Illuminating Messengers:An Update and Outlook on RNA Visualization in Bacteria

To be able to visualize the abundance and spatiotemporal features of RNAs in bacterial cells would permit obtaining a pivotal understanding of many mechanisms underlying bacterial cell biology. The first methods that allowed observing single mRNA molecules in individual cells were introduced by Bertrand et al. (1998) and Femino et al. (1998). Since then, a plethora of techniques to image RNA molecules with the aid of fluorescence microscopy has emerged. Many of these approaches are useful for the large eukaryotic cells but their adaptation to study RNA, specifically mRNA molecules, in bacterial cells progressed relatively slow. Here, an overview will be given of fluorescent techniques that can be used to reveal specific RNA molecules inside fixed and living single bacterial cells. It includes a critical evaluation of their caveats as well as potential solutions

The evolution of gene regulation research in Lactococcus lactis

Lactococcus lactis is a major microbe. This lactic acid bacterium (LAB) is used worldwide in the production of safe, healthy, tasteful and nutritious milk fermentation products. Its huge industrial importance has led to an explosion of research on the organism, particularly since the early 1970s. The upsurge in the research on L. lactis coincided not accidentally with the advent of recombinant DNA technology in these years. The development of methods to take out and re-introduce DNA in L. lactis, to clone genes and to mutate the chromosome in a targeted way, to control (over)expression of proteins and, ultimately, the availability of the nucleotide sequence of its genome and the use of that information in transcriptomics and proteomics research have enabled to peek deep into the functioning of the organism. Among many other things, this has provided an unprecedented view of the major gene regulatory pathways involved in nitrogen and carbon metabolism and their overlap, and has led to the blossoming of the field of L. lactis systems biology. All of these advances have made L. lactis the paradigm of the LAB. This review will deal with the exciting path along which the research on the genetics of and gene regulation in L. lactis has trodden

On the spatial organization of mRNA, plasmids, and ribosomes in a bacterial host overexpressing membrane proteins

By using fluorescence imaging, we provide a time-resolved single-cell view on coupled defects in transcription, translation, and growth during expression of heterologous membrane proteins in Lactococcus lactis. Transcripts encoding poorly produced membrane proteins accumulate in mRNA-dense bodies at the cell poles, whereas transcripts of a well-expressed homologous membrane protein show membrane-proximal localization in a translation-dependent fashion. The presence of the aberrant polar mRNA foci correlates with cessation of cell division, which is restored once these bodies are cleared. In addition, activation of the heat-shock response and a loss of nucleoid-occluded ribosomes are observed. We show that the presence of a native-like N-terminal domain is key to SRP-dependent membrane localization and successful production of membrane proteins. The work presented gives new insights and detailed understanding of aberrant membrane protein biogenesis, which can be used for strategies to optimize membrane protein production

N-terminal domain of membrane proteins influences mRNA localization and protein production.

<p><b>(A)</b> Schematic representation of the transmembrane domains, as predicted by TMHMM, of BcaP (1) and PS1Δ9 (2) and chimeric proteins in which a portion of N-terminal domains of PS1Δ9 (PS1Δ9^N) is replaced by those of BcaP (3) and vice versa (4). <b>(B)</b> mRNA localization in representative cells expressing <i>bcaP</i>^<i>N</i>-<i>PS1Δ9</i>_<i>12bs</i> (3) or <i>PS1Δ9</i>^<i>N</i><i>-bcaP</i>_<i>12bs</i> (4) transcripts. <b>(C)</b> The percentage of cells (n = 400) without polar mRNA of the proteins depicted in A. <b>(D)</b> Schematic representation of PS1Δ9 (red bars) fused via its N-terminal domain and a flexible linker containing a TEV protease site (blue dot) to the newly developed fusion partner BLS (for <u>B</u>caP <u>L</u>eading <u>S</u>egment; grey bars). <b>(E)</b> Dotblots of cytoplasmic fractions (C) and membrane fractions (M) of <i>L</i>. <i>lactis</i> NZ9000 cells grown in GCDM* or GCDM* supplemented with 1.5% casitone, and expressing the proteins shown in A and D. <b>(F)</b> Quantification of dotblot signals. Averaged signals are displayed as percentages of the signals obtained for BcaP expression. <b>(G)</b> Representative image of cells expressing <i>PS1Δ9</i>_<i>12bs</i> or <i>BLS-PS1Δ9</i>_<i>12bs</i> mRNA as visualized by FISH. Numbers in B, C, E, and F refer to the construct numbers in A and D.</p

Polar mRNA clusters are spatially unrelated to protein aggregation seeds.

<p><b>(A)</b> Single-cell <i>bcaP-gfp</i>_<i>12bs</i> mRNA content plotted as a function of intracellular BcaP-GFP protein abundance. Red encircled dots: Cells with polar <i>bcaP-gfp</i>_<i>12bs</i> clusters. <b>(B)</b> Single-cell <i>PS1Δ9-gfp</i>_<i>12bs</i> mRNA content plotted as a function of intracellular PS1Δ9-GFP protein abundance. Red encircled dots: Cells with polar <i>PS1Δ9-gfp</i>_<i>12bs</i> clusters. <b>(C)</b> Co-visualization of <i>bcaP-gfp</i>_<i>12bs</i> or <i>PS1Δ9-gfp</i>_<i>12bs</i> mRNA (FISH) with their protein products. Bottom panels: false-colored overlays of mRNA (red) and protein (cyan). Images were subjected to deconvolution to enhance the contrast. <b>(D)</b> Time course of fold changes (FC) of DnaK-GFP expression in <i>L</i>. <i>lactis</i> LG029, a strain in which <i>dnaK</i> was replaced with <i>dnaK-gfp</i>, after induction of expression of PS1Δ9 (red squares) or BcaP (black squares) compared to control cells (dashed grey line). <b>(E)</b> Co-visualization of DnaK-GFP and overexpressed <i>PS1Δ9</i>_<i>12bs</i> or <i>bcaP</i>_<i>12bs</i> mRNA in <i>L</i>. <i>lactis</i> LG029. Right panels: False colored-overlays of DnaK-GFP (cyan) and mRNA (red). <b>(F)</b> DnaK-GFP levels in single <i>L</i>. <i>lactis</i> LG029 cells displayed as a function of levels of <i>bcaP</i>_<i>12b</i>s mRNA (grey filled circles) or <i>PS1Δ9</i>_<i>12bs</i> mRNA (black dots). Black dots encircled in red: Cells with polar <i>PS1Δ9</i>_<i>12bs</i> clusters. Scale bar = 2 μm. Yellow lines indicate cell contours.</p

Effect of polar clustering and ribosome binding on degradation and localization of overexpressed mRNAs.

<p><b>(A)</b><i>L</i>. <i>lactis</i> LG010 cells with MS2-GFP-tagged <i>bcaP</i>_<i>12bs</i> or <i>PS1Δ9</i>_<i>12bs</i> mRNA treated or not with Cm and Ery. <b>(B)</b> FISH images of <i>L</i>. <i>lactis</i> NZ9000 cells expressing <i>bcaP</i>_<i>12bs</i> or <i>PS1Δ9</i>_<i>12bs</i> mRNAs with or without functional RBS, and visualized by <i>ms2</i> probe hybridization. <b>(C)</b> The percentage of cells (n = 500) expressing <i>bcaP</i>_<i>12bs</i> or <i>PS1Δ9</i>_<i>12bs</i> with (+) or without (-) RBS and containing mRNA clusters. Error bars = standard errors. <b>(D)</b> Location maps constructed from FISH images of 500 cells showing the predominant distribution of <i>bcaP</i>_<i>12bs</i> or <i>PS1Δ9</i>_<i>12bs</i> with or without RBS. <b>(E)</b> Snap shots of time-lapse microscopy of <i>L</i>. <i>lactis(rpsB</i>::<i>rpsB-eYFP)</i> cells expressing <i>bcaP</i>_<i>12bs</i> or <i>PS1Δ9</i>_<i>12bs</i>. <b>(F)</b> Boxplots of the ratio between the S2-eYFP protein spread along the long cell axis and total cell length obtained from 100 cells expressing BcaP or PS1Δ9, as well as representative pictures of the corresponding S2-eYFP distributions. ***: A Student’s <i>t</i>-test was performed to test for significance (p<0.005). <b>(G)</b> Time (hr) required for individual <i>PS1Δ9</i>_<i>12bs</i>-expressing cells (n = 100) to restore growth after transfer to agarose pads lacking nisin, as determined by time-lapse microscopy. <b>(H)</b> Examples of <i>PS1Δ9</i>_<i>12bs</i> and <i>SUT1</i>_<i>12bs</i> transcripts remaining in cells after 32 min treatment with rifampicin. <b>(I)</b> RnY-GFP localization in fixed <i>L</i>. <i>lactis</i> LG024a cells expressing nothing (-), <i>bcaP</i>_<i>12bs</i>, or <i>PS1Δ9</i>_<i>12bs</i>. Strain LG024a constitutively expresses <i>rnY-gfp</i> ectopically from its own promoter. The fusion gene was introduced additional to <i>rnY</i> to not interfere with essential mRNA breakdown.</p

Increased uracil density in <i>L</i>. <i>lactis</i> genes encoding membrane proteins.

<p><b>(A)</b> The relative codon bias for all hydrophobic amino acids in <i>L</i>. <i>lactis MG1363</i> (left bars) and <i>E</i>. <i>coli K12</i> (right bars). The red bars indicate the codon(s) with the highest uracil content. <b>(B)</b> Boxplots show that the uracil density of transcripts encoding membrane proteins is increased in both species. A Student’s <i>t</i>-test was performed: the uracil density was significantly different (p < 0.0001; indicated by ****) between the two types of transcripts for both <i>E</i>. <i>coli</i> and <i>L</i>. <i>lactis</i>.</p

pNZ8048 plasmids predominantly localize in the chromosomal area, not with mRNA-dense polar clusters.

<p>Plasmids tagged with <i>parS</i>/ParB-GFP were co-visualized with overexpressed <i>bcaP</i>_<i>12bs</i> mRNA <b>(A)</b> or <i>PS1Δ9</i>_<i>12bs</i> mRNA <b>(B)</b> using FISH. Right panels show false-colored images in which mRNA and plasmids are represented in red and cyan, respectively. Images were subjected to deconvolution to enhance the contrast. Scale bar = 2 μm. Yellow lines indicate cell contours. <b>(C)</b> ParB-GFP in all of the captured cells (± 200 cells per experiment) were traced with the ImageJ PeakFinder plug-in and jointly projected into one half of a model cell as described earlier. The intracellular distribution of ParB-GFP foci under various gene expression scenarios (empty vector control (-), BcaP or PS1Δ9 expression) is represented as location maps to highlight the predominant sites of plasmid localization averaged over all cells. <b>(D)</b> Intensity profiles of overexpressed mRNA and co-visualized plasmids created from the location maps to illustrate differences in distribution of pLG-BcaP (black solid line), pLG-PS1Δ9 (red solid line), <i>bcaP</i>_<i>12bs</i> (black dotted line), and <i>PS1Δ9</i>_<i>12bs</i> (red dotted line) along the X-axis of the model cells. Scale bars in microscopy images represent 2 μm.</p

Time-resolved analysis of <i>PS1Δ9</i>_<i>12bs</i> mRNA abundance and localization using time-lapse microscopy.

<p><b>(A)</b><i>PS1Δ9</i>_<i>12bs</i> mRNA expression in <i>L</i>. <i>lactis</i> LG010 cells was induced following a standard regime after which the cells were transferred to a microscopy slide carrying a thin layer of 1.5% agarose dissolved in GCDM* lacking nisin, for time-lapse fluorescence microscopy analysis. Shown are the portions of 100 tracked cells that contain polar <i>PS1Δ9</i>_<i>12bs</i> mRNA clusters that do not divide after transfer (ND), that grow very poorly and adopt swollen and deviating cell shapes (-), that grow at a moderate rate (+/-) and that grow at a normal rate (+). <b>(B)</b> Histogram displaying the time it takes for 100 individual cells to remove MS2-GFP-tagged <i>PS1Δ9</i>_<i>12bs</i> mRNA clusters from their poles, as visualized with time-lapse microscopy. <b>(C)</b> Box plots displaying the initial MS2-GFP expression levels, which is directly correlated to the mRNA level, in <i>PS1Δ9</i>_<i>12bs</i>-expressing cells that either resumed growth (+) or remained in a non-growing state (-). A Students <i>t</i>-test was performed to test for significance (p<0.005). <b>(D)</b> Schematic representation of the post-transfer sequence of patterns adopted by MS2-GFP-tagged <i>PS1Δ9</i>_<i>12bs</i> mRNA (colored red). <b>(E)</b> Histogram displaying the time it takes for 100 individual cells to stop nisin A-driven transcription of <i>bcaP</i>_<i>12bs</i> mRNA upon transfer of induced liquid cultures to agarose pads with growth medium lacking nisin A, as determined from MS2-GFP distributions in cells using time-lapse microscopy. <b>(F)</b> MS2-GFP expression (right axis) monitored in <i>L</i>. <i>lactis</i> LG010 cells after standard induction, expressing nothing (empty vector control; grey), BcaP (black), or PS1Δ9 (red) and their averaged growth (blue line, left axis). The MS2-GFP fluorescence values are normalized for OD₆₀₀.</p