-β-hydroxybutyric acid produced in fet-batch culture of Protomonas extorquens

Summary To control molecular weight of poly--hydroxybutyric acid (PHB) produced in a fedbatch culture of Protomonas extorquens, the effects of cultural temperature, pH, molar ratio of methanol and ammonia, and concentration of methanol in the medium on polymerization were inverstigated. Change of methanol concentration affected average molecular weight of PHB. When the cultivation was carried out at 0.05 g/l of methanol, average molecular weight of PHB reached above 8×10⁵. On the other hand, in the case of 32 g/l of methanol average molecular weight of PHB was less than 0.5×10⁵. Although every sample had a wide molelcular weight distribution, it became possible to control voluntarily average molecular weight of PHB.     

Globally asymptotic properties of proliferating cell populations

This paper presents a general model for the cell division cycle in a population of cells. Three hypotheses are used: (1) There is a substance (mitogen) produced by cells which is necessary for mitosis; (2) The probability of mitosis is a function of mitogen levels; and (3) At mitosis each daughter cell receives exactly one-half of the mitogen present in the mother cell. With these hypotheses we derive expressions for the and curves, the distributions of mitogen and cell cycle times, and the correlation coefficients between mother-daughter (_md) and sister-sister (_ss) cell cycle times.The distribution of mitogen levels is shown to be given by the solution to an integral equation, and under very mild assumptions we prove that this distribution is globally asymptotically stable. We further show that the limiting logarithmic slopes of (t) and (t) are equal and constant, and that _md0 while _ss0. These results are in accord with the experimental results in many different cell lines. Further, the transition probability model of the cell cycle is shown to be a simple special case of the model presented here.     

Growth characteristics of small and large free-living and attached bacteria in Lake Constance

The growth characteristics of small (0.2–1.0m) and large (1.0–3.0 (m) free-living and attached bacteria were studied in Lake Constance by comparing the spatial and seasonal dynamics of their biomass turnover time (ratio of biomass/production). The biomass of small free-living bacteria usually turned over significantly faster than that of large free-living bacteria throughout the water column. The turnover of attached bacterial biomass was characterized by large fluctuations. Occasionally, in aphotic water layers, it was as long as that of large free-living bacteria, but when large amounts of decaying organic particles were present, it was shorter than that of small free-living cells. Biomass turnover times of free-living bacteria were in the same range as their generation times, which were estimated from the increase in bacterial abundance in 3m prefiltered samples. The biomass turnover time of actively metabolizing bacteria was comparable to the generation time of actively metabolizing cells. These results indicate that the biomass turnover time is a useful indicator of the growth of different bacterial fractions, as it reflects their different amounts of participation in microbial processes of aquatic ecosystems.     

Mercury,cadmium, zinc,copper and selenium in harbour porpoise (Phocoena phocoena) from West Greenland

Muscle, liver, kidney and skin samples taken from 78 harbour porpoises (Phocoena phocoena) were analysed for mercury, cadmium, zinc, copper and selenium. The highest concentrations of mercury were found in the liver (geometric mean 4.17 g/g wet weight), whilst the highest concentrations of cadmium were in the kidney (g.m. 13.2 g/g ww). The levels of cadmium were more than ten times higher than in harbour porpoises from the North Sea and the British NW coast, whilst the mercury levels were about the same. The importance of the cadmium content in the prey is discussed, but this attempt did not revealed the differences. Very high levels of zinc (g.m. 359 g/g ww) and selenium (g.m. 28.6 g/g ww) were found in skin samples, respectively seven and ten times more than in liver. A significant correlation was found between age and the level of mercury and cadmium in all organs. The concentration of mercury and selenium in liver and skin samples and of cadmium and zinc in kidney samples were highly correlated.     

Production of poly-β-hydroxybutyrate by Azotobacter vinelandii UWD in media containing sugars and complex nitrogen sources

Summary Vigorously aerated batch cultures of Azotobacter vinelandii UWD formed < 1 g poly--hydroxybutyrate (PHB)/l in media containing pure sugars and 3 g PHB/l in media containing cane molasses, corn syrup or malt extract. However, > 7 g PHB/l was formed when the medium contained 5% beet molasses. Increased yields of PHB were promoted in the media containing pure or unrefined sugars by the addition of complex nitrogen sources. The greatest effect was obtained with 0.05–0.2% fish peptone (FP), proteose peptone no. 3 or yeast extract. Peptones caused a 1.6-fold increase in residual non-PHB biomass and up to a 25-fold increase in PHB content. Hence the increased PHB formation was not simply due to stimulation of culture growth. The amount of PHB per cell protein formed by UWD in media containing FP was greatest in glucose = corn syrup > malt extract > sucrose = fructose = cane molasses > maltose, as carbon sources. The addition of FP to medium containing beet molasses did not stimulate PHB yield. The peptone effect was most significant in well-aerated cultures, which were fixed nitrogen and consuming glucose at a high rate. An explanation for the peptone effect on PHB yield stimulation is proposed.     

Investigations into the number of respiring bacteria in groundwater from sandy and gravelly deposits

Samples were collected from organically polluted and unpolluted groundwater of sandy and gravelly deposits. After filtration onto polycarbonate filters (0.2m pore size) the number of respiring bacteria was recorded by microscopically counting cells containing red INT-formazan spots, which characterize respiring bacteria. The total number of bacteria was simultaneously recorded by epifluorescence microscopy after staining with acridine orange. The number of respiring bacteria in the groundwater samples (55–490×10³/cm³) is within the range of values for other aquatic biotopes, but as the total number of bacteria in groundwater was usually higher, the proportion of respiring groundwater bacteria (0.66–7. 4%) was lower. Mainly larger bacteria, rods, and bacteria on particles could be identified as being active, whereas hardly any respiratory activity could be detected among small cocci and free interstitial bacteria. If the supply of dissolved organic matter (DOM) is adequate, the biomass of respiring bacteria correlates well with oxygen concentration, but there is no direct correlation between DOM concentration in groundwater and active bacterial biomass. Nor could any relationship be observed between the biomass of total and respiring bacteria, or between the quantity of respiring bacteria and heterotrophic bacterial activity.     

Changes in membrane functions during short-term starvation of Vibrio fluvialis strain NCTC 11328

The marine bacterium Vibrio fluvialis strain NCTC 11328 responded to starvation conditions by forming ultramicrocells of dwarf bacteria. The viability of starved cells began to decrease after 2–3 days. During this time the respiratory potential of the bacteria decreased by four- or fivefold, most probably as a result of a decrease in the specific activity of NADH and succinate dehydrogenases. Although respiratory potential in starving cells was lower than in growing cells, bacteria starved for 1 or 2 days maintained a proton motive force that was slightly larger than that of growing bacteria. Starved bacteria contained substantial concentrations of ATP although the UTP and GTP concentrations were much lower in starved than in growing cells. Two or three proteins that were not present in membranes of growing cells, were evident in the membranes of starved bacteria.Abbreviations MMS modified Morita's salts - MMSGC modified Morita's salts plus 20 mM glucose and 0.1% (w/v) casamino acids - MMST modified Morita's salts buffered with 50 mM tricine, (pH 8.5) - NM broth nutrient modified Morita's salts - CFU colony-forming unit - TPP tetraphenylphosphonium - STM 0.1 M tricine, (pH 8.0) plus 0.25 M sucrose and 0.02 M magnesium acetate - DCPIP dichlorophenolindophenol - CCCP carbonyl cyanidem-chlorophenylhydrazone - PMF proton motive force     

Grazing of attached bacteria by heterotrophic microflagellates

Four species of heterotrophic microflagellates were examined for their ability to graze attached and unattached bacteria. The species tested displayed pronounced differences in their ability to graze the bacteriumPseudomonas halodurans attached to chitin particles. Two species of microflagellates (Monas andCryptobia sp.) efficiently grazed unattached bacteria but showed little or no ability to graze attached or aggregated cells. In contrast,Rhynchomonas nasuta andBodo sp. showed marked preferences for attached and aggregated bacteria and a limited ability to graze unattached cells. The density of attached bacteria was reduced by an order of magnitude due to grazing byBodo andR. nasuta, even though the density of unattached bacteria was 5–90× the density of attached cells. The maximum densities attained by microflagellates in the cultures were related to the density of unattached bacteria forMonas andCryptobia but not forBodo andR. nasuta. Growth of the latter two species appeared to be related to the density of attached or aggregated bacteria. Based on the results of these experiments, it is concluded that the pelagic existence of microflagellates that graze attached bacteria may be strongly linked to the distribution of suspended particles and their associated bacteria. In addition, the removal of attached bacteria by microflagellates can significantly affect the density of bacteria attached to particles in the plankton. This activity may have important implications for the controversy concerning the relative importance of attached and free-living bacteria in the plankton.     

The availability of microorganisms attached to sediment particles as food for Hydrobia ventrosa Montagu (Gastropoda: Prosobranchia)

Summary The deposit-feeding prosobranch Hydrobia ventrosa Montagu feeds most rapidly upon sediment particles that pass through a 10 m sieve. Ingestion rate decreases with particles 80–125 m, then increases with larger particles, which are fed upon by scraping fine material from their surfaces. Hydrobia is capable of digesting diatoms and bacteria from sediment particles, but with generally lower efficiencies than reported when fed pure cultures. Digestion of microorganisms appears to be constrained by ability of the snail to detach cells from sediment particles; only those cells detached from sediment seem to be available for digestion. In contrast, the amphipod Corophium volutator is capable of utilizing most of the diatoms not digested by Hydrobia. For a given sediment, a constant number of microorganisms appear to be safe from digestion by H. ventrosa, and bacteria and microalgae over this amount constitutes the available food.     

Increased poly-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activity in recombinant Escherichia coli

A second promoter (P1) was inserted to the PHA (poly--hydroxyalkanoate) operon (pSP2) of Esherichia coli DH5 with an optimal E. coli ribosome binding site and a trc strong promoter (pSJS1) to obtain poly--hydroxybutyrate (PHB) with long chain length. When the inducer, IPTG was added to the culture at 0.4 mM, the average molecular weight was 1.1 × 10⁶ Da. However, an even greater increase of the PHB average molecular weight to 2.5 × 10⁷ Da was observed without IPTG being added.     

Dynamics of Activity of the Key Enzymes of Polyhydroxyalkanoate Metabolism in Ralstonia eutropha B5786

The dynamics of accumulation of polyhydroxybutyrate (PHB) and the activities of key enzymes of PHB metabolism (-ketothiolase, acetoacetyl-CoA reductase, PHB synthase, D-hydroxybutyrate dehydrogenase, and PHB depolymerase) in the hydrogen bacterium Ralstonia eutropha B5786 were studied under various conditions of carbon nutrition and substrate availability. The highest activities of -ketothiolase, acetoacetyl-CoA reductase, and PHB synthase were recorded during acceleration of PHB synthesis. The activities of enzymes catalyzing PHB depolymerization (PHB depolymerase and D-hydroxybutyrate dehydrogenase) were low, being expressed only upon stimulated endogenous PHB degradation. The change of carbon source (CO₂ or fructose) did not affect the time course of the enzyme activity significantly.     

Growth and comparative physiology ofKlebsiella oxytoca attached to granular activated carbon particles and in liquid media

Experiments were performed to evaluate the comparative growth and physiology ofKlebsiella oxytoca grown attached to granular activated carbon particles (GAC) and in liquid medium. Laboratory studies showed that when this organism attached to GAC, the growth rate was enhanced more than 10 times in the presence of glutamate, a substrate that adsorbed to the surface. No differences were observed if the substrate was glucose, which did not adsorb to GAC. Cellular [³H]thymidine uptake was used to estimate DNA biosynthesis. Attached bacteria grown in a minimal nutrient medium containing 20.0 mg/liter glutamate took up 5 times more [³H]thymidine than cells grown in suspension. [³H]uridine was used as a measure of RNA turnover. Attached cells were shown to assimilate 11 times more [³H]uridine than cells in liquid media. Cell size measurements were performed by differential filtration. Cells grown in a minimal medium with 20.0 mg/liter glutamate decreased in size over time, with 62% of the total number passing through a 1.0m filter after 9 days incubation. In the same period, 39% of a cell population that was grown on GAC passed through a 1.0m filter. These studies indicate that GAC provides an interfacial environment for the enhanced growth ofK. oxytoca when glutamate is the substrate.     

High-cell-density production of poly-β-hydroxybutyrate (PHB) from methanol by Methylobacterium extorquens: production of high-molecular-mass PHB

Methylobacterium extorquens ATCC 55366 was successfully cultivated at very high cell densities in a fed-batch fermentation system using methanol as a sole carbon and energy source and a completely minimal culture medium for the production of poly--hydroxybutyrate (PHB). Cell biomass levels were between 100 g/l and 115 g/l (dry weight) and cells contained between 40% and 46% PHB on a dry-weight basis. PHB with higher molecular mass values than previously reported for methylotrophic bacteria was obtained under certain conditions. Shake-flask and fermentor experiments showed the importance of adjusting the mineral composition of the medium for improved biomass production and higher growth rates. High-cell-density cultures were obtained without the need for oxygen-enriched air; once the oxygen transfer capacity of the fermentor was reached, methanol was thereafter added in proportion to the amount of available dissolved oxygen, thus preventing oxygen limitation. Controlling the methanol concentration at a very low level (less than 0.01 g/l), during the PHB production phase, led not only to prevention of oxygen limitation but also to the production of very high-molecular-mass PHB, in the 900–1800 kDa range. Biomass yields relative to the total methanol consumed were in the range 0.29–0.33 g/g, whereas PHB yields were in the range 0.09–0.12 g/g. During the active period of PHB synthesis, PHB yields relative to the total methanol consumed were between 0.2 g/g and 0.22 g/g. M. extorquens ATCC 55366 appears to be a promising organism for industrial PHB production.     

Bacterial exopolymer and PHB production in fluctuating ground-water habitats

Abstract The physiological response of a strain of ground-water bacteria to short-term fluctuations in the quality of percolating water was followed in a continuous-flow water-saturated soil column. The cells reduced their production of uronic acids-characterized extracellular polysaccharides by more than 50% when pure ground water was enriched with phosphate and glucose but resumed it gradually as the poor growth conditions returned. Bacteria that were sorbed to the mineral particles in the soil column produced more exopolymers-about six times more in pure ground water-than free-living bacteria, but the composition of the polymer was the same. Glucose, mannose and galacturonic acid accounted for about 90% of the carbohydrate. The cells stored about five times more poly-β-hydroxybutyric acid (PHB) in pure than in enriched ground water, and sorbed cells had five times higher concentrations than free-living cells. The quality variations of the ground water were also reflected in the population size of bacteria, which differed by two to four orders of magnitude between poor and enriched conditions.     

Ilyobacter delafieldii sp. nov., a metabolically restricted anaerobic bacterium fermenting PHB

Enrichments from an estuarine sediment with crotonate as substrate resulted in the isolation of a motile, gram-negative, obligately anaerobic rod with pointed ends, designated strain 10cr1. The organism was asporogenous, did not reduce sulfur, sulfate, thiosulfate, nitrate, oxygen or fumarate, and had a mol %G+C ratio of 29. Strain 10cr1 was able to ferment crotonate, 3-hydroxybutyrate, lactate, pyruvate, and poly--hydroxybutyric acid (PHB). Acetate, propionate, butyrate, CO₂ and H₂ were the fermentation products. When grown on PHB there was accumulation of 3-hydroxybutyrate once growth had ceased, indicating degradation of PHB to the monomer. The 3-hydroxybutyrate formed during growth of the culture was fermented to acetate, butyrate and H₂. Experimental evidence suggested the production of an extracellular PHB depolymerase. The cells were not attached to the PHB granules. This is the first isolation of an anaerobic bacterium capable of degrading exogenous PHB. This strain is described as a new species, Ilyobacter delafieldii sp. nov., and strain 10cr1 (=DSM 5704) is designated as the type (and at present, only) strain.Abbreviations G+C guanine plus cytosine - OD optical density - PHB poly--hydroxybutyric acid - specific growth rate - HPLC high-performance liquid chromatography - YE yeast extract     

In vivo growth conditions suppress the expression of ganglioside GM2 and favour that of lacto series gangliosides in the human glioma D-54MG cell line

The human glioma D-54MG cell line grownin vitro primarily expresses ganglio series gangliosides, particularly GM2. Subcutaneous injection of these cells into nude mice produced xenografts with an increased content of the human glioma-associated lacto series gangliosides, primarily 3-isoLM1, an alteration that was dose dependent, with the highest dose (1×10⁸) resulting in a phenotype that was most like that of the inoculum. After one passagein vivo, the lacto series dominated and reached a proportional level that was kept throughout the 10 passages. The mRNA levels of the GM2-synthase clearly coincided with GM2 expression and was 20 times higher in cells grownin vitro than in those grownin vivo. These results support the view that ganglioside expression in human gliomas is strongly influenced by environmental factors. Abbreviations: The gangliosides have been designated according to Svennerholm (Eur J Biochem (1977)79: 11–21) GM3, II³NeuAc-LacCer; GM2, II³NeuAc-GgOse₃Cer; GM1, II³NeuAc-GgOse₄Cer; GD3, II³(NeuAc)₂-LacCer; GD2, II³(NeuAc)₂-GgOse₃Cer; GD1a, IV³NeuAc, II³NeuAc-GgOse₄Cer; GD1b, II³(NeuAc)₂-GgOse₄Cer; GT1b, IV³NeuAc, II³(NeuAc)₂-GgOse₄Cer; 3-LM1, IV³NeuAc-nLcOse₄Cer; 3-isoLM1, IV³NeuAc-LcOse₄Cer; 3,6-isoLD1, IV³NeuAc, III⁶NeuAc-LcOse₄Cer; 38-LM1, IV³(NeuAc)₂-nLcOse₄Cer. MAb(s), monoclonal antibody (ies); the designation LM1 is used when both 3-isoLM1 and 3-LM1 and LD1, when both 36-isoLD1 and 38-LD1 are included.     

High frequency transformation of Alcaligenes eutrophus producing poly-β-hydroxybutyric acid by electroporation

Summary A strain of Alcaligenes eutrophus producing poly--hydroxybutyric acid was successfully transformed by the electroporation. The plasmid used was a broad host range plasmid pKT230 conferring kanamycin resistance. The optimum yield of transformant was 0.8×10²/g DNA when 50 l competent cells at 10¹⁰/ml were pulsed by 11.5 kV/cm for 5 ms with 1 g DNA. Plasmid DNA in the A. eutrophus transformant was stably maintained as a monomeric structure.