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In filtered natural seawater supplemented with potassium glutamate, the ability of Escherichia coli MC4100 cells to grow on a complex medium was enhanced as a logarithmic function of the external glutamate concentration. By comparison, a glutamate-respiring strain of E. coli exhibited a greater decline in culturability in seawater, suggesting a protective influence of the accumulated amino acid. Potassium glutamate increased the uptake of 14C-glycinebetaine by E. coli MC4100 cells in seawater and enhanced the protective effects of the betaine against culturability loss, possibly by increasing the expression of the ProU transport system. This bacterium apparently was able to synthesize glutamate because a protective effect (i.e. a lower culturability loss) was observed in seawater when supplemented with precursor compounds (2-oxoglutarate and glutamine). The combination of 2-oxoglutarate and glutamine resulted in the greatest protection of cells, possibly due to the synthesis of glutamate through glutamine 2-oxoglutarate amino transferase activity. The possible influence of glutamate and its precursors on survival of E. coli cells in the natural marine environment is considered, since glutamate, glutamine and betaines have been found in marine coastal waters and sediments.

On the basis of phenotypical characteristics and analysis of 16S rRNA sequence, a new species belonging to a new genus is described, and the name Marinobacter hydrocarbonoclasticus is proposed. This organism, isolated from Mediterranean seawater near a petroleum refinery, is a gram-negative, aerobic, rod-shaped bacterium. It grows at NaCl concentrations of 0.08 to 3.5 M and uses various hydrocarbons as the sole source of carbon and energy. Its DNA has a guanine-plus-cytosine content of 52.7 mol%. The 16S rRNA analysis shows a clear affiliation between M. hydrocarbonoclasticus and the gamma group of the phylum Proteobacteria. A close phylogenetic relationship appears among the species Marinomonas vaga, Oceanospirillum linum, Halomonas elongata, and Pseudomonas aeruginosa. Because of the impossibility of finding a single most closely related species, we suggest that this bacterium be assigned to a new genus, at least temporarily. The possibility of a revision of this status when new data appear is, however, not excluded. The type strain is M. hydrocarbonoclasticus SP.17 (= ATCC 49840).

Sensitivity of Escherichia coli cells in seawater, considered in terms of culturability loss, was examined after different growth periods in a mineral medium supplemented with glucose (M9) at 37 degrees C under aerobic or anaerobic conditions. Their sensitivity varied considerably during the different growth phases and differed when cells were grown under aerobic or anaerobic conditions. Sensitivity of aerobic cells rapidly increased during the lag phase, then decreased during the exponential phase and became minimal during the stationary phase. Coliforms isolated from human faeces showed a similar sensitivity after incubation in wastewater at 37 degrees C for 3 h. The sensitivity phase was completely eliminated when cells were incubated with chloramphenicol. Variation of sensitivity in anaerobic cells according to their growth phase was comparable with that found for aerobic cells which had been left in seawater for a long period (6 d). However, for shorter periods in this medium (1-2 d), cells grown until the mid-exponential phase remained resistant to seawater. During the second half of the growth phase, they were as sensitive as aerobic cells at lag phase. Escherichia coli cells grown under anaerobic conditions, such as found in the intestine, progressively adapt to aerobic conditions after their transfer into aerated seawater and their sensitivity to seawater increases. On a practical level, these observations show that it is necessary to control accurately the age of cells before inoculation in seawater microcosms to conserve a comparative value in results. The importance of this factor is vital as all variations in sensitivity of cells to seawater according to their prior growth phase proved to be logarithmic functions of time.

The authors have compared the survival in seawater of Salmonella paratyphi B and Pseudomonas aeruginosa cells grown at low or high osmolarity, in the presence of organic osmolytes: glycine betaine, choline, proline, and glutamate. The four substrates enhanced the survival potential of S. paratyphi B while only glycine betaine protected P. aeruginosa. In addition only S. paratyphi B cells were more resistant after a preliminary growth at high osmolarity. Both bacteria were sensitive to osmotic down-shock, sensitization of S. paratyphi B being inversely proportional (p greater than or equal to 0.01) to the osmolarity of the medium used to wash cells. The transit in wastewater, at low osmolarity, can therefore modify the behavior of these pathogens in the marine environment.

The contribution of the major outer membrane porins OmpF and OmpC to the maintenance of viability and culturability of Escherichia coli cells in seawater was analyzed using isogenic mutant strains lacking one or both porins. Cells that possessed OmpF and OmpC survived better than those lacking one or both of them. However, the results differed, depending on whether the cells were adapted to high osmolarity or not before transfer to seawater. When cells were grown at low osmolarity, survival was largely influenced by porins, the OmpF+ strains surviving better than those lacking this porin. Addition of an OmpF plasmid to OmpF- OmpC- cells also improved their viability. When grown at high osmolarity, the role of porins was less critical since both the viability and culturability of the cells increased. However, cells that expressed only OmpC showed the most dramatic loss of viability. Cells lacking both OmpF and OmpC exhibited a higher loss of viability and culturability in seawater. Regarding the influence of porins on survival, these results show that the conditions that prevail during the growth of cells before their transfer to seawater are highly influential: cells that express the porin corresponding to the growth conditions they are in at the time of transfer survive better.

The high resistance of Escherichia coli grown in saline media to seawater was suppressed by an osmotic down-shock. The shock released several molecules into the medium, including potassium, glutamate, and glycine betaine when cells were previously grown in the presence of this osmolyte. Incubation of such sensitized cells in a solution containing K+ (80 mM) and glutamate (50 mM) at pH 7.4 restored their resistance to seawater up to a level close to that observed initially. The protective effect was partly due to the rapid accumulation of K+; a significant exponential relationship between intracellular concentration of K+ and resistance to seawater was observed. Glutamate was accumulated more slowly and progressively completed the action of K+. These data emphasize the specific influence of potassium glutamate on osmotically stressed E. coli cells. They confirm that regulation of osmotic pressure and, probably, of intracellular pH strongly enhances survival of E. coli in seawater. Osmotic fluctuations in waters carrying enteric bacteria from intestines to seawater, together with variations in their K+ and amino acid contents, could modify the ability of cells to survive in marine environments. These results demonstrate the need to strictly control conditions (K+ content, temperature) used to wash cells before their transfer to seawater microcosms. They suggest that the K+ and glutamate contents of media in which E. coli cells are transported to the sea can influence their subsequent survival in marine environments.

The conjugative transfer of plasmid RP4 between two strains of Escherichia coli in a sterile marine sediment was enhanced by the presence of glycine betaine (frequency increased 20 to 40 times). The conjugation was also facilitated by the osmoprotection of donor cells. Glycine betaine is a universal osmolyte and has been found in marine sediments at high concentrations. So this phenomenon could have epidemiological and sanitary importance by increasing the possibility of dissemination of some plasmids present in enterobacteria in natural marine deposits.

Transfer of Vibrio parahaemolyticus (biotype 2) from sediments to water and from water to benthic marine organisms was studied experimentally using a streptomycin-resistant strain. Transmission by trophic pathways was also studied using reconstituted marine food chains (Mytilus edulis, Nereis diversicolor, Carcinus maenas, Scorpaena porcus, Mus musculus). Water colonization by sediments could be observed only at temperatures above 16 degrees C. Sediments could well constitute a disseminating reservoir for these germs, their cycle in water being dependent of the cycle followed in the sediments. Contamination of animal organisms is essentially effected by a direct mean, either water or sediments; transfer by trophic pathways being negligible. Infection of land consumers (mice) is linked quantitatively to the nature of the last marine organism of the food chain since bacteria can flourish in the digestive tract of certain animals (Carcinus maenas).

A lipid antibiotic was extracted from whole cells of the marine diatom Chaetoceros lauderi (Ralfs) and roughly identified as an unsaturated fatty acid by thin layer chromatography. A number of Gram-positive terrestrial bacteria and several Gram-negative marine bacilli were sensitive to this antibiotic, whereas Gram-negative enteric bacteria were resistant. Solar irradiation led to its partial disappearance and to the production of more polar antibiotics with a stronger antibacterial activity. This observation is compared to similar data culled from literature, and its significance in a natural environment is discussed.

Six marine bacteria which synthesize macromolecular antibiotics were isolated from neritic waters on the French Mediterranean coast, and their frequency recorded over two successive years. They appeared in relatively large numbers during the period August to December, and can be identified as marine pseudomonads; however, the low guaninecytosine ratio of their DNA, lack of catalase and specific self-inhibition are not compatible with the characteristics of the genus Pseudomonas. Two produced violacein, usually synthesized by bacteria belonging to the genus Chromobacterium. Their taxonomic position is discussed.

Sixteen violet-pigmented heterotrophic bacilli were isolated from Mediterranean coastal waters. Morphological and physiological studies showed that they have a number of characteristics specific to the genus Chromobacterium. However, the absence of catalase, the presence of oxidase, and, more especially, the low percentage of bases guanine and cytosine in their DNA exclude them from the genus. The specificity of some characters assigned to Chromobacterium can thus be discussed. Several features allow us to consider such bacteria as being related to the genus Alteromonas; their consistency within the 16 strains investigated leads us to consider them as a single species, for which the specific name Alteromonas luteo-violaceus (sp. nov.) is proposed.

Authors tried to determine some factors of incertitude in the anlaysis of the bacterial contamination of seawater. Such analysis depends on the reliability of the methods used for bacterial numeration, and on the heterogeneity of water in the investigated area. The general numeration of organisms (GNO) and the numeration of coliforms (NC) were taken into account. The turbidity of the sample, and the operator's manner were shown to be factors of variation for the GNO. An interaction between the operator and the amount of the anlaysed sample seemed to influence NC. In a natural environment, variations with time were the most important. They were partly due to hydrological and meteorological fluctuations, but sometimes seemed to be random. During a day cycle, the bacterial contamination trended to decrease when the solar radiance was maximum. Influence of short scale variations in the location of sampling was generally random, yet with a slight increase of contamination in the more stagnant waters.

The synthesis of several types of antibiotics was investigated in four strains of violet-pigmented bacteria belonging to the species Alteromonas luteo-violaceus. Two of the strains simultaneously produce an antibiotic polyanionic polysaccharide, weakly bound to the cells and diffusing throughout the medium, and two intracellular brominated bactericidal substances. The third strain only synthesizes the polyanionic antibiotic. The fourth one is totally inactive. The macromolecular antibiotic, probably responsible for the autointoxication of the bacteria in their cultures, acts at the respiratory level; it induces an increase of oxygen uptake and the production of peroxides by test bacteria. Thus, its activity is inhibited by catalase and peroxidase.

The red prodigiosin-like pigment from Alteromonas rubra was shown to be a mixture of prodigiosin (pigment 1) and a new cyclic isomer (pigment 2). The new structure was elucidated by mass and nuclear magnetic resonance spectra. Careful examinations of the prodigiosins produced by Serratia marcescens, Vibrio psychoerythrus, and an unidentified red bacterium (LL-100-6) failed to disclose any of the new pigment, pigment 2.

The adaptation of enteric bacteria in seawater has previously been described in terms of nutrient starvation. In the present paper, we bring experimental arguments suggesting that survival of these microorganisms could also depend on their ability to overcome the effects of osmotic stress. We analyzed the influence of osmoregulatory mechanisms (potassium transport, transport and accumulation of organic osmolytes) on the survival of Escherichia coli in seawater microcosms by using mutants lacking components of the osmotic stress response. Long-term protection was afforded to cells by growth in a medium whose osmotic pressure was increased by either NaCl, LiCl, or saccharose. Achievement of the protection state depended at least partly on osmoregulatory mechanisms, but differed when these were activated or induced during prior growth or in resting cells suspended in phosphate buffer or in seawater. When achieved during growth, K+ transport, glycine-betaine (GBT) synthesis or transport, and trehalose synthesis helped increase the ability to survive in seawater. Protection by GBT was also obtained with resting cells in a phosphate buffer at high osmotic pressure. However, when added only to the seawater, GBT did not change the survival ability of cells no matter what their osmoregulation potential. These results showed that the survival of E. coli cells in seawater depends, at least partly, on whether they possess certain genes which enable them to regulate osmotic pressure and whether they can be stimulated to express those genes before or after their release into the environment. This expression requires nutrients as the substrates from which the corresponding gene products are made.

A toxigenic strain of Escherichia coli displayed important structural modifications when placed in seawater which naturally lacked nutritive elements, as observed by electron microscopy. These include cell wall and cell body distortion, modification of the membranes, central segregation of the chromosome, and retraction of the cytoplasm. These modifications were accompanied by a decrease in cell protein content of approximately 40%. Certain cytoplasmic membrane proteins were lost, and new ones appeared. The development of these changes was considerably slower in cells that had previously been grown in a seawater medium. This suggests that osmotic regulation mechanisms, which enable E. coli to survive much longer in marine conditions, may have a protective influence.

Some metabolic modifications of Escherichia coli cells during starvation in seawater were studied in laboratory microcosms. The apparent die-off of this bacterium under such conditions, as observed by comparing the enumeration of CFU in conventional freshwater media and direct epifluorescence counts, was partially prevented when cells were previously grown in salted organic medium or on seawater-wastewater agar. beta-Galactosidase activity of starved cells disappeared gradually with time, even though some other enzymatic activities, such as that of alkaline phosphatase, increased. Moreover, some modifications of sensitivity to antibiotics, heavy metals, and bacteriophages in seawater- and wastewater-grown cells suggested that the cells undergo structural changes under natural marine conditions. These results provide additional experimental data indicating the possible active adaptation of E. coli cells to seawater.

By use of experimental microcosms, it was demonstrated that the survival of Escherichia coli in nutrient-free seawater depended on the age of cells and on some physicochemical conditions during their prior growth. Cells grown in a bacteriological medium, with an acid or an alkaline pH, at high temperature (44 degrees C), or in the absence of oxygen were more sensitive to exposure to seawater of low nutrient content. In contrast, some complex media allowed production of cells adapting more rapidly to seawater. Cells grown in urine were far more sensitive than those grown in all bacteriological media tested. The sensitivity of all cells was highest when they were harvested during the early exponential phase of growth.

In sterile nutrient-free seawater, the number of Pseudomonas aeruginosa culturable cells decreased progressively over time and the bacteria developed cells capable of passing through a 0.45 micron pore membrane. This development was more rapid in non-autoclaved, stirred seawater and the recovery of filterable cells varied depending on the membrane type used. Minicells were observed under an electron microscope. They yielded normal cells in bacteriological media with analogous colonies and an unchanged antibiotic resistance profile. Some biochemical characters, such as gelatinase or urease activity, were however modified in the filterable cells.