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Volume 11, no. 5, e01774-20, 2020, [https://doi.org/10.1128/mBio.01774-20][1]. Professor Birgitta Henriques-Normark’s last name was missing a hyphen between Henriques and Normark in the original article. The byline should appear as shown above. Also, the affiliations and affiliation letters are

Nucleoid-associated proteins (NAPs) facilitate chromosome organization in bacteria, but the precise mechanism remains elusive. H-NS is a NAP that also plays a major role in silencing pathogen genes. We used genetics, single-particle tracking in live cells, superresolution microscopy, atomic force microscopy, and molecular dynamics simulations to examine H-NS/DNA interactions in single cells. We discovered a role for the unstructured linker region connecting the N-terminal oligomerization and C-terminal DNA binding domains. In the present work we demonstrate that linker amino acids promote engagement with DNA. In the absence of linker contacts, H-NS binding is significantly reduced, although no change in chromosome compaction is observed. H-NS is not localized to two distinct foci; rather, it is scattered all around the nucleoid. The linker makes DNA contacts that are required for gene silencing, while chromosome compaction does not appear to be an important H-NS function.

After Salmonella is phagocytosed, it resides in an acidic vacuole. Its cytoplasm acidifies to pH 5.6; acidification activates pathogenicity island 2 (SPI-2). SPI-2 encodes a type three secretion system whose effectors modify the vacuole, driving endosomal tubulation. Using super-resolution imaging in single bacterial cells, we show that low pH induces expression of the SPI-2 SsrA/B signaling system. Single particle tracking, atomic force microscopy, and single molecule unzipping assays identified pH-dependent stimulation of DNA binding by SsrB. A so-called phosphomimetic form (D56E) was unable to bind to DNA in live cells. Acid-dependent DNA binding was not intrinsic to regulators, as PhoP and OmpR binding was not pH-sensitive. The low level of SPI-2 injectisomes observed in single cells is not due to fluctuating SsrB levels. This work highlights the surprising role that acid pH plays in virulence and intracellular lifestyles of Salmonella; modifying acid survival pathways represents a target for inhibiting Salmonella., eLife digest Salmonellae are a group of bacteria that can cause vomiting and diarrhea if we consume contaminated food. Once in the bowel, the bacteria get inside our cells, where they stay in a compartment called the vacuole. This environment is very acidic, and the inside of the microbes also becomes more acidic in response. This change helps Salmonella to switch on genes that allow them to survive and infect humans, but it is still unclear how this mechanism takes place. To investigate this question, Liew, Foo et al. harnessed a recent technique called super-resolution imaging, which lets scientists see individual molecules in a cell. First, the technique was used to count a protein called SsrB as well as the enzyme that activates it, SsrA. The role of SsrB is to bind to DNA and turn on genes involved in making proteins that help Salmonella thrive. These studies revealed that the levels of SsrA/B proteins increased three-fold in an acidic environment. Then, Liew, Foo et al. followed SsrB inside cells, knowing that fast-moving particles are free in solution, while slow-moving particles are typically bound to DNA. In acidic conditions, the proportion of SsrB bound to DNA doubled. Finally, further experiments revealed that when the environment was acidic, SsrB became five times more likely to bind to DNA. Taken together, the results suggest that acidic conditions trigger a cascade of events which switch on genetic information that allows Salmonella to survive. If SsrB could be prevented from responding to acid stress, it could potentially stop Salmonella from surviving inside host cells. This knowledge should be applied to drive new treatment strategies for Salmonella and other microbes that infect human cells.

Two-component regulatory systems drive signal transduction in bacteria. The simplest of these employs a membrane sensor kinase and a cytoplasmic response regulator. Environmental sensing is typically coupled to gene regulation. The histidine kinase EnvZ and its cognate response regulator OmpR regulate expression of outer membrane proteins (porins) in response to osmotic stress. We used hydrogen:deuterium exchange mass spectrometry to identify conformational changes in the cytoplasmic domain of EnvZ (EnvZc) that were associated with osmosensing. The osmosensor localized to a seventeen amino acid region of the four-helix bundle of the cytoplasmic domain and flanked the His(243) autophosphorylation site. High osmolality increased autophosphorylation of His(243), suggesting that these two events were linked. The transmembrane domains were not required for osmosensing, but mutants in the transmembrane domains altered EnvZ activity. A photoactivatable fusion protein composed of EnvZc fused to the fluorophore mEos2 (EnvZc-mEos2) was as capable as EnvZc in supporting OmpR-dependent ompF and ompC transcription. Over-expression of EnvZc reduced activity, indicating that the EnvZ/OmpR system is not robust. Our results support a model in which osmolytes stabilize helix one in the four-helix bundle of EnvZ by increased hydrogen bonding of the peptide backbone, increasing autophosphorylation and downstream signaling. The likelihood that additional histidine kinases use similar cytoplasmic sensing mechanisms is discussed.

Two-component signaling systems are a major strategy employed by bacteria, and to some extent, yeast and plants, to respond to environmental stress. The EnvZ/OmpR system in E. coli responds to osmotic and acid stress and is responsible for regulating the protein composition of the outer membrane. EnvZ is a histidine kinase located in the inner membrane. Upon activation, it is autophosphorylated by ATP and subsequently, it activates OmpR. Phosphorylated OmpR binds with high affinity to the regulatory regions of the ompF and ompC porin genes to regulate their transcription. We set out to visualize these two-components in single bacterial cells during different environmental stress conditions and to examine the subsequent modifications to the bacterial nucleoid as a result. We created a chromosomally-encoded, active, fluorescent OmpR–PAmCherry fusion protein and compared its expression levels with RNA polymerase. Quantitative western blotting had indicated that these two proteins were expressed at similar levels. From our images, it is evident that OmpR is significantly less abundant compared to RNA polymerase. In cross-sectional axial images, we observed OmpR molecules closely juxtaposed near the inner membrane during acidic and hyposomotic growth. In acidic conditions, the chromosome was compacted. Surprisingly, under acidic conditions, we also observed evidence of a spatial correlation between the DNA and the inner membrane, suggesting a mechanical link through an active DNA–OmpR–EnvZ complex. This work represents the first direct visualization of a response regulator with respect to the bacterial chromosome.

Despite their small size and the lack of compartmentalization, bacteria exhibit a striking degree of cellular organization, both in time and space. During the last decade, a group of new microscopy techniques emerged, termed super-resolution microscopy or nanoscopy, which facilitate visualizing the organization of proteins in bacteria at the nanoscale. Single-molecule localization microscopy (SMLM) is especially well suited to reveal a wide range of new information regarding protein organization, interaction, and dynamics in single bacterial cells. Recent developments in click chemistry facilitate the visualization of bacterial chromatin with a resolution of ~20 nm, providing valuable information about the ultrastructure of bacterial nucleoids, especially at short generation times. In this chapter, we describe a simple-to-realize protocol that allows determining precise structural information of bacterial nucleoids in fixed cells, using direct stochastic optical reconstruction microscopy (dSTORM). In combination with quantitative photoactivated localization microscopy (PALM), the spatial relationship of proteins with the bacterial chromosome can be studied. The position of a protein of interest with respect to the nucleoids and the cell cylinder can be visualized by super-resolving the membrane using point accumulation for imaging in nanoscale topography (PAINT). The combination of the different SMLM techniques in a sequential workflow maximizes the information that can be extracted from single cells, while maintaining optimal imaging conditions for each technique.

A series of engineered linear analogues [coded as F6, W6, Y6, A6, S6 and C(Acm)6] were modeled, designed, synthesized and structurally characterized by mass spectra, circular dichroism, hydrophobicity analysis and molecular modeling. We have screened antimicrobial activity, hemolysis to rabbit erythrocytes, and cytotoxicity to human conjunctival epithelial cells. No significant hemolytic effect was observed for hBD3 or from five of the six analogues [F6, Y6, A6, S6 and C(Acm)6] over the range of 3-100 microg mL(-1). The six linear analogues have reduced cytotoxicity to human conjunctival epithelial cells over the range of 6-100 microg mL(-1) compared to hBD3. By tuning the overall hydrophobicity of linear hBD3 analogues, reduced cytotoxicity and hemolysis were obtained while preserving the antimicrobial properties. The decreased cytotoxicity of the linear analogues is suggested to be structurally related to the removal of disulfide bridges, and the flexible structure of the linear forms, which seem to be associated with loss of secondary structure. These results suggest a new approach for guiding the design of new linear analogues of defensin peptides with strong antibiotic properties and reduced cytotoxicity to mammalian cells.

The cornea is the major refracting optical element of the eye and therefore critical for forming a retinal image. The exposed surface of the eye is protected from pathogens by the innate immune system whose components include defensins, naturally occurring peptides with antimicrobial properties, and the physical barrier formed by the outer epithelial layer of the cornea. The proteomic approach has revealed that tear levels of defensins are correlated with the course of healing of an experimental corneal wound. Tears were collected from New Zealand White rabbits prior to (day 0) and daily for 5 days (days 1-5) following a standard unilateral 6 mm diameter corneal epithelial abrasion. Tear protein profiles obtained from wounded and contra-lateral control eyes were compared using SELDI ProteinChip technology. Peptides and proteins of interest were purified by RP-HPLC and characterized by nanoESI-MS/MS. Mass spectra of tears on post-wound day 1, revealed 13 peaks whose level decreased and five that increased. During wound healing the tear protein profile correlated with wound closure. An important finding was that the levels of rabbit defensins (NP-1 and NP-2), which were elevated after wounding returned to normal levels by the time the corneal abrasion healed. Relative quantification of NP-2 in tear fluid prior to (day 0) and after corneal wounding (days 1- 3) was determined using iTRAQ technology. A corneal wound eliminates the barrier function of innate immunity and puts the cornea at risk from microbial attack until the epithelial cells restore the surface barrier. The increased availability of defensins in the tears during healing suggests that these peptides could protect the cornea from microbial attack during a period of increased vulnerability.

Oct4 and Sox2 are two essential transcription factors that co-regulate target genes for the maintenance of pluripotency. However, it is unclear whether they interact prior to DNA binding or how the target sites are accessed in the nucleus. By generating fluorescent protein fusions of Oct4 and Sox2 that are functionally capable of producing iPSCs (induced pluripotent stem cells), we show that their interaction is dependent on the presence of cognate DNA-binding elements, based on diffusion time, complex formation and lifetime measurements. Through fluorescence correlation spectroscopy, the levels of Oct4 and Sox2 in the iPSCs were quantified in live cells and two diffusion coefficients, corresponding to free and loosely bound forms of the protein, were distinguished. Notably, the fraction of slow-diffusing molecules in the iPSCs was found to be elevated, similar to the profile in embryonic stem cells, probably due to a change in the nuclear milieu during reprogramming. Taken together, these findings have defined quantitatively the amount of proteins pertinent to the pluripotent state and revealed increased accessibility to the underlying DNA as a mechanism for Oct4 and Sox2 to find their target binding sites and interact, without prior formation of heterodimer complexes.

The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (K(D)) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42(G12V) which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42(T17N). While Cdc42(G12V) binds to IQGAP1 with an apparent K(D) of approximately 100 nM, Cdc42(T17N) has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and K(D) from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions.

Oct4 and Sox2 are key transcription factors (TFs) essential for maintaining pluripotency as well as the self-renewal ability of embryonic stem cells (ESC). Oct4 and Sox2 have been previously described to synergistically control pluripotent-specific expression of a number of genes. It has already been reported that Oct4 and Sox2 cooperatively bind with an oct-sox cis-regulatory element and thereby regulates the transcription of a number of important target genes such as Fgf4, Oct4, Nanog and Sox2. Presently, there is no quantitative assay characterizing the cooperative regulation of the TF complex. In this study, we focus on a quantitative investigation regarding the binding pathway of ternary complex formation and how the cooperativity works. We are using a combination of biophysical (eg. single-wavelength fluorescence cross-correlation spectroscopy) and biochemical assays to confirm the functionality of our protein constructs and to demonstrate that Sox2 has a crucial role in helping Oct4 to bind to the oct-sox element whereas Oct4 does not promote Sox2 binding. We further establish that Oct4 and Sox2 are only able to form a heterodimer in the presence of the oct-sox element and do not interact without DNA. Such quantitative measurements will provide deeper insight into the Oc4-Sox2 regulatory network and move us towards a systems level understanding of the regulation of pluripotency.

The RhoGTPase Cdc42 coordinates cell morphogenesis, cell cycle, and cell polarity decisions downstream of membrane-bound receptors through distinct effector pathways. Cdc42-effector protein interactions represent important elements of cell signaling pathways that regulate cell biology in systems as diverse as yeast and humans. To derive mechanistic insights into cell signaling pathways, it is vital that we generate quantitative data from in vivo systems. We need to be able to measure parameters such as protein concentrations, rates of diffusion, and dissociation constants (KD) of protein-protein interactions in vivo. Here we show how single wavelength fluorescence cross-correlation spectroscopy in combination with Förster resonance energy transfer analysis can be used to determine KD of Cdc42-effector interactions in live mammalian cells. Constructs encoding green fluorescent protein or monomeric red fluorescent protein fusion proteins of Cdc42, an effector domain (CRIB), and two effectors, neural Wiskott-Aldrich syndrome protein (N-WASP) and insulin receptor substrate protein (IRSp53), were expressed as pairs in Chinese hamster ovary cells, and concentrations of free protein as well as complexed protein were determined. The measured KD for Cdc42V12-N-WASP, Cdc42V12-CRIB, and Cdc42V12-IRSp53 was 27, 250, and 391 nm, respectively. The determination of KD for Cdc42-effector interactions opens the way to describe cell signaling pathways quantitatively in vivo in mammalian cells.

Fluorescence Correlation Spectroscopy (FCS) is a powerful technique to assess molecule dynamics and interactions on a single molecule level. It has been routinely used to harvest biomedical information both in vitro and in vivo. Numerous intracellular measurements have been reported in cytoplasm, nucleoplasm and plasma membrane. However, it is now generally accepted that the Petri-Dish based cell culture system cannot represent the essential physiological environment of a living organism and 3D cultures are only a partial solution. Here, we adapted single wavelength fluorescence cross-correlation spectroscopy (SW-FCCS) to work in a living animal model. We chose zebrafish as the optical transparency of early stage zebrafish embryo makes microscopic techniques suitable and established genetic tools enable one to easily express foreign genes within the embryo. First we investigated the penetration depth in the embryo body as tissue tends to cause light scattering and decrease signal to noise ratio if working deep beneath skin. We practiced one- and two-photon excitation and obtained FCS curves up to 80 μm and 200 μm, respectively. Then we characterized the diffusion coefficients of genetically expressed EGFP in muscle fibers and motor neurons and also investigated the mobility of a membrane expressed G protein coupled receptor-CXCR4b. Finally, we measured the dissociation constant (KD) between a small Rho-GTPase and an actin-binding scaffolding protein, in living zebrafish embryos and the results were compared with data obtain from CHO-K1 cultured cells. We showed that molecular dynamics and interactions can be studied in small living animals that provide a genuine physiological environment and questions of developmental biology on a single molecule level are directly accessible by FCS.

The EnvZ/OmpR two component regulatory system in bacteria regulate the porin genes ompF and ompC in response to the changes in osmolality of the environment. At low osmolality, levels of phosphorylated EnvZ (EnvZ-P) are presumably low, leading to a low amount of OmpR-P. This in turns produces the porin OmpF. On the other hand, at high osmolality, EnvZ-P, and thus OmpR-P levels increase. This represses ompF and produces OmpC. While the general mechanism is known mainly from genetics and biochemical studies, it is difficult to monitor dynamic changes such as the effect of a sudden change of osmolarity and study how that alters the interaction between EnvZ, OmpR and DNA. Thus we employ single-molecule-sensitive fluorescence spectroscopy to study their interaction under differing osmolalities. The cytoplasmic portion of EnvZ (EnvZc) has been shown to be the osmo-sensor (Wang et al., EMBO J, 2012). Thus we purified a cysteine EnvZc mutant (EnvZc294C) and labeled it with Alexa568 at its lone cysteine residue. Using fluorescence correlation spectroscopy (FCS), we showed that EnvZc294C labeled with Alexa 568 (EnvZc294C-A568) binds to OmpR with a dissociation constant Kd of 92 ± 33 nM in solution. It was also observed that two OmpR molecules bind to a single EnvZc294C-A568 dimer in a cooperative manner. Fluorescence cross-correlation spectroscopy (FCCS) experiments indicated that OmpR labeled with Alexa488 (OmpR-A488) binds to EnvZc294C-A568. With these preliminary results, we are currently anchoring EnvZc294C-A568 onto supported lipid bilayers followed by addition of OmpR-A488. These experiments will explore the effect of changing osmolality, in the presence or absence of ATP and/or DNA, on the interaction between EnvZc, OmpR and DNA by mimicking the organization of proteins in living bacteria. Supported by Mechanobiology RCE from the Ministry of Education, Singapore and VA5IO1BX000372 to LJK.

The EnvZ/OmpR two-component regulatory system in bacteria regulates the porins OmpF and OmpC in response to changes in osmolality of the environment. The cytoplasmic portion of the sensor kinase EnvZ (EnvZc) has been shown to be the osmo-sensor (Wang et al., EMBO J, 2012). At high osmolality, the four-helix bundle domain of the EnvZc dimer is relaxed, increasing the autophosphorylation of EnvZ. On the other hand, at low osmolality, the four-helix bundle domain is stretched, leading to lower autophosphorylation. After EnvZ is autophosphorylated, the phosphoryl group is transferred to the DNA binding protein OmpR. OmpR∼P binds to the ompF and ompC promoters to regulate their expression. Since the osmo-sensing capability of EnvZ is associated with stretch-relaxation dynamics, we wanted to understand and characterize the mechanical stability of EnvZ in the absence and presence of ATP- or OmpR/OmpR∼P-binding. In our initial experiments, we used magnetic tweezers to stretch a single EnvZc molecule. This gives two unfolding events from the dimerization and ATP binding domains. Although there is a wealth of in vitro biochemical studies of EnvZ and OmpR, an understanding of the signal transduction from EnvZ to OmpR in vivo is poorly understood. We used single-particle tracking of an OmpR-PAmcherry fusion to examine the relative abundance of DNA-bound/free OmpR obtained from the mobility of the OmpR-PAmCherry tracks. This was then used to determine how the fraction of bound/free OmpR changed in response to environmental stress. Our preliminary results indicate that the bound fraction of OmpR is localized at certain cellular positions, which could be the location of the genes regulated by OmpR. Supported by Mechanobiology RCE from the Ministry of Education, Singapore and VA-IO1BX000372 to LJK.