Structural and functional characterization of MrpR, the master repressor of the Bacillus subtilis prophage SPß.

Prophages control their lifestyle to either be maintained within the host genome or enter the lytic cycle. Bacillus subtilis contains the SPß prophage whose lysogenic state depends on the MrpR (YopR) protein, a key component of the lysis-lysogeny decision system. Using a historic B. subtilis strain harboring the heat-sensitive SPß c2 mutant, we demonstrate that the lytic cycle of SPß c2 can be induced by heat due to a single nucleotide exchange in the mrpR gene, rendering the encoded MrpRG136E protein temperature-sensitive. Structural characterization revealed that MrpR is a DNA-binding protein resembling the overall fold of tyrosine recombinases. MrpR has lost its recombinase function and the G136E exchange impairs its higher-order structure and DNA binding activity. Genome-wide profiling of MrpR binding revealed its association with the previously identified SPbeta repeated element (SPBRE) in the SPß genome. MrpR functions as a master repressor of SPß that binds to this conserved element to maintain lysogeny. The heat-inducible excision of the SPß c2 mutant remains reliant on the serine recombinase SprA. A suppressor mutant analysis identified a previously unknown component of the lysis-lysogeny management system that is crucial for the induction of the lytic cycle of SPß.

Identification of Gip as a novel phage-encoded gyrase inhibitor protein of Corynebacterium glutamicum.

By targeting key regulatory hubs of their host, bacteriophages represent a powerful source for the identification of novel antimicrobial proteins. Here, a screening of small cytoplasmic proteins encoded by the CGP3 prophage of Corynebacterium glutamicum resulted in the identification of the gyrase-inhibiting protein Cg1978, termed Gip. Pull-down assays and surface plasmon resonance revealed a direct interaction of Gip with the gyrase subunit A (GyrA). The inhibitory activity of Gip was shown to be specific to the DNA gyrase of its bacterial host C. glutamicum. Overproduction of Gip in C. glutamicum resulted in a severe growth defect as well as an induction of the SOS response. Furthermore, reporter assays revealed an RecA-independent induction of the cryptic CGP3 prophage, most likely caused by topological alterations. Overexpression of gip was counteracted by an increased expression of gyrAB and a reduction of topA expression at the same time, reflecting the homeostatic control of DNA topology. We postulate that the prophage-encoded Gip protein plays a role in modulating gyrase activity to enable efficient phage DNA replication. A detailed elucidation of the mechanism of action will provide novel directions for the design of drugs targeting DNA gyrase.

HrrSA orchestrates a systemic response to heme and determines prioritization of terminal cytochrome oxidase expression.

Heme is a multifaceted molecule. While serving as a prosthetic group for many important proteins, elevated levels are toxic to cells. The complexity of this stimulus has shaped bacterial network evolution. However, only a small number of targets controlled by heme-responsive regulators have been described to date. Here, we performed chromatin affinity purification and sequencing to provide genome-wide insights into in vivo promoter occupancy of HrrA, the response regulator of the heme-regulated two-component system HrrSA of Corynebacterium glutamicum. Time-resolved profiling revealed dynamic binding of HrrA to more than 200 different genomic targets encoding proteins associated with heme biosynthesis, the respiratory chain, oxidative stress response and cell envelope remodeling. By repression of the extracytoplasmic function sigma factor sigC, which activates the cydABCD operon, HrrA prioritizes the expression of genes encoding the cytochrome bc1-aa3 supercomplex. This is also reflected by a significantly decreased activity of the cytochrome aa3 oxidase in the ΔhrrA mutant. Furthermore, our data reveal that HrrA also integrates the response to heme-induced oxidative stress by activating katA encoding the catalase. These data provide detailed insights in the systemic strategy that bacteria have evolved to respond to the versatile signaling molecule heme.

Biosensor-based growth-coupling and spatial separation as an evolution strategy to improve small molecule production of Corynebacterium glutamicum.

Evolutionary engineering is a powerful method to improve the performance of microbial cell factories, but can typically not be applied to enhance the production of chemicals due to the lack of an appropriate selection regime. We report here on a new strategy based on transcription factor-based biosensors, which directly couple production to growth. The growth of Corynebacterium glutamicum was coupled to the intracellular concentration of branched-chain amino acids, by integrating a synthetic circuit based on the Lrp biosensor upstream of two growth-regulating genes, pfkA and hisD. Modelling and experimental data highlight spatial separation as key strategy to limit the selection of 'cheater' strains that escaped the evolutionary pressure. This approach facilitated the isolation of strains featuring specific causal mutations enhancing amino acid production. We envision that this strategy can be applied with the plethora of known biosensors in various microbes, unlocking evolution as a feasible strategy to improve production of chemicals.

Toxic but tasty - temporal dynamics and network architecture of heme-responsive two-component signaling in Corynebacterium glutamicum.

Heme is an essential cofactor and alternative iron source for almost all bacterial species but may cause severe toxicity upon elevated levels and consequently, regulatory mechanisms coordinating heme homeostasis represent an important fitness trait. A remarkable scenario is found in several corynebacterial species, e.g. Corynebacterium glutamicum and Corynebacterium diphtheriae, which dedicate two paralogous, heme-responsive two-component systems, HrrSA and ChrSA, to cope with the Janus nature of heme. Here, we combined experimental reporter profiling with a quantitative mathematical model to understand how this particular regulatory network architecture shapes the dynamic response to heme. Our data revealed an instantaneous activation of the detoxification response (hrtBA) upon stimulus perception and we found that kinase activity of both kinases contribute to this fast onset. Furthermore, instant deactivation of the PhrtBA promoter is achieved by a strong ChrS phosphatase activity upon stimulus decline. While the activation of detoxification response is uncoupled from further factors, heme utilization is additionally governed by the global iron regulator DtxR integrating information on iron availability into the regulatory network. Altogether, our data provide comprehensive insights how TCS cross-regulation and network hierarchy shape the temporal dynamics of detoxification (hrtBA) and utilization (hmuO) as part of a global homeostatic response to heme.

Impact of CO2/HCO3- Availability on Anaplerotic Flux in Pyruvate Dehydrogenase Complex-Deficient Corynebacterium glutamicum Strains.

The pyruvate dehydrogenase complex (PDHC) catalyzes the oxidative decarboxylation of pyruvate, yielding acetyl coenzyme A (acetyl-CoA) and CO2 The PDHC-deficient Corynebacterium glutamicum ΔaceE strain therefore lacks an important decarboxylation step in its central metabolism. Additional inactivation of pyc, encoding pyruvate carboxylase, resulted in a >15-h lag phase in the presence of glucose, while no growth defect was observed on gluconeogenetic substrates, such as acetate. Growth was successfully restored by deletion of ptsG, encoding the glucose-specific permease of the phosphotransferase system (PTS), thereby linking the observed phenotype to the increased sensitivity of the ΔaceE Δpyc strain to glucose catabolism. In this work, the ΔaceE Δpyc strain was used to systematically study the impact of perturbations of the intracellular CO2/HCO3- pool on growth and anaplerotic flux. Remarkably, all measures leading to enhanced CO2/HCO3- levels, such as external addition of HCO3-, increasing the pH, or rerouting metabolic flux via the pentose phosphate pathway, at least partially eliminated the lag phase of the ΔaceE Δpyc strain on glucose medium. In accordance with these results, inactivation of the urease enzyme, lowering the intracellular CO2/HCO3- pool, led to an even longer lag phase, accompanied by the excretion of l-valine and l-alanine. Transcriptome analysis, as well as an adaptive laboratory evolution experiment with the ΔaceE Δpyc strain, revealed the reduction of glucose uptake as a key adaptive measure to enhance growth on glucose-acetate mixtures. Taken together, our results highlight the significant impact of the intracellular CO2/HCO3- pool on metabolic flux distribution, which becomes especially evident in engineered strains exhibiting low endogenous CO2 production rates, as exemplified by PDHC-deficient strains.IMPORTANCE CO2 is a ubiquitous product of cellular metabolism and an essential substrate for carboxylation reactions. The pyruvate dehydrogenase complex (PDHC) catalyzes a central metabolic reaction contributing to the intracellular CO2/HCO3- pool in many organisms. In this study, we used a PDHC-deficient strain of Corynebacterium glutamicum, which additionally lacked pyruvate carboxylase (ΔaceE Δpyc). This strain featured a >15-h lag phase during growth on glucose-acetate mixtures. We used this strain to systematically assess the impact of alterations in the intracellular CO2/HCO3- pool on growth in glucose-acetate medium. Remarkably, all measures enhancing CO2/HCO3- levels successfully restored growth. These results emphasize the strong impact of the intracellular CO2/HCO3- pool on metabolic flux, especially in strains exhibiting low endogenous CO2 production rates.

Generation of a Prophage-Free Variant of the Fast-Growing Bacterium Vibrio natriegens.

The fast-growing marine bacterium Vibrio natriegens represents an emerging strain for molecular biology and biotechnology. Genome sequencing and quantitative PCR analysis revealed that the first chromosome of V. natriegens ATCC 14048 contains two prophage regions (VNP1 and VNP2) that are both inducible by the DNA-damaging agent mitomycin C and exhibit spontaneous activation under standard cultivation conditions. Their activation was also confirmed by live cell imaging of an mCherry fusion to the major capsid proteins of VNP1 and VNP2. Transmission electron microscopy visualized the release of phage particles belonging to the Siphoviridae family into the culture supernatant. Freeing V. natriegens from its proviral load, followed by phenotypic characterization, revealed an improved robustness of the prophage-free variant toward DNA-damaging conditions, reduced cell lysis under hypo-osmotic conditions, and an increased pyruvate production compared to wild-type levels. Remarkably, the prophage-free strain outcompeted the wild type in a competitive growth experiment, emphasizing that this strain is a promising platform for future metabolic engineering approaches.IMPORTANCE The fast-growing marine bacterium Vibrio natriegens represents an emerging model host for molecular biology and biotechnology, featuring a reported doubling time of less than 10 minutes. In many bacterial species, viral DNA (prophage elements) may constitute a considerable fraction of the whole genome and may have detrimental effects on the growth and fitness of industrial strains. Genome analysis revealed the presence of two prophage regions in the V. natriegens genome that were shown to undergo spontaneous induction under standard cultivation conditions. In this study, we generated a prophage-free variant of V. natriegens Remarkably, the prophage-free strain exhibited a higher tolerance toward DNA damage and hypo-osmotic stress. Moreover, it was shown to outcompete the wild-type strain in a competitive growth experiment. In conclusion, our study presents the prophage-free variant of V. natriegens as a promising platform strain for future biotechnological applications.

Silencing of cryptic prophages in Corynebacterium glutamicum.

DNA of viral origin represents a ubiquitous element of bacterial genomes. Its integration into host regulatory circuits is a pivotal driver of microbial evolution but requires the stringent regulation of phage gene activity. In this study, we describe the nucleoid-associated protein CgpS, which represents an essential protein functioning as a xenogeneic silencer in the Gram-positive Corynebacterium glutamicum CgpS is encoded by the cryptic prophage CGP3 of the C. glutamicum strain ATCC 13032 and was first identified by DNA affinity chromatography using an early phage promoter of CGP3. Genome-wide profiling of CgpS binding using chromatin affinity purification and sequencing (ChAP-Seq) revealed its association with AT-rich DNA elements, including the entire CGP3 prophage region (187 kbp), as well as several other elements acquired by horizontal gene transfer. Countersilencing of CgpS resulted in a significantly increased induction frequency of the CGP3 prophage. In contrast, a strain lacking the CGP3 prophage was not affected and displayed stable growth. In a bioinformatics approach, cgpS orthologs were identified primarily in actinobacterial genomes as well as several phage and prophage genomes. Sequence analysis of 618 orthologous proteins revealed a strong conservation of the secondary structure, supporting an ancient function of these xenogeneic silencers in phage-host interaction.

A prophage-encoded actin-like protein required for efficient viral DNA replication in bacteria.

In host cells, viral replication is localized at specific subcellular sites. Viruses that infect eukaryotic and prokaryotic cells often use host-derived cytoskeletal structures, such as the actin skeleton, for intracellular positioning. Here, we describe that a prophage, CGP3, integrated into the genome of Corynebacterium glutamicum encodes an actin-like protein, AlpC. Biochemical characterization confirms that AlpC is a bona fide actin-like protein and cell biological analysis shows that AlpC forms filamentous structures upon prophage induction. The co-transcribed adaptor protein, AlpA, binds to a consensus sequence in the upstream promoter region of the alpAC operon and also interacts with AlpC, thus connecting circular phage DNA to the actin-like filaments. Transcriptome analysis revealed that alpA and alpC are among the early induced genes upon excision of the CGP3 prophage. Furthermore, qPCR analysis of mutant strains revealed that both AlpA and AlpC are required for efficient phage replication. Altogether, these data emphasize that AlpAC are crucial for the spatio-temporal organization of efficient viral replication. This is remarkably similar to actin-assisted membrane localization of eukaryotic viruses that use the actin cytoskeleton to concentrate virus particles at the egress sites and provides a link of evolutionary conserved interactions between intracellular virus transport and actin.

Impact of LytR-CpsA-Psr Proteins on Cell Wall Biosynthesis in Corynebacterium glutamicum.

Proteins of the LCP (LytR, CpsA, Psr) family have been shown to inherit important roles in bacterial cell wall biosynthesis. However, their exact function in the formation of the complex cell wall structures of the Corynebacteriales, including the prominent pathogens Mycobacterium tuberculosis and Corynebacterium diphtheriae, remains unclear. Here, we analyzed the role of the LCP proteins LcpA and LcpB of Corynebacterium glutamicum, both of which localize at regions of nascent cell wall biosynthesis. A strain lacking lcpB did not show any growth-related or morphological phenotype under the tested conditions. In contrast, conditional silencing of the essential lcpA gene resulted in severe growth defects and drastic morphological changes. Compared to the wild-type cell wall, the cell wall of this mutant contained significantly less mycolic acids and a reduced amount of arabinogalactan. In particular, rhamnose, a specific sugar component of the linker that connects arabinogalactan and peptidoglycan, was decreased. Complementation studies of the lcpA-silencing strain with several mutated and truncated LcpA variants suggested that both periplasmic domains are essential for function whereas the cytoplasmic N-terminal part is dispensable. Successful complementation experiments with proteins of M. tuberculosis and C. diphtheriae revealed a conserved function of LCP proteins in these species. Finally, pyrophosphatase activity of LcpA was shown in an in vitro assay. Taken together, our results suggest that LCP proteins are responsible for the transfer of arabinogalactan onto peptidoglycan in actinobacterial species and support a crucial function of a so-far-uncharacterized C-terminal domain (LytR_C domain) which is frequently found at the C terminus of the LCP domain in this prokaryotic phylum. IMPORTANCE: About one-third of the world's population is infected with Mycobacterium tuberculosis, and multiple-antibiotic resistance provokes the demand for novel antibiotics. The special cell wall architecture of Corynebacteriales is critical for treatments because it is either a direct target or a barrier that the drug has to cross. Here, we present the analysis of LcpA and LcpB of the closely related Corynebacterium glutamicum, the first of which is an essential protein involved in cell wall biogenesis. Our work provides a comprehensive characterization of the impact of LCP proteins on cell wall biogenesis in this medically and biotechnologically important class of bacteria. Special focus is set on the two periplasmic LcpA domains and their contributions to physiological function.

Live cell imaging of SOS and prophage dynamics in isogenic bacterial populations.

Almost all bacterial genomes contain DNA of viral origin, including functional prophages or degenerated phage elements. A frequent but often unnoted phenomenon is the spontaneous induction of prophage elements (SPI) even in the absence of an external stimulus. In this study, we have analyzed SPI of the large, degenerated prophage CGP3 (187 kbp), which is integrated into the genome of the Gram-positive Corynebacterium glutamicum ATCC 13032. Time-lapse fluorescence microscopy of fluorescent reporter strains grown in microfluidic chips revealed the sporadic induction of the SOS response as a prominent trigger of CGP3 SPI but also displayed a considerable fraction (∼30%) of RecA-independent SPI. Whereas approx. 20% of SOS-induced cells recovered from this stress and resumed growth, the spontaneous induction of CGP3 always led to a stop of growth and likely cell death. A carbon source starvation experiment clearly emphasized that SPI only occurs in actively proliferating cells, whereas sporadic SOS induction was still observed in resting cells. These data highlight the impact of sporadic DNA damage on the activity of prophage elements and provide a time-resolved, quantitative description of SPI as general phenomenon of bacterial populations.

Light-Controlled Cell Factories: Employing Photocaged Isopropyl-ß-d-Thiogalactopyranoside for Light-Mediated Optimization of lac Promoter-Based Gene Expression and (+)-Valencene Biosynthesis in Corynebacterium glutamicum.

Precise control of microbial gene expression resulting in a defined, fast, and homogeneous response is of utmost importance for synthetic bio(techno)logical applications. However, even broadly applied biotechnological workhorses, such as Corynebacterium glutamicum, for which induction of recombinant gene expression commonly relies on the addition of appropriate inducer molecules, perform moderately in this respect. Light offers an alternative to accurately control gene expression, as it allows for simple triggering in a noninvasive fashion with unprecedented spatiotemporal resolution. Thus, optogenetic switches are promising tools to improve the controllability of existing gene expression systems. In this regard, photocaged inducers, whose activities are initially inhibited by light-removable protection groups, represent one of the most valuable photoswitches for microbial gene expression. Here, we report on the evaluation of photocaged isopropyl-ß-d-thiogalactopyranoside (IPTG) as a light-responsive control element for the frequently applied tac-based expression module in C. glutamicum In contrast to conventional IPTG, the photocaged inducer mediates a tightly controlled, strong, and homogeneous expression response upon short exposure to UV-A light. To further demonstrate the unique potential of photocaged IPTG for the optimization of production processes in C. glutamicum, the optogenetic switch was finally used to improve biosynthesis of the growth-inhibiting sesquiterpene (+)-valencene, a flavoring agent and aroma compound precursor in food industry. The variation in light intensity as well as the time point of light induction proved crucial for efficient production of this toxic compound. IMPORTANCE: Optogenetic tools are light-responsive modules that allow for a simple triggering of cellular functions with unprecedented spatiotemporal resolution and in a noninvasive fashion. Specifically, light-controlled gene expression exhibits an enormous potential for various synthetic bio(techno)logical purposes. Before our study, poor inducibility, together with phenotypic heterogeneity, was reported for the IPTG-mediated induction of lac-based gene expression in Corynebacterium glutamicum By applying photocaged IPTG as a synthetic inducer, however, these drawbacks could be almost completely abolished. Especially for increasing numbers of parallelized expression cultures, noninvasive and spatiotemporal light induction qualifies for a precise, homogeneous, and thus higher-order control to fully automatize or optimize future biotechnological applications.

Transcription factor-based biosensors in biotechnology: current state and future prospects.

Living organisms have evolved a plethora of sensing systems for the intra- and extracellular detection of small molecules, ions or physical parameters. Several recent studies have demonstrated that these principles can be exploited to devise synthetic regulatory circuits for metabolic engineering strategies. In this context, transcription factors (TFs) controlling microbial physiology at the level of transcription play a major role in biosensor design, since they can be implemented in synthetic circuits controlling gene expression in dependency of, for example, small molecule production. Here, we review recent progress on the utilization of TF-based biosensors in microbial biotechnology highlighting different areas of application. Recent advances in metabolic engineering reveal TF-based sensors to be versatile tools for strain and enzyme development using high-throughput (HT) screening strategies and adaptive laboratory evolution, the optimization of heterologous pathways via the implementation of dynamic control circuits and for the monitoring of single-cell productivity in live cell imaging studies. These examples underline the immense potential of TF-based biosensor circuits but also identify limitations and room for further optimization.

Screening of an Escherichia coli promoter library for a phenylalanine biosensor.

In recent years, the application of transcription factor-based biosensors for the engineering of microbial production strains opened up new opportunities for industrial biotechnology. However, the design of synthetic regulatory circuits depends on the selection of suitable transcription factor-promoter pairs to convert the concentration of effector molecules into a measureable output. Here, we present an efficient strategy to screen promoter libraries for appropriate parts for biosensor design. To this end, we pooled the strains of the Alon library containing about 2000 different Escherichia coli promoter-gfpmut2 fusions, and enriched galactose- and L-phenylalanine-responsive promoters by toggled rounds of positive and negative selection using fluorescence-activated cell sorting (FACS). For both effectors, responsive promoters were isolated and verified by cultivation in microtiter plates. The promoter of mtr, encoding an L-tryptophan-specific transporter, was identified as suitable part for the construction of an L-phenylalanine biosensor. In the following, we performed a comparative analysis of different biosensor constructs based on the mtr promoter. The obtained data revealed a strong influence of the biosensor architecture on the performance characteristics. For proof-of-principle, the mtr sensor was applied in a FACS high-throughput screening of an E. coli MG1655 mutant library for the isolation of L-phenylalanine producers. These results emphasize the developed screening approach as a convenient strategy for the identification of effector-responsive promoters for the design of novel biosensors.

Impact of spontaneous prophage induction on the fitness of bacterial populations and host-microbe interactions.

Bacteriophages and genetic elements, such as prophage-like elements, pathogenicity islands, and phage morons, make up a considerable amount of bacterial genomes. Their transfer and subsequent activity within the host's genetic circuitry have had a significant impact on bacterial evolution. In this review, we consider what underlying mechanisms might cause the spontaneous activity of lysogenic phages in single bacterial cells and how the spontaneous induction of prophages can lead to competitive advantages for and influence the lifestyle of bacterial populations or the virulence of pathogenic strains.

Phosphatase activity of the histidine kinases ensures pathway specificity of the ChrSA and HrrSA two-component systems in Corynebacterium glutamicum.

The majority of bacterial genomes encode a high number of two-component systems controlling gene expression in response to a variety of different stimuli. The Gram-positive soil bacterium Corynebacterium glutamicum contains two homologous two-component systems (TCS) involved in the haem-dependent regulation of gene expression. Whereas the HrrSA system is crucial for utilization of haem as an alternative iron source, ChrSA is required to cope with high toxic haem levels. In this study, we analysed the interaction of HrrSA and ChrSA in C. glutamicum. Growth of TCS mutant strains, in vitro phosphorylation assays and promoter assays of P(hrtBA) and P(hmuO) fused to eyfp revealed cross-talk between both systems. Our studies further indicated that both kinases exhibit a dual function as kinase and phosphatase. Mutation of the conserved glutamine residue in the putative phosphatase motif DxxxQ of HrrS and ChrS resulted in a significantly increased activity of their respective target promoters (P(hmuO) and P(hrtBA) respectively). Remarkably, phosphatase activity of both kinases was shown to be specific only for their cognate response regulators. Altogether our data suggest the phosphatase activity of HrrS and ChrS as key mechanism to ensure pathway specificity and insulation of these two homologous systems.

Biosensor-driven adaptive laboratory evolution of l-valine production in Corynebacterium glutamicum.

Adaptive laboratory evolution has proven a valuable strategy for metabolic engineering. Here, we established an experimental evolution approach for improving microbial metabolite production by imposing an artificial selective pressure on the fluorescent output of a biosensor using fluorescence-activated cell sorting. Cells showing the highest fluorescent output were iteratively isolated and (re-)cultivated. The L-valine producer Corynebacterium glutamicum ΔaceE was equipped with an L-valine-responsive sensor based on the transcriptional regulator Lrp of C. glutamicum. Evolved strains featured a significantly higher growth rate, increased L-valine titers (~25%) and a 3-4-fold reduction of by-product formation. Genome sequencing resulted in the identification of a loss-of-function mutation (UreD-E188*) in the gene ureD (urease accessory protein), which was shown to increase L-valine production by up to 100%. Furthermore, decreased L-alanine formation was attributed to a mutation in the global regulator GlxR. These results emphasize biosensor-driven evolution as a straightforward approach to improve growth and productivity of microbial production strains.

Spatiotemporal microbial single-cell analysis using a high-throughput microfluidics cultivation platform.

Cell-to-cell heterogeneity typically evolves due to a manifold of biological and environmental factors and special phenotypes are often relevant for the fate of the whole population but challenging to detect during conventional analysis. We demonstrate a microfluidic single-cell cultivation platform that incorporates several hundred growth chambers, in which isogenic bacteria microcolonies growing in cell monolayers are tracked by automated time-lapse microscopy with spatiotemporal resolution. The device was not explicitly developed for a specific organism, but has a very generic configuration suitable for various different microbial organisms. In the present study, we analyzed Corynebacterium glutamicum microcolonies, thereby generating complete lineage trees and detailed single-cell data on division behavior and morphology in order to demonstrate the platform's overall capabilities. Furthermore, the occurrence of spontaneously induced stress in individual C. glutamicum cells was investigated by analyzing strains with genetically encoded reporter systems and optically visualizing SOS response. The experiments revealed spontaneous SOS induction in the absence of any external trigger comparable to results obtained by flow cytometry (FC) analyzing cell samples from conventional shake flask cultivation. Our microfluidic setup delivers detailed single-cell data with spatial and temporal resolution; complementary information to conventional FC results.

Analysis of SOS-induced spontaneous prophage induction in Corynebacterium glutamicum at the single-cell level.

The genome of the Gram-positive soil bacterium Corynebacterium glutamicum ATCC 13032 contains three integrated prophage elements (CGP1 to -3). Recently, it was shown that the large lysogenic prophage CGP3 (∼187 kbp) is excised spontaneously in a small number of cells. In this study, we provide evidence that a spontaneously induced SOS response is partly responsible for the observed spontaneous CGP3 induction. Whereas previous studies focused mainly on the induction of prophages at the population level, we analyzed the spontaneous CGP3 induction at the single-cell level using promoters of phage genes (Pint2 and Plysin) fused to reporter genes encoding fluorescent proteins. Flow-cytometric analysis revealed a spontaneous CGP3 activity in about 0.01 to 0.08% of the cells grown in standard minimal medium, which displayed a significantly reduced viability. A PrecA-eyfp promoter fusion revealed that a small fraction of C. glutamicum cells (∼0.2%) exhibited a spontaneous induction of the SOS response. Correlation of PrecA to the activity of downstream SOS genes (PdivS and PrecN) confirmed a bona fide induction of this stress response rather than stochastic gene expression. Interestingly, the reporter output of PrecA and CGP3 promoter fusions displayed a positive correlation at the single-cell level (ρ = 0.44 to 0.77). Furthermore, analysis of the PrecA-eyfp/Pint2-e2-crimson strain during growth revealed the highest percentage of spontaneous PrecA and Pint2 activity in the early exponential phase, when fast replication occurs. Based on these studies, we postulate that spontaneously occurring DNA damage induces the SOS response, which in turn triggers the induction of lysogenic prophages.

IpsA, a novel LacI-type regulator, is required for inositol-derived lipid formation in Corynebacteria and Mycobacteria.

BACKGROUND: The development of new drugs against tuberculosis and diphtheria is focused on disrupting the biogenesis of the cell wall, the unique architecture of which confers resistance against current therapies. The enzymatic pathways involved in the synthesis of the cell wall by these pathogens are well understood, but the underlying regulatory mechanisms are largely unknown. RESULTS: Here, we characterize IpsA, a LacI-type transcriptional regulator conserved among Mycobacteria and Corynebacteria that plays a role in the regulation of cell wall biogenesis. IpsA triggers myo-inositol formation by activating ino1, which encodes inositol phosphate synthase. An ipsA deletion mutant of Corynebacterium glutamicum cultured on glucose displayed significantly impaired growth and presented an elongated cell morphology. Further studies revealed the absence of inositol-derived lipids in the cell wall and a complete loss of mycothiol biosynthesis. The phenotype of the C. glutamicum ipsA deletion mutant was complemented to different extend by homologs from Corynebacterium diphtheriae (dip1969) and Mycobacterium tuberculosis (rv3575), indicating the conserved function of IpsA in the pathogenic species. Additional targets of IpsA with putative functions in cell wall biogenesis were identified and IpsA was shown to bind to a conserved palindromic motif within the corresponding promoter regions. Myo-inositol was identified as an effector of IpsA, causing the dissociation of the IpsA-DNA complex in vitro. CONCLUSIONS: This characterization of IpsA function and of its regulon sheds light on the complex transcriptional control of cell wall biogenesis in the mycolata taxon and generates novel targets for drug development.