Bacterial DNA repair and molecular search

Surveillance and repair of DNA damage is necessary in all kinds of life. Different types of DNA damage require different repair mechanisms, but these mechanisms are often similar in all domains of life. The most serious type of damage, double stranded DNA breaks, are for example repaired in conceptually similar ways in both bacteria and eukaryotes. When this kind of breaks are repaired by homologous recombination, a homology to the site of the break must be found. Sometimes, this homology can be located far away from the break necessitating a search. Considering the large amount of heterologous DNA present, the complexity of this search is enormous. If and how this search can proceed has been unclear even in simple and well characterized organisms as E. coli. In this thesis, microscopy together with microfluidics are used to show that DNA repair by homologous recombination occurs even between homologies separated by several micrometers. We also see that it finishes well within the time of a cell generation, with the enigmatic search phase being as quick as eight or possibly even three minutes. Since this time is much faster than expected, we present a physical model demonstrating how homology search on this time scale is indeed plausible. Based on these results, we conclude that homologous repair using distantly located templates is likely to be a physiologically relevant mechanism of DNA repair. Microscopy together with image analysis by deep learning also provides a new method of detecting DNA damage in real time. Combined with tracking of cell lineages, it reveals that DNA damage in E. coli is repaired efficiently enough that the resulting fitness cost is close to none. With the same methods we also study the effect of deletions of several DNA repair enzymes, and largely confirms their previous characterizations. Among these, we confirm that the intriguing RecN protein is important but not absolutely necessary in DSB repair, that it acts early, and possibly aids in physically shaping the structure mediating the search. In addition to this, it is shown how DNA transcription and translation modulates the shape of the E. coli nucleoid. We observe how strong a transcription of a gene within a few minutes moves the gene towards the periphery of the cell where the concentration of ribosomes is higher, a movement possibly also aided by protein translation. We also present MINFLUX, a microscope for both nanometer scale localization of single fluorophores as well as in vivo single particle tracking with unprecedented trace length and resolution. Using this, the E. coli small ribosomal subunit could be observed to quickly shift between fast and slow diffusion states which might represent probing and discarding of RNAs suitable for translation

Bacterial DNA repair and molecular search

Surveillance and repair of DNA damage is necessary in all kinds of life. Different types of DNA damage require different repair mechanisms, but these mechanisms are often similar in all domains of life. The most serious type of damage, double stranded DNA breaks, are for example repaired in conceptually similar ways in both bacteria and eukaryotes. When this kind of breaks are repaired by homologous recombination, a homology to the site of the break must be found. Sometimes, this homology can be located far away from the break necessitating a search. Considering the large amount of heterologous DNA present, the complexity of this search is enormous. If and how this search can proceed has been unclear even in simple and well characterized organisms as E. coli. In this thesis, microscopy together with microfluidics are used to show that DNA repair by homologous recombination occurs even between homologies separated by several micrometers. We also see that it finishes well within the time of a cell generation, with the enigmatic search phase being as quick as eight or possibly even three minutes. Since this time is much faster than expected, we present a physical model demonstrating how homology search on this time scale is indeed plausible. Based on these results, we conclude that homologous repair using distantly located templates is likely to be a physiologically relevant mechanism of DNA repair. Microscopy together with image analysis by deep learning also provides a new method of detecting DNA damage in real time. Combined with tracking of cell lineages, it reveals that DNA damage in E. coli is repaired efficiently enough that the resulting fitness cost is close to none. With the same methods we also study the effect of deletions of several DNA repair enzymes, and largely confirms their previous characterizations. Among these, we confirm that the intriguing RecN protein is important but not absolutely necessary in DSB repair, that it acts early, and possibly aids in physically shaping the structure mediating the search. In addition to this, it is shown how DNA transcription and translation modulates the shape of the E. coli nucleoid. We observe how strong a transcription of a gene within a few minutes moves the gene towards the periphery of the cell where the concentration of ribosomes is higher, a movement possibly also aided by protein translation. We also present MINFLUX, a microscope for both nanometer scale localization of single fluorophores as well as in vivo single particle tracking with unprecedented trace length and resolution. Using this, the E. coli small ribosomal subunit could be observed to quickly shift between fast and slow diffusion states which might represent probing and discarding of RNAs suitable for translation

RecA mediated homology search finds segregated sister locus in minutes after a double stranded break

The search for a homologous template is a central and critical step in the repair of DNA breaks by homologous recombination. However, it is still unclear how the search process is carried out within a cell. Here, we image double-stranded break (DSB) repair in living E. coli growing in a microfluidic device and show that two segregated homologous sequences find each other less than 9 min after an induced DSB. To characterize the mechanisms of the search process, we use a new RecA fluorescent fusion that rapidly forms structures after DSBs. Initially, RecA forms a bright cluster at the site of the DNA damage and then extends to form a thin, flexible, and dynamic filament. Based on our observations, we propose a model where the 1D ssDNA-RecA filament stretches along the cell, and the homology search is mediated by diffusion of the repair template that can interact with any segment of the filament. This model reduces the complexity of the search from 3D to 2D and quantitatively predicts that genome-wide search for homology can be finished in minutes, in agreement with our measurements

RecA mediated homology search finds segregated sister locus in minutes after a double stranded break

The search for a homologous template is a central and critical step in the repair of DNA breaks by homologous recombination. However, it is still unclear how the search process is carried out within a cell. Here, we image double-stranded break (DSB) repair in living E. coli growing in a microfluidic device and show that two segregated homologous sequences find each other less than 9 min after an induced DSB. To characterize the mechanisms of the search process, we use a new RecA fluorescent fusion that rapidly forms structures after DSBs. Initially, RecA forms a bright cluster at the site of the DNA damage and then extends to form a thin, flexible, and dynamic filament. Based on our observations, we propose a model where the 1D ssDNA-RecA filament stretches along the cell, and the homology search is mediated by diffusion of the repair template that can interact with any segment of the filament. This model reduces the complexity of the search from 3D to 2D and quantitatively predicts that genome-wide search for homology can be finished in minutes, in agreement with our measurements

Rationally designed Spot 42 RNAs with an inhibition/toxicity profile advantageous for engineering E. coli

Bacterial regulatory small RNAs (sRNAs) have shown promise for gene knock-down studies and metabolic engineering. However, some mRNAs might be difficult to target due to poor binding by the Hfq chaperone, individual synthetic sRNAs can have off-target effects, potential sRNA toxicities have not been studied globally, and a consensus on optimal sRNA design has yet to emerge. Here, Spot 42 sRNA is validated as an excellent scaffold by showing that its over-expression minimally affects the growth rate of Escherichia coli, and that inhibition is reliably achieved for all eight tested protein targets by designing antisense to target the first few codons. Two related sRNAs that could not be cloned, possibly due to lethality of the encoded sRNAs, became clonable when an eight-nucleotide sequence was inserted directly upstream of the antisense region. Global fitness costs for E. coli of the designer sRNAs were measured and found to be variable but tolerable. Importantly for utility, there was no correlation between target inhibition and cellular toxicity. As a proof of concept for applications, suppression of the UAG stop codon was improved by knock down of translation release factor 1 (RF1)

RecA finds homologous DNA by reduced dimensionality search

Homologous recombination is essential for the accurate repair of double-stranded DNA breaks (DSBs)1. Initially, the RecBCD complex2 resects the ends of the DSB into 3' single-stranded DNA on which a RecA filament assembles3. Next, the filament locates the homologous repair template on the sister chromosome4. Here we directly visualize the repair of DSBs in single cells, using high-throughput microfluidics and fluorescence microscopy. We find that, in Escherichia coli, repair of DSBs between segregated sister loci is completed in 15 ± 5 min (mean ± s.d.) with minimal fitness loss. We further show that the search takes less than 9 ± 3 min (mean ± s.d) and is mediated by a thin, highly dynamic RecA filament that stretches throughout the cell. We propose that the architecture of the RecA filament effectively reduces search dimensionality. This model predicts a search time that is consistent with our measurement and is corroborated by the observation that the search time does not depend on the length of the cell or the amount of DNA. Given the abundance of RecA homologues5, we believe this model to be widely conserved across living organisms

Transcription and translation contribute to gene locus relocation to the nucleoid periphery in E. coli

Transcription by RNA polymerase (RNAP) is coupled with translation in bacteria. Here, we observe the dynamics of transcription and subcellular localization of a specific gene locus (encoding a non-membrane protein) in living E. coli cells at subdiffraction-limit resolution. The movement of the gene locus to the nucleoid periphery correlates with transcription, driven by either E. coli RNAP or T7 RNAP, and the effect is potentiated by translation

RHS-elements function as type II toxin-antitoxin modules that regulate intra-macrophage replication of Salmonella Typhimurium

RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages.

Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology

Background: Coral reefs are colored by eukaryotic chromoproteins (CPs) that are homologous to green fluorescent protein. CPs differ from fluorescent proteins (FPs) by intensely absorbing visible light to give strong colors in ambient light. This endows CPs with certain advantages over FPs, such as instrument-free detection uncomplicated by ultra-violet light damage or background fluorescence, efficient Forster resonance energy transfer (FRET) quenching, and photoacoustic imaging. Thus, CPs have found utility as genetic markers and in teaching, and are attractive for potential cell biosensor applications in the field. Most near-term applications of CPs require expression in a different domain of life: bacteria. However, it is unclear which of the eukaryotic CP genes might be suitable and how best to assay them. Results: Here, taking advantage of codon optimization programs in 12 cases, we engineered 14 CP sequences (meffRed, eforRed, asPink, spisPink, scOrange, fwYellow, amilGFP, amajLime, cjBlue, mefiBlue, aeBlue, amilCP, tsPurple and gfasPurple) into a palette of Escherichia coil BioBrick plasmids. BioBricks comply with synthetic biology's most widely used, simplified, cloning standard. Differences in color intensities, maturation times and fitness costs of expression were compared under the same conditions, and visible readout of gene expression was quantitated. A surprisingly large variation in cellular fitness costs was found, resulting in loss of color in some overnight liquid cultures of certain high-copy-plasmid-borne CPs, and cautioning the use of multiple CPs as markers in competition assays. We solved these two problems by integrating pairs of these genes into the chromosome and by engineering versions of the same CP with very different colors. Conclusion: Availability of 14 engineered CP genes compared in E coil, together with chromosomal mutants suitable for competition assays, should simplify and expand CP study and applications. There was no single plasmid-borne CP that combined all of the most desirable features of intense color, fast maturation and low fitness cost, so this study should help direct future engineering efforts