Effects of Polychlorinated Biphenyls on Macromolecular Synthesis by a Heterotrophic Marine Bacterium

Growth rates and final cell yields of a polychlorinated biphenyl (PCB)-sensitive pseudomonad isolated from the open ocean were reduced in a dose-dependent manner by 10 to 100 μg of Aroclor 1254 per liter, a commercial mixture of PCB isomers added to its culture medium. Effects on growth rates were detected within 1 h (approximately one doubling time) of treatment. By 4 h posttreatment, the amounts of deoxyribonucleic acid and ribonucleic acid per cell in exponentially growing populations treated with sublethal doses of Aroclor were detectably lower than in appropriate controls. Corresponding cell protein values were slightly higher than in controls. Selective degradation of cell proteins or nucleic acids was not detected in cells whose growth was totally suppressed for 4 h by PCBs. Cells whose growth rate was inhibited 20 to 50% by Aroclor synthesized protein at normal rates for periods in excess of 5 h from the time the chlorinated hydrocarbons were added. In contrast, rates per cell of adenine uptake and adenine incorporation into deoxyribonucleic acid and total nucleic acids by the cells treated with PCBs were significantly lower than in control cells. Intracellular adenine pools of cells whose growth was inhibited to 20% of the control rate by PCBs were 30% smaller and appeared to require a longer interval to equilibrate than those of untreated cells. This may indicate impaired transport and/or efflux of this nucleic acid precursor through the membrane of affected cells. Inhibition of nucleic acid synthesis in this sensitive bacterium by PCBs could explain the observed inhibitory effects of the chlorinated hydrocarbons on its growth.     

Ag + -Sensitive NADH Dehydrogenas in the Na+-Motive Respiratory Chain of the Marine Bacterium Vibrio alginolyticus

Components of the Na⁺ -motive NADH : quinone oxi-doreductase segment in the respiratory chain of Vibrio alginolyticus were examined in membranes prepared from wild type, Na⁺ -pump-defective mutants, and a spontaneous revertant. Ag⁺ distinguished two kinds of respiratory NADH dehydrogenases. The Na⁺ -pump-defective mutants lacked Ag⁺ -sensitive NADH dehydrogenase activity. Incubation of the Ag⁺ -sensitive NADH dehydrogenase with solubilized membrane proteins of the mutant led to the reconstitution of Na⁺ -motive NADH : quinone oxidoreductase activity. We think that Ag⁺ -sensitive NADH dehydrogenase is an essential component of the respiratory Na⁺ pump of this organism.     

Protease Formation by a Marine Psychrophilic Bacterium

Proteolytic activity was found in the culture fluids of numerous psychrophilic bacteria isolated from terrestrial or marine samples. Among these organisms, a marine psychrophilic bacterium, Pseudomonas sp. No. 548, showed the highest proteolytic activity. This organism required salts of sea water for both growth and protease formation. The optimum temperature for the growth of this organism was 20°C. The formation of protease was the greatest at 5°C and decreased with increasing temperature. The extracellular protease system was fractionated into at least two components having proteolytic activities by chromatography with DEAE-cellulose. Increasing culture temperature tended to increase the activity ratio of Fraction I to Fraction II. Some cultural conditions for protease formation were investigated.     

Production of a Pyrrole Antibiotic by a Marine Bacterium

Evidence is presented for the isolation and identification of bacteria able to synthesize an unusual antibiotic containing five bromine atoms per molecule. The identification and taxonomic position of these bacteria was made by use of a computer in conjunction with traditional methods. These microorganisms and closely related strains have been isolated on various occasions from tropical water in the vicinity of Puerto Rico. One bacterium, a pseudomonad, has been given the name Pseudomonas bromoutilis because of its distinctive capability. The antibiotic has been extracted, purified, and obtained in crystal form, and its structure has been determined. Although clinical tests of its properties were not encouraging, it may be of significant value and interest from an ecological standpoint.     

Novel Epibiotic Thiothrix Bacterium on a Marine Amphipod

Comparative analysis of the 16S rRNA gene and fluorescent in situ hybridization (FISH) was used to identify epibiotic filamentous bacteria living on the marine amphipod crustacean Urothoe poseidonis. The epibionts belong to the gamma proteobacteria and represent a novel marine phylotype within the genus Thiothrix. FISH and denaturing gradient gel electrophoresis revealed that the Thiothrix filaments are present on the majority of the amphipods examined.     

Lipopolysaccharide from a Gram-Negative Marine Bacterium

A lipopolysaccharide molecule was isolated from a marine bacterium. The molecule seems to be composed of lipid A and the hexoses, glucose and galactose.     

Attachment Stimulates Exopolysaccharide Synthesis by a Bacterium

This study examined the hypothesis that solid surfaces may stimulate attached bacteria to produce exopolymers. Addition of sand to shake-flask cultures seemed to induce exopolymer synthesis by a number of subsurface isolates, as revealed by optical microscopy. Several additional lines of evidence indicated that exopolymer production by attached cells (in continuous-flow sand-packed columns) was greater than by their free-living counterparts. Total carbohydrates and extracellular polysaccharides, both normalized to cell protein, were greater (2.5- and 5-fold, respectively) for attached cells than for free-living cells. Also, adsorption of a polyanion-binding dye to the exopolymer fraction was sixfold greater for attached cells than for unattached cells. When surface-grown cells were resuspended in fresh medium, exopolymer production decreased to the level characteristic of unattached cells, which ruled out the possibility that attached cells comprised a subpopulation of sticky mucoid variants. The mechanism by which attachment stimulated exopolymer synthesis did not involve changes of the specific growth rate, growth stage, or limiting nutrient.     

Osmotic Effects on Membrane Permeability in a Marine Bacterium

When cells of Alteromonas haloplanktis 214 (ATCC 19855) were preloaded with α-[¹⁴C]aminoisobutyric acid or the K⁺ in the cells was labeled with ⁴²K by incubation in a buffered salt solution containing 0.05 M MgSO₄, 0.01 M KCl, and 0.3 M NaCl, the cells retained their radioactivity when resuspended in the same salt solution. When NaCl was omitted from the solution, 80 to 90% of the radioactivity was lost from the cells. Cells suspended at intermediate concentrations of NaCl also lost radioactivity. New steady-state levels of the intracellular solutes were established within 15 s of suspending the cells; the percentage of radioactivity retained at each level decreased proportionately as the osmolality of the NaCl in the suspending solution decreased. With minor variations in effectiveness, MgCl₂, LiCl, and sucrose could substitute for NaCl on an equiosmolal basis for the retention of radioactivity by the cells. KCl, RbCl, and CsCl were appreciably less effective as replacements for NaCl, particularly when their osmolalities in the suspending solutions were low. The amount of α-[¹⁴C]aminoisobutyric acid taken up by the cells at the steady-state level increased to a maximum as the NaCl concentration in the suspending medium increased to 0.3 M. At suboptimal levels of NaCl, either LiCl or sucrose could substitute for NaCl in increasing the steady-state levels. The results obtained indicate that the porosity of the cytoplasmic membrane of this organism is determined by the difference between the osmotic pressure of the cytoplasm and the suspending medium. The lesser effectiveness of K⁺, Rb⁺, and Cs⁺ than Na⁺, Li, or Mg²⁺ in permitting the retention of solutes by the cells is attributed to the greater penetrability of the hydrated ions of the former group through the dilated pores of a stretched cytoplasmic membrane.     

Isolation and Characterization of a Fucoidan-Degrading Marine Bacterium

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from the algal bodies of Cladosiphon okamuranus (class Phaeophyceae, order Chordariales, family Chordariaceae) with a yield of 2.0% of the wet weight of the alga. To obtain enzymes that digest the fucoidan, we screened bacteria in the gut contents of the sea cucumber Stichopus japonicus for their ability to decrease the fucoidan in their culture media, and successfully isolated one bacterial strain that could decrease it. The bacterial strain was gram-negative and possessed menaquinone 7 as the predominant respiratory quinone, and the GC content of its genomic DNA was 52%. The results of the phylogenetic analysis of its 16S ribosomal DNA sequence indicated that the bacterial strain was a member of the division Verrucomicrobia. However, as the bacterial strain is phylogenetically and phenotypically distinct from verrucomicrobial species described previously, the strain was assumed to be a new member of the division Verrucomicrobia. When the bacterial strain was cultivated in an algal fucoidan-containing medium, the strain decreased fucoidan from C. okamuranus (44%), Nemacystus decipiens (19%), Laminaria japonica (31%), Kjellmaniella crassifolia (23%), sporophyl of Undaria pinnatifida (22%), Fucus vesiculosus (42%), and Ascophyllum nodosum (61%).     

Anaerobic Degradation of Ethylbenzene by a New Type of Marine Sulfate-Reducing Bacterium

Anaerobic degradation of the aromatic hydrocarbon ethylbenzene was studied with sulfate as the electron acceptor. Enrichment cultures prepared with marine sediment samples from different locations showed ethylbenzene-dependent reduction of sulfate to sulfide and always contained a characteristic cell type that formed gas vesicles towards the end of growth. A pure culture of this cell type, strain EbS7, was isolated from sediment from Guaymas Basin (Gulf of California). Complete mineralization of ethylbenzene coupled to sulfate reduction was demonstrated in growth experiments with strain EbS7. Sequence analysis of the 16S rRNA gene revealed a close relationship between strain EbS7 and the previously described marine sulfate-reducing strains NaphS2 and mXyS1 (similarity values, 97.6 and 96.2%, respectively), which grow anaerobically with naphthalene and m-xylene, respectively. However, strain EbS7 did not oxidize naphthalene, m-xylene, or toluene. Other compounds utilized by strain EbS7 were phenylacetate, 3-phenylpropionate, formate, n-hexanoate, lactate, and pyruvate. 1-Phenylethanol and acetophenone, the characteristic intermediates in anaerobic ethylbenzene degradation by denitrifying bacteria, neither served as growth substrates nor were detectable as metabolites by gas chromatography-mass spectrometry in ethylbenzene-grown cultures of strain EbS7. Rather, (1-phenylethyl)succinate and 4-phenylpentanoate were detected as specific metabolites in such cultures. Formation of these intermediates can be explained by a reaction sequence involving addition of the benzyl carbon atom of ethylbenzene to fumarate, carbon skeleton rearrangement of the succinate moiety (as a thioester), and loss of one carboxyl group. Such reactions are analogous to those suggested for anaerobic n-alkane degradation and thus differ from the initial reactions in anaerobic ethylbenzene degradation by denitrifying bacteria which employ dehydrogenations.     

Glutamate Functions in Osmoregulation in a Marine Bacterium

Beneckea harveyi, growing in either minimal or complex media increased the total amino acid pool with increasing salinity of the medium. Glutamate was the predominate amino acid involved.     

Influence of permeant acids and bases on active Na+ extrusion by Chlorella

Chlorella cells were enriched with Na⁺ by incubation in 3.5 mM ²²Na labelled NaOH for 2–3 h. The active Na⁺ extrusion from these cells into media containing 6.5 × 10⁻⁴ M salts of permeant acids (sodium acetate, phenylacetate, propionate, isobutyrate) or bases (ammonium and methylammonium chloride) was studied under illumination and aeration at pH 6.0 and and 7.8. The carboxylates depressed and the hydrochlorides stimulated Na⁺ efflux efflux. The effect of the carboxylates could be attributed to acidification of the cell interior due to penetration of the undissociated acids. The strong effect of the hydrochlorides could only partially be explained by penetration of the free bases. When the external medium contained only low concentrations of K⁺, Na⁺ export was increasingly inhibited with time. This effect was attibuted to acidification of the cell interior by Na⁺/H⁺ exchange. The results can be explained either by secondary active Na⁺/H⁺ antiport or by competition between Na⁺ and H⁺ at the inner site of a primary active extrusion pump, some observations favouring the latter assumption.     

Na+-Dependent Transport of Amino Acids and Its Significance for Growth of a Lysine-producing Bacterium

A lysine-producing mutant Brevibacterium flavum HUT 8052, a threonine plus methionine (or threonine plus homoserine) auxotroph, grew rapidly as nearly as the wild strain in a medium supplemented with NaCl (60 µg/ml), threonine (100 µg/ml), and methionine (33.3 µg/ml). With NaCl concentrations less than 20 µg/ml, the mutant grew little or very slowly, The peculiar growth behavior of the mutant including the above phenomenon could be reasonably explained by the finding of Na⁺-dependent amino acids transport and the feedback inhibition of homoserine dehydrogenase by threonine in the bacterium.

The threonine transport was stimulated by Na⁺ and Li⁺. though the latter being less effective. The transport of threonine was inhibited by serine. The uptake of serine, isoleucine, leucine and valine was also stimulated by Na⁺     

Taxonomic Study of a Marine Bacterium Producing Guluronate Lyase

A marine bacterium that secretes a novel guluronate lyase that is heat- and denaturants-durable, has been isolated from the intestine contents of a red sea bream, Pagrus major. To characterize this marine bacterium, we attempted taxonomic study with respect to eleven characteristics used for the specification with an electron microscope photograph. The findings showed that the marine bacterium is a member of the genus Vibrio.     

Low-affinity Na+ sites on (Na+ +K+)-ATPase modulate inhibition of Na+-ATPase activity by vanadate

Na+-ATPase activity is extremely sensitive to inhibition by vanadate at low Na+ concentrations where Na+ occupies only high-affinity activation sites. Na+ occupies low-affinity activation sites to reverse inhibition of Na+-ATPase and (Na+, K+)-ATPase activities by vanadate. This effect of Na+ is competitive with respect to both vanadate and Mg2+. The apparent affinity of the enzyme for vanadate is markedly increased by K+. The principal effect of K+ may be to displace Na+ from the low-affinity sites at which it activates Na+-ATPase activity.     

Microcalorimetric Measurements of Glucose Metabolism by Marine Bacterium Vibrio alginolyticus

Microcalorimetric measurements of heat production from glucose by Vibrio alginolyticus were made to assess the viability of calorimetry as a technique for studying the metabolism of marine bacteria at organic nutrient concentrations found in marine waters. The results show that the metabolism of glucose by this bacterium can be measured by calorimetry at submicromolar concentrations. A linear correlation between glucose concentration and total heat production was observed over a concentration range of 8 mM to 0.35 μM. It is suggested that these data indicate a constant efficiency of metabolism for this bacterium over the wide range of glucose concentrations studied.     

NADPH Production from NADP+ by a Formate-utilizing Methanogenic Bacterium

Resting cells of the methanogen strain HU, a formate-utilizing methanogenic bacterium, was able to utilize formate or hydrogen as electron donor for the production of NADPH from NADP⁺ under suitable conditions. In the presence of 0.2% Triton X-100 and 0.3 m potassium phosphate, pH 9.0 at 30°C, the resting cells could convert ca. 60% of the exogenous NADP⁺ into NADPH yielding ca. 6 g NADPH/liter. Phosphate ions greatly enhanced the NADPH production.     

Synthesis of Na+/K+ ATPase by the preimplantation rabbit blastocyst

The rates of incorporation of [35S]methionine into Na+/K+ ATPase, actin (beta- and gamma-isoforms), and total protein of the preimplantation rabbit blastocyst were determined between Days 4 and 7 of development. Blastocyst proteins were metabolically radiolabelled with [35S]methionine and subsequently analysed by co-isolation with purified Na+/K+ ATPase using two-dimensional polyacrylamide gel electrophoresis, immunoprecipitation, immunoblotting, fluorography, and liquid scintillation spectroscopy. The rate of [35S]methionine incorporation into acid-soluble total protein increased 24-fold between Days 4 and 6 post coitum (p.c.), then diminished approximately 79% on Day 7. In-vitro incorporation of [35S]methionine was linear at each stage of blastocyst development. [35S]methionine incorporation rates were unaffected by low free intracellular methionine concentration (less than 0.06 mM) and stage-related differences in blastocoele volume. Analysis of beta- and gamma-actin synthesis revealed patterns of [35S]methionine incorporation rates which were similar to those of total protein. In contrast, synthesis of blastocyst Na+/K+ ATPase was characterized by a 90-fold increase (P less than 0.001) in the rate of [35S]methionine incorporation between Days 4 and 6 p.c. The results demonstrate that Na+/K+ ATPase is actively synthesized at a high and increasing rate during preimplantation development in the rabbit at a period which is characterized by rapid fluid accumulation by the blastocyst.     

Inhibition of Settlement by Larvae of Balanus amphitrite and Ciona intestinalis by a Surface-Colonizing Marine Bacterium

In an attempt to isolate bacteria with inhibitory effects against settlement by larvae of sessile invertebrates, 40 marine bacterial isolates were screened for effects against laboratory-reared barnacle larvae (Balanus amphitrite) and ascidian larvae (Ciona intestinalis). Five isolates displayed non-pH-dependent inhibitory effects against the larvae. The initial characterization of a toxic component released from an isolate, designated D2 (CCUG 26757), and its effect on laboratory-reared barnacle and ascidian larvae were studied. D2 is a facultative, anaerobic, gram-negative bacterium isolated from the surface of C. intestinalis from waters off the Swedish west coast at a depth of 10 m. Results suggest that the toxic component is released by D2 during the stationary phase. Aged biofilms were more toxic to the larvae than unaged films. The biologically active compound was in the supernatant of D2 and was heat stable and <500 Da in molecular mass. No evidence of protein or peptide moieties was found. On the basis of two phase and chromatography separations, the component is polar and neutral and contains or binds to carbohydrate moieties. Metaperiodate treatment increased toxicity; undiluted supernatant from a 24-h growth culture of D2 killed barnacle and ascidian larvae within a few hours of exposure, whereas after metaperiodate treatment, the larvae were killed in approximately 30 min.