Luminous bacteria cultured from fish guts in the Gulf of Oman.

The incidence of culturable luminous bacteria in Omani market fish guts was correlated to habitat type amongst 109 species of fish. Isolated representative luminous bacteria were compared to known species using the Biolog system (95 traits/isolate) and cluster analysis, which showed that the main taxa present in fish guts were clades related to Vibrio harveyi and Photobacterium species with sporadic incidence of P. phosphoreum. The luminous isolates from gut of the slip-mouth (barred pony fish), Leiognathus fasciatus, were mainly a type related to Photobacterium but phenotypically different from known species. These luminous gut bacteria were identical with the bacteria in the light organ, indicating that the light organ supplies a significant quantity of luminous bacteria to the gut. In many of the fish that lack light organs, luminous bacteria were also the dominant bacterial type in the gut, while in some others luminous bacteria were encountered sporadically and at low densities, reflecting the incidence of culturable luminous bacteria in seawater. Pelagic fish contained the highest incidence of culturable luminous bacteria and reef-associated fish the lowest. No correlation was found between the incidence of culturable luminous bacteria and the degree to which fish produce a melanin-covered gut.

Differentiation of marine luminous bacteria using commercial identification plates.

Differentiation of marine luminous bacteria using Biology GN plate combined with API 20e or the BBL Crystal ID plate inoculated with cell suspensions in artificial seawater was accomplished by comparison to type species using cluster analysis. Inoculum density affected the results from Biolog GN plates, but had less of an effect on the reactions obtained from API 20e strip or BBL Crystal ID plate. In a few cases, combination of the Biolog GN traits along with either the API 20e or Crystal ID traits was necessary to differentiate some marine luminous bacteria.

Shewanella woodyi sp. nov., an exclusively respiratory luminous bacterium isolated from the Alboran Sea.

Thirty-four strains of nonfermentative, respiratory, luminous bacteria were isolated from samples of squid ink and seawater from depths of 200 to 300 m in the Alboran Sea. Although these strains had a few properties similar to properties of Shewanella (Alteromonas) hanedai, they did not cluster phenotypically with any previously described bacterium. The nucleotide sequence of a 740-bp segment of luxA was not homologous with other known luxA sequences but clustered with the luxA sequences of Shewanella hanedai, Vibrio logei, Vibrio fischeri, and Photobacterium species. The 16S RNA gene from two strains was sequenced and was found to be most closely related to the S. hanedai 16S RNA gene. Based on the differences observed, we describe the new isolates as members of new species, Shewanella woodyi sp. nov. Strain ATCC 51908 (= MS32) is the type strain of this new species.

Association of luminous bacteria with artificial and natural surfaces in arabian gulf seawater.

Luminous bacteria in seawater around the islands of Bahrain are predominantly Vibrio harveyi and have the capability to adhere to artificial fibrous surfaces. Phytoplankton did not appear to have any specific relationship with luminous bacteria, but macroalgae were shown to possess an enhanced concentration of luminous bacteria.

Stabilization of luciferase intermediates by fatty amines, amides, and nitriles.

Long-chain aliphatic amides, mono- and diamines, mono- and dialcohols, and nitriles were found to inhibit the bacterial luciferase reaction by binding with an enzyme intermediate (II, the luciferase-bound 4 alpha-flavin hydroperoxide). Inhibition was determined by measuring the decay rates of the inhibitor-intermediate II complex at different inhibitor concentrations. The data fit a model which was used to estimate the KI. At high concentrations, a plot of the decay rate (k) vs 1/[I] produced a straight line; extrapolation of this to 1/[I] = 0 yields an estimate of the decay rate at infinite inhibitor concentration which we defined as the inhibitor-enzyme-substrate stabilization constant, kESI.

Bovine serum albumin interacts with bacterial luciferase.

Bovine serum albumin (BSA) affects the amount of light obtained from bacterial luciferase by competing with luciferase for one of the luciferase substrates, the aldehyde. At low aldehyde concentrations BSA behaves as an inhibitor, but at high aldehyde concentrations BSA relieves substrate inhibition. BSA reversibly binds decanal with a Ksi = 3.36 mumol/l, approximately half the affinity of luciferase for decanal (KM = 1.5 mumol/l). BSA also increased the rate of intermediate II dark decay. The data suggest that this involves a direct protein-protein (BSA-luciferase) interaction.

A cyanide-aldehyde complex inhibits bacterial luciferase.

Cyanide at high (millimolar) concentrations inhibited in the in vitro Vibrio harveyi luciferase reaction. Cyanide reacted with free aldehyde to form an inhibitor. Inhibitor formation was accelerated by alkaline conditions and bovine serum albumin.

HCO(3) Fixation by Naturally Occurring Tufts and Pure Cultures of Thiothrix nivea.

Naturally occurring tufts of the mixotroph Thiothrix nivea blanketed the East Everglades (Dade County, Fla.) Chekika artesian well and runoff areas. The rate of HCO(3) fixation by these Thiothrix tufts was determined to be 14.0 +/- 5.4 nmol of HCO(3) per min per mg of dry weight, which reflected a growth rate of 5.0%/h. The addition of 10 mM glucose, ribose, acetate, or pyruvate or 0.05% Casamino Acids (Difco Laboratories, Detroit, Mich.) did not appear to alter the HCO(3) fixation rate. Whereas 1 mM acetate or 10 mM lactate, ethanol, glycerol, alpha-ketoglutarate, succinate, fumarate, or citrate slightly stimulated HCO(3) fixation, 5 to 10 mM malate inhibited HCO(3) fixation by 90%. Pure Thiothrix cultures isolated from Chekika fixed HCO(3) at rates as high as 29.9 +/- 2.8 nmol of HCO(3) per min per mg of dry weight in the presence of growth medium. Malate did not have a suppressive effect but rather slightly stimulated in vivo HCO(3) fixation.

Luciferase-dependent oxygen consumption by bioluminescent vibrios.

Oxygen uptake due to luciferase in two luminous Vibrio species was estimated in vivo by utilizing inhibitors having specificities for luciferase (decanol) and cytochromes (cyanide). Cyanide titration of respiration revealed a component of oxygen uptake less sensitive to cyanide which was completely inhibitable by low concentrations of decanol. From this it was estimated that in vivo luciferase is responsible for less than 12% (Vibrio harveyi) or 20% (Vibrio fischeri) of the total respiration. From these data in vivo bioluminescent quantum yields are estimated to be not lower than 1.7 and 2.6%, respectively.

Poising of the arginine pool and control of bioluminescence in Beneckea harveyi.

Arginine dramatically stimulates bioluminescence in the marine bacterium Beneckea harveyi growing in minimal media, an effect that is due to increases in both the synthesis and expression of luciferase. To elucidate the mechanism of this phenomenon, studies were made of the transport and metabolism of arginine in B. harveyi. The transport of arginine and lysine involves two kinetically distinct transport systems for the uptake of arginine and lysine. In contrast, ornithine is transported only by a system common to all three amino acids. The internal amino acid pools were measured in mutants that do not show stimulation of bioluminescence by arginine and in wild-type cells that do. In minimal media, the internal arginine pools are undetectably low. Furthermore, exogenously added labeled arginine is rapidly transported and converted to citrulline and argininosuccinate. The results can be accommodated by a model in which the internal arginine is poised at a very low concentration; the stimulatory effect of exogenous arginine on luciferase biosynthesis occurs at the transcriptional level, and the actual mediator can be either arginine or argininyl transfer ribonucleic acid.

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.

Bacterial luciferase subunits are synthesized in equal quantities.

Synthesis of luciferase, an alpha beta dimer, occurs during a relatively short period of time near the end of exponential growth of Beneckea harveyi. The rates of synthesis of the individual alpha and beta chains of luciferase are compared by quantitating the molar ratio of total cellular alpha to that of beta at different growth stages. The addition of exogenous alpha or beta subunit to crude lysates of cells taken prior to and during the luciferase induction produces no increase in luciferase activity. This result, together with previous evidence that there are no antigenically cross-reacting precursors, allows us to conclude that luciferase alpha and beta chains are synthesized pari passu, i.e. in equal proportions, and exist primarily in the alpha beta dimeric form. It is also shown that insoluble sedimentable cellular materials contain no detectable luciferase subunits.

Sand beach bacteria: enumeration and characterization.

Bacteria in the water-saturated sand of a relatively unpolluted sand beach were enumerated by direct microscope and viable counting. The number of interstitial bacteria was estimated to be a significant fraction of the total number of bacteria present. Three hundred sixty-two strains were isolated and submitted to cultural and biochemical tests. Fermentational abilities and the production of indole suggested that a significant number of these bacteria were symbiotically associated with resident metazoans.

Calcium requirement and magnesium stimulation of Escherichia coli L-form induction.

Calcium was found to be required for l-form penicillin induction of Escherichia coli K-12 W1485, and magnesium was found to be stimulatory.