The physiological responses of Pseudomonas putida KT2442 to phosphate starvation were examined with respect to cell morphology, qualitative demonstration of the accumulation of the intracellular storage component poly-3-hydroxyalkanoate (PHA), cellular ATP and ribosome content, and the rate of total protein synthesis. Upon prolonged incubation under phosphate-limiting conditions, the number of viable cells decreased by two to three orders of magnitude during the first 3 weeks. However, after this decline, viability of the cultures remained remarkably constant for many weeks. The cells remained rod-shaped under phosphate starvation conditions with a tendency to swell in parallel with the accumulation of PHA. Protein synthesis and ribosome concentration were gradually reduced, and ATP levels dropped to very low values after the onset of starvation; later, however, there was a return to near-normal ATP concentrations. Evidence was obtained that the strong selective pressure imposed by phosphate deprivation forces the selection of mutants with a competitive advantage. These mutants are able to grow, possibly utilizing nutrients derived from dead cells, and eventually take over the cultures. One frequently encountered mutant formed smaller colonies on rich solidified medium and displayed an altered cell morphology. This mutant was isolated and further characterized. By employing a bioluminescence-based marker system, we demonstrated that this mutant is able to replace wild-type cells in mixed culture experiments. Thus, long-term phosphate-deprived cultures represent dynamic regimes that can undergo population shifts.
AnbaJ., BidaudM., VasilM. L., LazdunskiA.1990; Nucleotide sequence of the Pseudomonas aeruginosa phoB gene, the regulatory gene for the phosphate regulon. J Bacteriol 172:4685–4689
De WegerL. A., DekkersL. C., van der BijA. J., LugtenbergJ. J.1993; Use of phosphate-reporter bacteria to study phosphate limitation in the rhizosphere and bulk soil. Mol Plant-Microbe Interact 7:32–38
FlSrdhK., CohenP. S., KjellebergS.1992; Ribosomes exist in large excess over the apparent demand for protein synthesis during carbon starvation in marine Vibrio sp. strain CCUG 15956. J Bacteriol 174:6780–6788
GivskovM., EberlL., MollerS., PoulsenL. K., MolinS.1994a; Response to nutrient starvation in Pseudomonas putida KT2442: analysis of general cross-protection, cell shape and macromolecular content. J Bacteriol 176:7–14
HancockR. E. W., PooleK., BenzR.1982; Outer membrane protein P of Pseudomonas aeruginosa: regulation by phosphate deficiency and formation of small anion-specific channels in lipid bilayer membranes. J Bacteriol 150:730–738
HuijbertsG. N. M., de RijkT. C., de WaardP., EgginkG.1994; 13C-Nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxy-alkanoate) synthesis. J Bacteriol 176:1661–1666
HuismanG. W., de LeeuwO., EgginkG., WitholtB.1989; Synthesis of poly-3-hydroxyalkanoates is a common feature of fluorescent pseudomonads. Appl Environ Microbiol 55:1949–1954
HuismanG. W., WoninkE., MeimaR., KazemierB., TerpstraP., WitholdB.1991; Metabolism of poly(3-hydroxyalkanoates) (PHAs) by Pseudomonas oleovorans: identification and sequences of genes and function of the encoded proteins in the synthesis and degradation of PHA. J Biol Chem 266:2191–2198
KlemmP., ChristiansenG.1987; Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae. Mol & Gen Genet 208:439–445
KristensenC. S., EberlL., Sanches-RomeroJ. M., GivskovM., MolinS., de LorenzoV.1995; Site-specific deletions of chromosomally located DNA segments with the multimer resolution system of broad-host-range plasmid RP4. J Bacteriol 177:52–58
McCannM. P., KidwellJ. P., MatinA.1991; The putative a factor KatF has a central role in development of starvation-mediated general resistance in Escherichia coli. J Bacteriol 173:4188–4194
MollerS., KristensenC. S., PoulsenL. K., CarstensenJ. M., MolinS.1995; Bacterial growth on surfaces: automated image analysis for quantification of growth rate-related parameters. Appl Environ Microbiol 61:741–748
NystromT., OlssenR. M., KjellebergS.1992; Survival, stress resistance and alterations in protein expression in the marine Vibrio sp. strain S14 during starvation for different individual nutrients. Appl Environ Microbiol 58:55–65
OliverJ. D., StringerW. F.1984; Lipid composition of a psychrophilic marine Vibrio sp. during starvation-induced morphogenesis. Appl Environ Microbiol 47:461–466
OstroffR. M., VasilM. L.1987; Identification of a new phospholipase C activity by analysis of an insertional mutation in the hemolytic phospholipase C structural gene of Pseudomonas aeruginosa. J Bacteriol 169:4597–4601
PooleK., HancockR. E. W.1984; Phosphate transport in Pseudomonas aeruginosa: involvement of a periplasmatic phosphate-binding protein. Eur J Biochem 144:607–612
SampleE.C., SoperR. J., RaezG. J.1980; Reactions of phosphate fertilizers in soils. The Role of Phosphate in Agriculture263310 Edited by KhasawnehF. E., SampleE. C., KamprathE. J. Madison, WI: American Society of Agronomy;
SiegeleD. A., AlmirdnM., KolterR.1993; Approaches to the study of survival and death in stationary phase Escherichia coli. Starvation in Bacteria151–169 Edited by KjellebergS. New York: Plenum Press;
StouthamerH. A.1973; A theoretical study of the amount of ATP required for synthesis of microbial cell material. Antonie van Eeeuwenhoek. J Microbiol Serol 39:545–565
TanakaK., TakayanasiY., FujitaN., IshihamaA., TakahashiH.1993; Heterogeneity of principal sigma factor in Escherichia coli: the rpoS gene product tx38, is a principal sigma factor of RNA polymerase in stationary phase Escherichia coli. Proc Natl Acad Sci USA 90:3511–3515
WannerB.1987; Phosphate regulation of gene expression in E. coli. Escherichia coli and Salmonella typhimurium: Cellular and Molecular BiologyVol. 21326–1333 Edited by NeidhardtF. C., IngrahamJ. L., MagasanikB., LowK. B., SchaechterM., UmbargerH. E. Washington, DC: American Society for Microbiology;
ZambranoM. M., SiegeleD. A., AlmirtinM., TormoA., KolterR.1993; Microbial competition: Escherichia coli mutants that take over stationary phase cultures. Science 259:1757–1760