Fate of Microcolonies Developing In Situ

<p>Microcolonies on days 4 and 5 of separate adaptive mutation experiments were sampled by touching with a needle, then the sample was spread on rich X-gal medium and scored for point-mutant or sectored Lac⁺ phenotype (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#s3" target="_blank">Materials and Methods</a>). The microcolonies were then left to grow into visible colonies (∼10⁷ cells), and the colonies sampled again and samples plated to score for sectoring (hatched bars, day 4 microcolony samples; black bars, day 5 microcolony samples). The numbers of each type of colony observed are shown next to the bars. One single stable microcolony produced a mixed visible colony, which may have been caused by overlap with another colony or microcolony.</p

Pol I Is Required for Adaptive Amplification and Not Point Mutation

<div><p>Strains were plated on lactose-minimal medium and Lac⁺ colonies counted daily (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#s3" target="_blank">Materials and Methods</a>). The plots are cumulative, showing the mean of 3–4 cultures with one SEM. Strains used: FC40 <i>dinB⁺ polA⁺</i><i>fadAB</i>::Tn<i>10</i>Kan<i>,</i> SMR3490 (squares); FC40 <i>dinB⁺ polA12</i>(Ts)<i>fadAB</i>::Tn<i>10</i>Kan<i>,</i> SMR3491 (diamonds); FC40 <i>dinB10 polA12</i>(Ts)<i>fadAB</i>::Tn<i>10</i>Kan<i>,</i> PJH308 and PJH309 (triangles, inverted triangles); and FC40 <i>dinB10 pol⁺ fadAB</i>::Tn<i>10</i>Kan<i>,</i> PJH310 (circles). All cultures were grown at 30 °C and the experiments conducted at 37 °C.</p> <p>(A) An example of the effect of <i>polA</i>(Ts) on the yield of <i>lac-</i>amplified colonies, showing a partial requirement for <i>polA</i> at a semi-permissive temperature. (B) and (C) show the effect of the <i>dinB10</i> mutation on adaptive <i>lac-</i>amplification and point mutation, respectively. The <i>dinB polA</i>(Ts) cells display the decreased <i>lac</i> amplification of the <i>polA</i> mutant (B), and the decreased point mutation characteristic of the <i>dinB</i> mutant (C), demonstrating that the decrease in <i>lac</i> amplification rate does not result from channeling of <i>lac</i>-amplified cells into a point mutation pathway. These data (C) also show that the absence of Pol I increases point mutation (reported previously, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-Harris1" target="_blank">Harris 1997</a>) in a completely DinB-dependent manner. This could occur via the absence of Pol I leading to SOS induction (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-Bates1" target="_blank">Bates et al. 1989</a>) and more DinB/Pol IV, or via relief of a competition between high-fidelity Pol I and error-prone Pol IV at the replisome. Neither of these ways should affect<i>lac</i> amplification, which is Pol IV–independent, as observed (B).</p></div

DNA Amplification Does Not Induce the SOS Response

<div><p>(A) Known <i>lac</i>-amplified and point-mutant (control) derivatives of SMR6039 were grown in liquid M9 medium containing either lactose or glycerol. Mid-logarithmic phase cells were harvested and scored microscopically for GFP fluorescence, using an Olympus BL51 microscope mounted with a mercury lamp UV source and a High Q Endow GFP emission fluorescence filter cube. Some 1,000–2,000 cells from each of 4–10 fields were scored per determination. Error bars indicate one SEM for 8–13 cultures as indicated.</p> <p>(B) Microcolonies were harvested and suspended in 500 μl of buffer, 50 μl of which was spread on LBH X-gal rif solid medium to determine sectoring in resulting colonies. The remainders were concentrated and examined microscopically for GFP fluorescence, counting 60–400 cells per microcolony. These experiments were plated at very low cell density to avoid significant numbers of background Lac⁻ cells being harvested with the microcolonies. Open bars, fraction of stable Lac⁺ isolates (32 microcolonies); black bars, fraction of sectored isolates (11 microcolonies). The two distributions do not differ (<i>p</i> = 0.8 by Student's <i>t</i>-test). These experiments were repeated, giving similar results.</p></div

Colony Morphologies

<div><p>(A) Point-mutant Lac⁺ colony showing solid blue color (the pale colonies are derived from Lac⁻ cells).</p> <p>(B) <i>lac</i>-amplified colonies showing sectoring caused by the instability of the amplified array. Cells from these colonies grow either into sectored blue colonies or, if they have lost the amplification, into white colonies, a phenotype that we call “unstable.”</p> <p>(C) A sectored colony that is not unstable in that it was found to contain only stable blue and stable white cfu upon retesting.</p> <p>(D) An example of a microcolony of the sort used in this work. The visible colony on the lower edge of the field has a diameter of 1.4 mm (>10⁸ cells).</p> <p>(E and F) Phase contrast (E) and green fluorescence (F) of the same field, showing two of 30 SMR6039 cells fluorescing.</p></div

Stringent Analysis of Whole Microcolonies: Analyzing All Cells in a Microcolony and Reducing Contamination by Unrelated Neighboring Bacteria

<p>To analyze all cells in a microcolony, very young microcolonies were harvested (10³–10⁴ cfu/microcolony; see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-g001" target="_blank">Figure 1</a>D; <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-t001" target="_blank">Table 1</a>). To reduce contamination with neighboring Lac⁻ bacteria and unrelated Lac⁺ microcolonies from the minimal-lactose selection plate, first, a minority of the Lac⁻ cells plated carried an antibiotic-resistance marker, and only resistant microcolonies were analyzed, and, second, sectored colonies observed were retested for instability (to eliminate sectored colonies that are not unstable, shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-g001" target="_blank">Figure 1</a>C, which may result from accidental overlap of blue and white cfu). Procedures described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#s3" target="_blank">Materials and Methods</a>, “whole microcolony analysis.” Cam^R, chloramphenicol resistant; Kan^R, kanamycin resistant; Tet^R, tetracycline resistant. Streptomycin resistance (not shown) was also used.</p

Stress-Response Models for Adaptive Amplification and Point Mutation

<p>Modified from <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-Lombardo1" target="_blank">Lombardo et al. (2004)</a> and <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-Rosenberg3" target="_blank">Rosenberg and Hastings (2004a)</a>. DSBR, DSB repair; hypermutation, increased mutation at <i>lac</i> and elsewhere. The origins of DSEs during starvation on lactose medium in this assay system are unknown, and possibilities are reviewed elsewhere (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-Rosenberg1" target="_blank">Rosenberg 2001</a>). On the F′ plasmid carrying the <i>lac</i> gene, DSEs may be frequent and may be derived from chronic single-strand nicks at the origin of transfer. However chromosomal mutation during starvation on lactose medium in these cells also requires DSB repair proteins (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-Bull1" target="_blank">Bull et al. 2001</a>), and so probably results from (lower levels of) DSEs generated in the chromosome, independently of the transfer origin, or perhaps by transient integration of the F′ into the chromosome (Hfr formation). Both adaptive point mutation and amplification are proposed to be outcomes of RpoS-dependent stress response, which both mechanisms require (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0020399#pbio-0020399-Lombardo1" target="_blank">Lombardo et al. 2004</a>), and to result from alternative error-prone ways of repairing DSBs.</p

ENDIA microbiome QC OTUs and metadata

<div>OTU tables as .biom files, and associated metadata, used in paper currently titled</div><div>"Influence of fecal collection conditions and 16S rRNA gene sequencing protocols at two centers on human gut microbiota analysis"</div><div>Sequence data is deposited with SRA with identifier SRP116702</div><div>The study is registered with NCBI as BioProject PRJNA393083 </div