The utilisation of alkylated amines by marine bacteria

Six species of bacteria and a marine yeast which are able to use alkylated amines as a sole source of nitrogen have been isolated from marine mud and tentatively assigned to genera. One isolate (Micrococcus sp.) has been studied in greater detail. This organism has a constitutive ability to utilise trimethylamine and the lesser methylated amines as a sole source of nitrogen. Growth and metabolic studies suggest that the methylated amines are metabolized by a pathway involving a stepwise demethylation process. The implications of the results obtained on the route of regeneration and recycling of amine nitrogen in the marine environment is discussed.     

Uptake of dissolved organics by marine bacteria as a function of fluid motion

A mass transfer analysis predicts that fluid motion can increase the assimilation of dissolved organics by attached compared to free-living microorganisms under certain conditions. To test this we examined the effect of advective flow and fluid shear on the uptake of model compounds (leucine and glucose) by natural assemblages of heterotrophic bacteria, collected from Roosevelt Inlet, Delaware Bay (USA), in 1989. We found that [³H]leucine uptake by cells held in fluid moving at 20 to 70 m d^–1 was eight times larger than uptake by cells at a velocity of 3 m d^–1. This effect was only observed at low leucine concentrations (ca. 1 nM), when uptake was likely not saturated. When we added leucine at concentrations expected to saturate leucine uptake (ca. 11 nM), fluid motion past cells did not affect uptake. Fluid flow past bacteria did not increase [³H]glucose uptake, and laminar shear rates of 0.5 to 2.1 s^–1 did not increase either glucose or leucine uptake by suspended bacteria. These results indicate that fluid motion increases bacterial uptake of certain lowmolecular-weight dissolved organics only when the microorganism exists in an advective flow field. As predicted from a mass transfer model, fluid shear rates in natural systems are too low to affect bacterial uptake of such compounds.     

Ammonium regeneration and carbon utilization by marine bacteria grown on mixed substrates

We examined the impact of exposing natural populations of marine bacteria (from seawater collected near Woods Hole, Massachusetts, USA) to multiple nitrogen and carbon sources in a series of batch growth experiments conducted from 1989 through 1990. The substrate C:N ratio (C:N_s) was varied from 1.5:1 to 10:1 either with equal amounts of NH ₄ ⁺ and different amino acids or an amino acid mixture, all supplemented with glucose to maintain the C:N_s ratio equal to that of the respective amino acid, or with combinations of glucose and NH ₄ ⁺ alone. A common feature of the experiments involving amino acids was the concurrent uptake of NH ₄ ⁺ and amino acids that persisted as long as a readily assimilable carbon source (glucose in our case) was taken up. There was no net regeneration of NH ₄ ⁺ , even though catabolism of amino acids occurred. Regeneration of NH ₄ ⁺ was evident only after glucose was completely utilized, which usually occurred at the end of exponential growth. The contribution of¹⁵NH ₄ ⁺ to total nitrogen uptake by the end of exponential growth varied from ~60 to 80% when individual amino acids were present and down to ~24% when the amino acid mixture was added. These estimates are conservative because we did not account for possible isotope dilution effects resulting from amino acid catabolism. When NH ₄ ⁺ and glucose were the sole nitrogen and carbon sources, there was a stoichiometric balance between glucose and NH ₄ ⁺ uptake over a wide range of C:N_s ratios, leading to a constant bacterial biomass C:N ratio (C:N_B) of ~4.5:1. As a result NH ₄ ⁺ usage varied from 50% when the C:N_s ratio was 3.6:1, to 100% when the C:N_s ratio was 10:1. Gross growth efficiency varied from ~60% when NH ₄ ⁺ plus glucose were added alone or with the amino acid mixture, to 47% when the individual amino acids were used in place of the mixture. It is thus evident that actively growing bacteria will act as sinks for nitrogen when a carbon source that can be assimilated easily is available to balance NH ₄ ⁺ uptake, even when amino acids are available and are being co-metabolized.     

Plasmid mediated antibiotic resistance in marine bacteria

This research work was conducted in Uppanar estuary to ascertain the role of plasmids in the antibiotic resistance of bacteria. Water and sediment samples were collected for a period of three months. When tested against 20 antibiotics 22 MAR strains were isolated from the samples, which were found resistant to 5-13 antibiotics. They belong to 7 genera and 10 species. Gram-negative bacteria namely Neisseria mucosa, N. sicca, Branhamella catarrhalis, Klebsiella ozaenae, Citrobacterintermedius, Pseudomonas fluorescens and Enterobacter aerogenes were isolated. Gram-positive bacteria were of Bacillus subtilis, B. megaterium and Micrococcus luteus. When plasmid curing was done using acredine orange, the resistance against penicillin-G, ampicillin, tetracycline, amoxycillin, kanamycin, and chloramphenicol were totally lost in all strains, which confirmed the role of plasmid in these strains against antibiotics. Ten strains belong to different species were selected for the plasmid isolation and electrophoresis was done. Presence of plasmids in all strains was confirmed and the molecular weight was in the range of 2850 to 3170 bp. The study revealed that MAR strains are common in Uppanar estuary and they are plasmid mediated. This environment is seemed to be deteriorating at an alarming rate.     

Respiration and physiological state in marine bacteria

The relationship between oxygen consumption (R) and respiratory electron-transport-system (ETS) activity was investigated in batch cultures of 5 species of marine bacteria, Vibrio adaptatus, V. anguillarum, a partially identified Vibrio sp. SA774, Serratia marinorubra, and Pseudomonas perfectomarinus. Although cellular levels of R and ETS varied widely among the species tested, the R:ETS ratios for growing or senescent populations were relatively constant among the species; these ratios were 5.02 in growth and 0.426 in senescence, with coefficients of variation of 29 and 20%, respectively. The lower senescent-phase R:ETS ratio was due to a depression of the respiration rates following growth termination. The regression log (R per cell) = 0.832 log (ETS per cell) + 0.727 for the growing populations was similar to that determined for marine zooplankton. The slight dependency of the R:ETS ratio on organism dry weight found for zooplankton was supported by our data. Planktonic respiration rates estimated from measured ETS-depth profiles in the eastern tropical North Pacific Ocean using the senescent-phase R:ETS ratio were similar to published oxygen consumption rates in the deep sea.Contribution No. M79-61 from the University of Washington's Department of Oceanography, and No. 79032 from the Bigelow Laboratory for Ocean Sciences.     

Arsenic metabolism in marine bacteria and yeast

Arsenic metabolism was studied for two marine microorganisms, a facultative anaerobic bacterium, Serratia marinorubra, and an obligately aerobic yeast, Rhodotorula rubra. Both were cultivated in media with (⁷⁴As) arsenate (As V), and the products of arsenate metabolism were determined qualitatively. Both the bacterium and the yeast produced arsenite (AS III) and methylarsonic acid [CH₃AsO(OH)₂]. In addition to the foregoing, only the yeast produced dimethylarsinic acid (CH₃)₂AsO(OH) and volatile alkylarsines. In contrast, the bacterium growing anaerobically with cobalamine as a cofactor did not synthesize gaseous forms of arsenic such as methylarsines. Neither organism synthesized arsoniumphospholipids such as those produced by marine phytoplankton or terrestrial fungi. The yeast did not accumulate arsenite, but instead transported some of it into the culture medium and methylated the remainder first to methylarsonic acid and then to dimethylarsinic acid. Finally, the latter compound was methylated further and volatile alkylarsines were formed. In contrast, the bacterium retained all products of arsenate metabolism intracellularly. Both the bacterium and the yeast, therefore, converted relatively toxic arsenate, the most abundant arsenic compound in seawater, to products that were presumably less toxic.     

Influence of bacteria on growth and hemolysin production by the marine dinoflagellate Amphidinium carterae

The effect of various bacteria on the growth of and hemolysin production by Amphidinium carterae was studied. The algal culture had an indigenous bacterial flora of Moraxella and Pseudomonas and these could not be eliminated by treatment with bactericidal antibiotics like gentamicin. Various bacteria like Micrococcus, Aeromonas, Vibrio, and Moraxella-like bacteria were added to A. carterae cultures to study their effect on growth and hemolysin production. Micrococcus and Aeromonas were found to improve the growth marginally. Hemolysin titres were considerably higher in A. carterae cultures supplemented with bacteria. Received: 27 May 1997 / Accepted: 22 July 1997     

Are some bacteria toxic for marine algae?

It was observed in vitro that some bacteria from different origins, including the sea, are capable of inhibiting the growth of several marine algae. However, there is too much difference between in vitro experiments and in situ environmental conditions to assume that bacterial poisons may rule a great deal of algac-bacteria relationships in the sea as a whole. In the author's opinion the importance of bacterial poison is limited to special and rare instances.     

Study of bacteria associated with marine algae in culture

Inhibitory action of some usual antibiotic, substances have been tested on 25 species of bacteria isolated from marine algae cultures. Penicillin, kanamycin, neomycin and streptomycin appear to be the most convenient antibiotics for eliminating or inhibiting polluting bacteria populations.     

Sulfide and thiosulfate-oxidizing bacteria in anoxic marine basins

The occurrence of sulfide and thiosulfate-oxidizing bacteria was examined in waters and sediments of the Black Sea and the Caraco Trench. Isolates obtained by enrichment-culture techniques exhibited facultative autotrophic-growth characteristics and oxidized thiosulfate or sulfide. Acid was not formed during growth in mineral medium. Most of the isolates were facultatively anaerobic, using nitrate as the terminal hydrogen acceptor. Strictly aerobic bacteria were found only in oxygenated waters. Comparison of the metabolic capabilities of the isolates during growth in sulfide and thiosulfate media with chemical parameters measured in the Black Sea and Cariaco Trench waters suggests that the biological oxidation of thiosulfate is the rate-limiting step in the oxidative turnover of sulfur compounds in the basins. Evidence for the oxidation of sulfide in the anoxic waters is presented, and possible explanations for the occurrence of this process in the absence of suitable electron acceptors (O₂. NO₃) are discussed. A maximum dark assimilation of carbon dioxide was located just below the oxygen-sulfide interface in the Cariaco Trench. Problems of interpreting these data are considered.Contribution No. 2958 of the Woods Hole Occanographic Institution. Supported by the National Science Foundation Grants GA 29665 and GA 33405.     

Study of bacteria associated with marine algae in culture

Seventy seven organic compounds have been tested for receptivity to attack by bacteria strains isolated from marine algae cultures. Bacteria utilize amino and organic acids more frequently than sugars and derivatives, especially when growth substances are not included in the experimental medium.     

Identification of marine bacteria affecting lithium adsorbents in seawater

Lithium manganese oxide–based adsorbents have been developed for the recovery of lithium from seawater. To maximize the recovery efficiency, it is important to prevent microfouling of lithium adsorbents by marine bacteria. To identify the marine bacteria that cause biofouling against the lithium adsorbents, lithium adsorbents were installed into a non-coated frame or a frame coated with an antifouling agent soaked in seawater. Microorganisms from the surface of lithium adsorbents were collected for 30 days at 10-day intervals, cultured in marine broth, isolated, and identified by 16S rDNA sequencing. Pseudoalteromonas and Vibrio were constituted to 35.6 and 28.8 % of total isolates, respectively, and were predominant in the non-coated frame, whereas Vibrio was poorly isolated (2.3 %) from the antifouling agent–coated frame. In this study, antifouling strategy for maximum lithium recovery efficiency in the marine area takes account of Pseudoalteromonas and Vibrio.     

Inorganic nitrogen metabolism in marine bacteria: Nitrate uptake and reduction in a marine pseudomonad

Growth experiments in batch cultures indicated that the uptake of nitrate by the marine pseudomonad PL1 was inhibited in the presence of ammonia provided that the ammonia concentration was higher than 1 mM. At ammonia concentrations of less than about 1 mM, however, both nitrate and ammonia were utilised simultaneously. The saturation constants for nitrate and ammonia uptake were both 2.6x10^-4 M, and similar to the Michaelis constants of nitrate reductase for nitrate (2.9x10^-4 M) and glutamine synthetase for ammonia (2x10^-4 M). Nitrate reductase activity linked to NADH was detected in chemostat-grown cultures with nitrate as nitrogen source, and in cultures containing limiting concentrations of nitrate and ammonia, ammonia or glutamate. Enzyme synthesis appeared to be repressed in cultures containing an excess of ammonia or glutamate. Chemostat cultures utilised ammonia or glutamate in preference to nitrate, while there was no marked preference between ammonia and glutamate.     

Kinetics of glucose and amino acid uptake by attached and free-living marine bacteria in oligotrophic waters

Kinetics of glucose and amino acid uptake by attached and free-living bacteria were compared in the upper 70 m of the oligotrophic north-western Mediterranean Sea. Potential uptake rates of amino acids were higher than those of glucose in all the samples analysed. Cell-specific potential uptake rates of attached bacteria were up to two orders of magnitude higher than those of total bacteria, both for amino acids and glucose (0.72–153 amol amino acids cell⁻¹ h⁻¹ and 0.05–58.42 amol glucose cell⁻¹ h⁻¹ for attached bacteria and 0.34–1.37 amol amino acids cell⁻¹ h⁻¹ and 0.07–0.22 amol glucose cell⁻¹ h⁻¹ for total bacteria). The apparent K _m values were also higher in attached bacteria than in total bacteria, both for amino acids and glucose. These results would reflect the presence of different uptake systems in attached and free-living bacteria, which is in accordance with the different nutrient characteristics of their microenvironments, ambient water and particles. Attached bacteria show transport systems with low affinity, which characterise a bacterial community adapted to high concentration of substrates. Received: 13 June 2000 / Accepted: 6 December 2000     

High recovery of culturable bacteria from the surfaces of marine algae

The culturability of heterotrophic marine bacteria obtained from the surfaces of two species of marine algae (Lobophora variegata andHalimeda copiosa) was assessed by comparing total DAPI-stained cell counts to colony-forming bacterial counts on two agar media. The colony-forming bacterial counts on a low-nutrient medium (LN) consisting of seawater and agar were significantly greater for both algal species than counts obtained on a high-nutrient medium (HN) similar in composition to that typically used for the isolation of heterotrophic marine bacteria. On average, 14 and 58%, respectively, of the total bacteria fromL. variegata andH. copiosa were culturable on LN. These recovery rates far exceed those typically reported for marine bacteria. Of 119 LN strains obtained in pure culture, 55% failed to grow on HN. The yeast extract component of HN (1.5 gl^-1) was responsible for the majority of the observed inhibition, suggesting that this nutrient can be highly toxic to marine bacteria. Eighty-nine percent of the strains inhibited by HN were capable of growth when the nutrients in this medium were diluted by a factor of 100 with seawater. Of 66 epiphytic strains, 30 (45%) initially inhibited by HN showed the ability to adapt to this medium after a period of laboratory handling. The initial inability of low-nutrient-adapted bacteria to grow on high-nutrient media may be due to nutrient shock. The results presented here indicate that the culturability of specific populations of marine bacteria can be dramatically improved by the use of low-nutrient media. Further, the importance of developing new medium formulations that eliminate traditional nutrients, some of which are clearly toxic to bacteria, is demonstrated.     

Inorganic nitrogen metabolism in marine bacteria: The intracellular free amino acid pools of a marine pseudomonad

The marine pseudomonad bacterium PL₁ contains an intracellular pool of free amino acids which consist mainly of glutamate with small amounts of glutamine and aspartate when grown in a nutrient medium containing 0.2 M NaCl. When the NaCl concentration of the growth medium is increased to 0.8 M, proline becomes a major component of the intracellular pool together with glutamate—at this molarity and under suitable nutrient conditions these amino acids comprise 20% of total bacterial amino acid nitrogen. When grown in a nutrient growth medium containing a constant level of NaCl, the intracellular pool size can vary by a factor of 4 depending on the concentration of carbon and nitrogen in the medium. Experiments show that the amino acid pool can act as a nitrogen reserve but has little function as a carbon reserve. At high NaCl concentrations there is a marked dependence for growth on the presence of sufficient potassium in the medium. However, no correlation between K⁺ and glutamate concentration in either nitrogen or K⁺-limited cultures has been found. None of the enzymes associated with glutamate biosynthesis was influenced by NaCl levels between 0.2 and 0.5 M. Neither Na⁺ or K⁺ stimulated the activity of these enzymes when tested in vitro.     

Effect of neem extract against the bacteria isolated from marine fish

Marine ornamental fishes are exceedingly valuable due to their high demand in domestic and international markets. There is a growing global interest to rear the fishes in captivity. But problem due to bacteria and fungi are the major hitch in captive condition. Since, the use of antibiotics is banned, an attempt was made to ascertain in vitro assay of the neem leaves extract against the bacterial pathogens isolated from infected fishes. Bacterial strains isolated from infected regions of the clown fishes Amphiprion sebae and A. ocellaris were identified as Aeromonas hydrophila, Enterobacter sp., E. coli, Pseudomonas aeruginosa, Proteus sp., Streptococcus sp., Vibrio cholerae, V. alginolyticus, V. parahaemolyticus and Yersinia enterocolitica. Ethanol and methanol extracts were highly inhibitory to the bacterial isolates when compared to other solvents. Ethanol extracts exhibited low minimum inhibitory concentration (75-250 microg ml(-1)) as compared to other extracts. The present finding revealed that the neem leaf extract significantly reduces the bacterial pathogens and their infection in marine ornamental fishes.     

Spatial distribution of bacteria associated with the marine sponge Tethya californiana

Microbial diversity and spatial distribution of the diversity within tissue of the marine sponge Tethya californiana was analyzed based on 16S rRNA gene sequences. One candidate division and nine bacterial phyla were detected, including members of all five subdivisions of Proteobacteria. Moreover, chloroplast-derived Stramenopiles- and Rhodophyta-affiliated 16S rRNA gene sequences were found and Stramenopiles represented the most abundant clones (30%) in the clone library. On the phylum-level, the microbial fingerprint of T. californiana showed a similar pattern as its Mediterranean relative T. aurantium. An interesting difference was that Cyanobacteria that were abundantly present in T. aurantium were not found in T. californiana, but that the latter sponges harbored phototrophic Stramenopiles instead. Surprisingly, the phototrophic microorganisms were evenly distributed over the inner and outer parts of the sponge tissue, which implies that they also reside in regions without direct light exposure. The other phyla were also present in both the outer cortex and the mesohyl of the sponges. These results were confirmed by analysis on the operational taxonomic unit level. This leads to the conclusion that from a qualitative point of view, spatial distribution of microorganisms in T. californiana tissue is quite homogeneous. Thirty-two percent of the operational taxonomic units shared less than 95% similarity with any other known sequence. This indicates that marine sponges are a rich source of previously undetected microbial life.     

Decomposition of organic and solubilisation of inorganic phosphorus compounds by bacteria isolated from a marine sandy beach

The ability to decompose organic and to solubilise inorganic phosphorus compounds was studied in heterotrophic bacteria isolated from a sandy beach in Sopot, southern Baltic coast. Bacteria able to hydrolyse DNA and phytin were most numerous, while only a small percentage of the studied strains was able to depolymerise glycerophosphate. The ranking of the potential of the studied bacteria to solubilise metallic phosphate salts as follows: calcium hydrogen phosphate>ferric phosphate>magnesium phosphate>calcium phosphate>aluminium phosphate. There were marked differences in the level of depolymerisation of organic compounds of phosphorus and solubilisation of inorganic phosphorus between bacteria inhabiting different parts of the studied beach, while there were generally no differences between the surface and subsurface sand layers. The activity of alkaline phosphatase was higher than that of acid phosphatase.Communicated by O. Kinne, Oldendorf/Luhe     

Possible strategy for the survival of marine bacteria under starvation conditions

A population of a psychrophilic marine vibrio (Ant-300) suspended at a low cell density in natural seawater (SW) or artificial seawater (ASW) showed an initial 200-fold increase in cell numbers. Ant-300 suspended in ASW at various densities showed a magnified initial increase in numbers as well as increased longevity as the population density decreased. The magnitude of the initial increase and the viability of the cells after 7 weeks continued incubation were the same whether the cells were suspended in SW, ASW amended with amino acids, or organic-free ASW. Continued incubation (long-term starvation) of a culture of Ant-300 at low cell densities in ASW showed that after 70 weeks over 15 times the orginal number of cells were still viable. When compared to the starvation survival of other bacceria, Ant-300 exceeds the longest reported starvation survival by at least 2.5 times. Our data indicate that Ant-300 is especially adapted for survival at low nutrient concentrations and low population densities due to a sustained increase in cell numbers that may represent a species survival mechanism for marine bacteria.Technical Paper No. 4493, Oregon Agricultural Experiment Station.