Nutrition and metabolism of marine bacteria. XVI. Formation of protoplasts, spheroplasts, and related forms from a gram-negative marine bacterium

When cells of a marine pseudomonad were washed and suspended in 0.5 m sucrose, they retained their rod shape, but thin sections, when examined in an electron microscope, revealed that the outer layer of the cell wall had separated a considerable distance from the cytoplasmic membrane. Treatment of such cells with lysozyme alone produced no obvious change, but treatment with ethylenediaminetetraacetic acid (EDTA) alone caused the outer wall to disappear. A combination of EDTA and lysozyme resulted in the rapid formation of spheres essentially free from hexosamine and indistinguishable from protoplasts of gram-positive bacteria. When cells were washed with 0.5 m NaCl and then suspended in 0.5 m sucrose, they also retained their rod shape, but in this case the outer layer separated from the cells completely and could be recovered from the suspending medium. Such cells were converted to protoplasts by the action of lysozyme alone. Cells washed and finally suspended in 0.5 m NaCl, when treated with EDTA and lysozyme, slowly became spherical. Thin sections revealed typical spheroplasts of gram-negative bacteria in which the outer wall remained intact. Protoplasts took up alpha-aminoisobutyric acid by a Na(+)-dependent process.     

Survival and nutritional requirements of three bacteria isolated from ultrapure water

Bacteria isolated previously from ultrapure water (UPW) systems were examined for their ability to survive in UPW, with the ultimate goal of elucidating potential carbon and energy sources for the bacteria. Two strains of Ralstonia pickettii isolated from different areas within the UPW system (pretreatment and polishing loop, and referred to as strains 3A1 and MF254A, respectively) and a strain of Bradyrhizobium sp. were compared to increase our understanding of the fundamental behavior of bacteria contaminating UPW. R. pickettii (3A1) grew significantly slower in R2A medium, with a final cell yield much lower than the isolate from the polishing loop. In addition, R. pickettii MF254A showed a broader substrate range than either strain 3A1 or Bradyrhizobium sp. In UPW, there appears to be a threshold cell concentration (approximately 10⁶ colony-forming units/ml), whereby the cell numbers remain constant for a prolonged period of 6 months or more. Below this concentration, rapid proliferation is observed until the threshold concentration is attained. Preliminary experiments suggested that nitrogen gas (frequently added to UPW storage tanks) may contribute to growth of Bradyrhizobium sp. Above the threshold concentration, the strain of Ralstonia sp. isolated from the polishing loop was capable of cryptic growth with heat-killed cells in UPW. However, cryptic growth was not observed when the cells supplied as nutrients were killed using UV254 light. Furthermore, cryptic growth did not appear to contribute significantly to proliferation of Bradyrhizobium sp. or Ralstonia sp. 3A1 (isolated from the pretreatment loop). We believe that cryptic growth may aid survival of the bacteria in UPW, but further experiments are warranted to prove this phenomenon conclusively. Journal of Industrial Microbiology & Biotechnology (2002) 29, 75–82 doi:10.1038/sj.jim.7000273 Received 23 January 2002/ Accepted in revised form 29 April 2002     

Phylogenetic identification and metabolism of marine dimethylsulfide-consuming bacteria

Microbial consumption is one of the main processes, along with photolysis and ventilation, that remove the biogenic trace gas dimethylsulfide (DMS) from the surface ocean. Although a few isolates of marine bacteria have been studied for their ability to utilize DMS, little is known about the characteristics or phylogenetic affiliation of DMS consumers in seawater. We enriched coastal and open-ocean waters with different carbon sources to stimulate different bacterial communities (glucose-consuming bacteria, methyl group-consuming bacteria and DMS consumers) in order to test how this affected DMS consumption and to examine which organisms might be involved. Dimethylsulfide consumption was greatly stimulated in the DMS addition treatments whereas there was no stimulation in the other treatments. Analysis of microbial DNA by two different techniques (sequenced bands from DGGE gels and clone libraries) showed that bacteria grown specifically with the presence of DMS were closely related to the genus Methylophaga. We also followed the fate of consumed DMS in some of the enrichments. Dimethylsulfide was converted mostly to DMSO in glucose or methanol enrichments, whereas it was converted mostly to sulfate in DMS enrichments, the latter suggesting use of DMS as a carbon and energy source. Our results indicate that unlike the biochemical precursor of DMS, dimethylsulfoniopropionate (DMSP), which is consumed by a broad spectrum of marine microorganisms, DMS seems to be utilized as a carbon and electron source by specialists. This is consistent with the usual observation that DMSP turns over at much higher rates than DMS.