To the two lots of serum. It lost all detectable viability after 7 days incubation in the first lot, decreasing from an estimated 1E8 to ,1E2 CFU/mL in both experiments. However, it grew throughout the time course in the second lot, increasing from 1.1E5/mL to 5.4E9/mL by day seven. The time courses of nutritional stimulation on pre-rRNA pools in serum-acclimated cells were evaluated. Each cell suspension in serum was divided into two aliquots and centrifuged. One pellet was retained as a non-stimulated (“0-hour”) control aliquot while the other was resuspended in TSB and incubated at 37uC for up to 4 hours. Samples were taken after 1, 2, and 4 hours of nutritional stimulation. Pre-rRNA and genomic DNA were quantified by qPCR with a common primer set. By normalizing pre-rRNA to genomic DNA in all samples, cellular pre-rRNA abundance could be compared between samples, with minimal effects from variations in nucleic acid extraction efficiency or the presence of PCR inhibitors. Ratios of pre-rRNA to gDNA (P:G) in stimulated and control samples were calculated from the results. Because of the inefficiency of RT-qPCR relative to qPCR, P:G ratios usually appeared to be 1 or less. In reality, pre-rRNA copies per genome number in the hundreds or more, as indicated by past experiments in which pre-rRNA and gDNA were detected by direct probe analyses [20]. Pre-rRNA stimulation correlated with viability in serum-derived bacteria. A. baumannii, which survived and grew in serum as determined by CFU plating, exhibited robust increases in the P:G ratio within 1 hour of 1527786 nutritional stimulation (bars in Figure 2A). During this 1-hour period genomic DNA increased marginally if at all (line in Figure 2A). The P:G ratio began to decline afterResults Specificity of RT-qPCR Assays for Pre-rRNAIn all four bacterial species, RT-qPCR reactions detected the 59 ETS1 region upstream of the small subunit (SSU) rRNA-encoding gene. Primer sets were designed to straddle the 59 mature rRNA terminus as described previously [18]. Primers for cDNA synthesis and reverse qPCR primers recognized semi-conserved sequences within the mature rRNA (16S), while forward primers recognized species-specific sequences within the ETS1. Therefore, successful amplification required intact pre-rRNA molecules (or gDNA) as templates. Figure 1A shows the primer sequences used for both manual and Hesperidin manufacturer semi-automated 15857111 pre-rRNA buy Met-Enkephalin measurements. When possible, reverse transcription and reverse PCR primers targeted mature SSU rRNA sequences with at least some phylogenetic specificity; however, each assay primarily derived its specificity from the forward primer targeting the hypervariable ETS1. All forward primers were predicted to be species-specific based on BLAST searches conducted against the NCBI non-redundant database. Consistent with this prediction, when PCR primer sets for A.Viability Testing by Pre-rRNA Analysishours post-stimulation, at which point the cells had already initiated active replication as indicated by genomic DNA signals. Relative to A. baumannii, the P:G ratio in S. aureus increased more slowly, peaking at 2 hours, with genomic DNA signals remaining low throughout the experiment (Figure 2B). S. aureus cells may have lysed in the serum, leaving behind a small number of intact cells. Some of these cells were viable as indicated by the plating results, and sufficient to yield robust pre-rRNA increases after nutritional stimulation. In contrast to A. baumannii and S. aureus, P. aer.To the two lots of serum. It lost all detectable viability after 7 days incubation in the first lot, decreasing from an estimated 1E8 to ,1E2 CFU/mL in both experiments. However, it grew throughout the time course in the second lot, increasing from 1.1E5/mL to 5.4E9/mL by day seven. The time courses of nutritional stimulation on pre-rRNA pools in serum-acclimated cells were evaluated. Each cell suspension in serum was divided into two aliquots and centrifuged. One pellet was retained as a non-stimulated (“0-hour”) control aliquot while the other was resuspended in TSB and incubated at 37uC for up to 4 hours. Samples were taken after 1, 2, and 4 hours of nutritional stimulation. Pre-rRNA and genomic DNA were quantified by qPCR with a common primer set. By normalizing pre-rRNA to genomic DNA in all samples, cellular pre-rRNA abundance could be compared between samples, with minimal effects from variations in nucleic acid extraction efficiency or the presence of PCR inhibitors. Ratios of pre-rRNA to gDNA (P:G) in stimulated and control samples were calculated from the results. Because of the inefficiency of RT-qPCR relative to qPCR, P:G ratios usually appeared to be 1 or less. In reality, pre-rRNA copies per genome number in the hundreds or more, as indicated by past experiments in which pre-rRNA and gDNA were detected by direct probe analyses [20]. Pre-rRNA stimulation correlated with viability in serum-derived bacteria. A. baumannii, which survived and grew in serum as determined by CFU plating, exhibited robust increases in the P:G ratio within 1 hour of 1527786 nutritional stimulation (bars in Figure 2A). During this 1-hour period genomic DNA increased marginally if at all (line in Figure 2A). The P:G ratio began to decline afterResults Specificity of RT-qPCR Assays for Pre-rRNAIn all four bacterial species, RT-qPCR reactions detected the 59 ETS1 region upstream of the small subunit (SSU) rRNA-encoding gene. Primer sets were designed to straddle the 59 mature rRNA terminus as described previously [18]. Primers for cDNA synthesis and reverse qPCR primers recognized semi-conserved sequences within the mature rRNA (16S), while forward primers recognized species-specific sequences within the ETS1. Therefore, successful amplification required intact pre-rRNA molecules (or gDNA) as templates. Figure 1A shows the primer sequences used for both manual and semi-automated 15857111 pre-rRNA measurements. When possible, reverse transcription and reverse PCR primers targeted mature SSU rRNA sequences with at least some phylogenetic specificity; however, each assay primarily derived its specificity from the forward primer targeting the hypervariable ETS1. All forward primers were predicted to be species-specific based on BLAST searches conducted against the NCBI non-redundant database. Consistent with this prediction, when PCR primer sets for A.Viability Testing by Pre-rRNA Analysishours post-stimulation, at which point the cells had already initiated active replication as indicated by genomic DNA signals. Relative to A. baumannii, the P:G ratio in S. aureus increased more slowly, peaking at 2 hours, with genomic DNA signals remaining low throughout the experiment (Figure 2B). S. aureus cells may have lysed in the serum, leaving behind a small number of intact cells. Some of these cells were viable as indicated by the plating results, and sufficient to yield robust pre-rRNA increases after nutritional stimulation. In contrast to A. baumannii and S. aureus, P. aer.
