A new approach to the analysis of hybridization of bacterial nucleic acids. Analysis of ribosomal ribonucleic acids of Bacillus subtilis

A new graphical analytical technique is described for the hybridization of bacterial RNA with denatured homologous DNA immobilized on cellulose nitrate membrane filters. To a constant amount of DNA, various amounts of bacterial RNA were added and the percentage of input RNA bound was plotted against the DNA/RNA weight ratio in a given experiment. When RNA samples were used that hybridize to denatured DNA as a single species, the resulting curves (RNA-hybridization-efficiency curves) could be analysed to show the percentage of the DNA capable of specifically binding the RNA and could also be used to detect the presence of minor RNA contaminants in a purified specimen. The method could also estimate the relative amounts of two species of RNA in a mixture when these were hybridized independently to different DNA cistrons or cistron groups. As an example of RNA that can be studied in this way, the 16s and 23s ribosomal RNA species of Bacillus subtilis were chosen. These each behave in DNA-RNA hybridization as a single species and bind independently to different groups of DNA cistrons. The results obtained from hybridization-efficiency curves were compared with those obtained by the more usual method of saturating the specific DNA regions with excess of ribosomal RNA (hybridization-saturation curves). It was confirmed by both approaches that 0.15 (+/-0.02)% of B. subtilis DNA would hybridize with 16s ribosomal RNA, 0.30 (+/-0.02)% would hybridize with 23s ribosomal RNA, and 0.46 (+/-0.02)% would hybridize with (16s+23s) ribosomal RNA. This agreement suggested that mass-action equilibria between hybridized and free RNA had a negligible effect on the hybridization curves over the range of DNA and RNA concentrations employed.     

Determination of synthesis rate and lifetime of bacterial mRNAs

A method has been developed to determine the synthesis rate and lifetime of bacterial mRNAs, either bulk mRNA or specific mRNAs, with a minimum of physiological disturbance. The method uses hybridization of pulse-labeled RNA to specific probes followed by an evaluation based on a computer simulation of the labeling kinetics of different classes of RNA. The method was applied to the determination of bulk mRNA in Escherichia coli growing in glucose minimal medium: 60% of the instantaneous rate of RNA synthesis, or 2.3% of the total amount of RNA, was found to be mRNA with an average lifetime of 1.0 +/- 0.2 min (= 0.7 min half-life).     

Assessment of the state of activity of individual bacterial cells by hybridization with a ribosomal RNA targeted fluorescently labelled oligonucleotidic probe

Abstract A fluorescently labelled oligonucleotide probe complementary to an evolutionarily conserved domain of the small subunit ribosomal RNA sequence has been used with an image analysis equipment to quantify cellular rRNA contents during the successive phase of a bacterial culture ( Escherichia coli ). The amount of hybridization increased steadily upon inoculation of stationary phase bacteria into a new medium, while cell divisions were delayed. This hybridization signal was abruptly reduced by 50% at the onset of the exponential growth phase during which it then remained stable. A further slow decrease took place during stationary phase. The amount of rRNA per cell is thus maximal immediately before the beginning of active cell division. The implications of these results are discussed in terms of bacterial ecology.     

Extraction of high-quality bacterial RNA from infected leaf tissue for bacterial in planta gene expression analysis by multiplexed fluorescent Northern hybridization

Plant pathogenic bacteria possess a large number of genes that allow them to grow and cause disease on plants. In planta gene expression analysis is important to understand the impact of these genes on bacterial virulence. A new mRNA-based approach using multiplexed Northern hybridization was developed. High-quality bacterial and plant total RNA was successfully isolated from leaf tissue infiltrated with Pseudomonas syringae . The procedure employs a new extraction buffer formulation containing glycine, sodium dodecylsulphate, cetyltrimethylammonium bromide, high-molecular-weight polyethylene glycol and β-mercaptoethanol. Cell lysis and classical acid–phenol extraction steps followed by LiCl precipitation yielded large amounts of total RNA of high purity and integrity. Multiplexing of DIG and chemically fluorescently labelled RNA probes was developed and expression data were normalized using the 23S rRNA gene as reference. The method was validated by studying in planta expression of the P. syringae genes mucD , cmaA , cfl , corR , corS and corP comprising a selection of highly expressed biosynthetic and low-expressed regulatory genes. The method was assessed regarding its sensitivity and might by useful for studying a variety of plant–microbe interactions.