PCR amplification of 16S rDNA was found to be highly biased, so that the rDNA from one species out of four was preferentially amplified. We present evidence that the observed PCR bias most likely occurs because the genomic DNA of some species contains segments outside the amplified sequence that inhibit the initial PCR steps. Attempts to overcome this bias by use of a ‘touch down’ PCR procedure or by performing PCR in the presence of denaturants or cosolvents such as acetamide, DMSO, or glycerol were unsuccessful. Since the PCR inhibiting interference from template flanking DNA segments evidently is dependent on the position of the primer sites, we suggest that community diversity analysis based on PCR amplification of 16S rDNA can be improved by extending the procedure from comparative analysis of 16S rDNA amplified by use of only one primer set to a procedure involving at least two different 16S rDNA PCR amplifications performed with different primer sets.

A fast routine method for estimating bacterial cell growth rates by using the metachromatic dye acridine orange is described. The method allows simultaneous estimates of cellular RNA and DNA contents of single cells. Acridine orange staining can be used as a nonspecific supplement to quantitative species-specific hybridizations with fluorescence-labelled ribosomal probes to estimate the single-cell concentration of RNA. By automated analysis of digitized images of stained cells, we determined four independent growth rate-related parameters: cellular RNA and DNA contents, cell volume, and the frequency of dividing cells in a cell population. These parameters were used to compare physiological states of liquid-suspended and surfacegrowing Pseudomonas putida KT2442 in chemostat cultures. The major finding is that the correlation between substrate availability and cellular growth rate found for the free-living cells was not observed for the surfacebound cells; in contrast, the data indicate an almost constant growth rate for attached cells which was independent of the dilution rate in the chemostat.

Pseudomonas putida KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere. Lysine is one of the major compounds in root exudates, and P. putida KT2440 uses this amino acid as a source of carbon, nitrogen, and energy. Lysine is channeled to ␦-aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products. We show that the davDT genes form an operon transcribed from a single 70 -dependent promoter. The relatively high level of basal expression from the davD promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be ␦-aminovaleric acid because in a mutant unable to metabolize lysine to ␦-aminovaleric acid, this compound, but not lysine, acted as an effector. Effective induction of the P. putida P davD promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters. Mutants in these ABC uptake systems retained basal expression from the davD promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.

An efficient approach for the insertion of fluorescent marker genes with sequence specificity into conjugative plasmids in Escherichia coli is described. For this purpose, homologous recombination of linear double-stranded targeting DNA was mediated by the bacteriophage V recombination functions using very short regions of homology. Initial manipulation of the IncFII target plasmids R1 and R1drd19 indicated that the linear targeting DNA should be devoid of all extraneous homologies to the target molecule for optimal insertion specificity. Indeed, a simple recombination assay proved that in the presence of additional homologous regions in the targeting DNA, strand exchanges occurred exclusively within the longest regions of homology. A versatile panel of vectors was created to facilitate convenient PCR amplification of targeting DNAs containing various combinations of different antibiotic resistance genes and fluorescent markers. The choice of 5P non-homologous extensions in primer pairs used for amplifying the marker cassettes determines the site specificity of the targeting DNA. This methodology is applicable to the modification of all plasmids that replicate in E. coli and is not restricted by plasmid size. ß

Sequence analysis of domains 3 and 4 of 23S rRNA from Pseudomonas fluorescens Ag1 was carried out to allow the design of a strain-specific rRNA oligonucleotide probe targeting this strain. The specificity of the probe, Ps-Ag1, was assessed by dot blot analysis and whole-cell hybridization, and it was found to be specific for P. fluorescens Ag1. The correlation between the ribosomal content of P. fluorescens Ag1 and growth rate was determined during balanced growth conditions with generation times ranging from 1.2 to 31.8 h. Hybridization of the rRNA-targeting probes combined with charged coupled device-enhanced microscopy was used to determine the rRNA content. The total RNA content per cell was determined by staining with acridine orange and charged coupled device-enhanced microscopy. After 2 h under carbon starvation conditions, the rRNA content per cell decreased to 45% of the content of an exponentially growing cell. After 1 day of carbon starvation, the rRNA content had decreased to 20%. When cells were grown at different temperatures, it was found that the rRNA content per cell was only dependent on the substrate in the temperature range from 5 to 30؇C. P. fluorescens Ag1 was used in a mesocosm release experiment. The strain could be detected by use of the oligonucleotide probe targeting rRNA for 8 days in the water column and for 10 days on solid surfaces. The standard curve correlating growth rate with rRNA content was used to estimate the physiological activity of P. fluorescens Ag1 in the mesocosm experiment.