Isolation and characterisation of the marine ultramicrobacteriumSphingomonassp. strain RB2256

Abstract

Indigenous heterotrophic marine bacteria are of great importance to global nutrient cycling. Predominant native bacteria are of ultramicrobacterial dimensions, are not associated with aggregates and must have truly remarkable abilities for substrate capture. Agar media are unsuited for the isolation of the dominant oceanic bacterioplankton. In contrast, the dilution culture technique [Button et al. (1993) Appl. Environ. Microbiol. 59, 881–891; Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160] leads to a successful enrichment of the dominant cell types. Up to 50% of the indigenous bacterial population in water obtained from Resurrection Bay was able to grow in dilution tubes containing only filtered, autoclaved natural sea water (FAS). Ultramicrobacteria (UMB), very small bacteria with small genomes, predominate in such cultures. Generally, dilution factors that resulted in inocula of approximately 2 cells per tube were optimal and prevented outgrowth of atypical large bacteria. Strain RB2256 [Schut et al. (1993) Appl. Environ. Microbiol. 59, 2150–2160], one of the UMB isolated by this dilution culture technique and tentatively identified as a marine Sphingomonas sp., was investigated in more detail. Although reverted from obligately oligotrophic to facultatively oligotrophic upon isolation, this strain possessed a number of traits assigned to a ‘model oligotroph’ and some unpredicted novel properties. The cells showed no miniaturisation upon starvation but consistently exhibited low cell volumes. They had a very low DNA content, were rich in protein, and contained only one copy of the rRNA operon. The cells were well adapted to the simultaneous utilisation of mixed substrates. A constitutive, high-affinity and binding protein-dependent uptake system for mixed amino acids was found that would allow realistic in situ generation times at the prevailing amino acid concentrations. Further studies on this same organism revealed that the cells appeared to be extremely resistant to various stress-inducing agents. High survival rates were observed after high-intensity heat shocks, treatments with H2O2 or with ethanol. Moreover, no marked differences were observed between starved or actively growing cells in this respect, particularly when cells were grown in chemostat. Application of the dilution culture technique to the field of subsurface microbiology could be adopted to study the occurrence of UMB in groundwater with a comparable and stable ‘low-nutrient-conditioned’ phenotype.