Single-cell Analysis of Bacillus subtilis Biofilms Using Fluorescence Microscopy and Flow Cytometry

Biofilm formation is a general attribute to almost all bacteria ^1-6. When bacteria form biofilms, cells are encased in extracellular matrix that is mostly constituted by proteins and exopolysaccharides, among other factors ^7-10. The microbial community encased within the biofilm often shows the differentiation of distinct subpopulation of specialized cells ^11-17. These subpopulations coexist and often show spatial and temporal organization within the biofilm ^18-21.Biofilm formation in the model organism Bacillus subtilis requires the differentiation of distinct subpopulations of specialized cells. Among them, the subpopulation of matrix producers, responsible to produce and secrete the extracellular matrix of the biofilm is essential for biofilm formation ^11,19. Hence, differentiation of matrix producers is a hallmark of biofilm formation in B. subtilis.We have used fluorescent reporters to visualize and quantify the subpopulation of matrix producers in biofilms of B. subtilis^15,19,22-24. Concretely, we have observed that the subpopulation of matrix producers differentiates in response to the presence of self-produced extracellular signal surfactin ²⁵. Interestingly, surfactin is produced by a subpopulation of specialized cells different from the subpopulation of matrix producers ¹⁵.We have detailed in this report the technical approach necessary to visualize and quantify the subpopulation of matrix producers and surfactin producers within the biofilms of B.subtilis. To do this, fluorescent reporters of genes required for matrix production and surfactin production are inserted into the chromosome of B. subtilis. Reporters are expressed only in a subpopulation of specialized cells. Then, the subpopulations can be monitored using fluorescence microscopy and flow cytometry (See Fig 1).The fact that different subpopulations of specialized cells coexist within multicellular communities of bacteria gives us a different perspective about the regulation of gene expression in prokaryotes. This protocol addresses this phenomenon experimentally and it can be easily adapted to any other working model, to elucidate the molecular mechanisms underlying phenotypic heterogeneity within a microbial community.     

Boundary cells regulate a switch in the expression of FGF3 in hindbrain rhombomeres

Background  

During formation of the vertebrate central nervous system, the hindbrain is organized into segmental units, called rhombomeres (r). These cell-lineage restricted segments are separated by a subpopulation of cells known as boundary cells. Boundary cells display distinct molecular and cellular properties such as an elongated shape, enriched extracellular matrix components and a reduced proliferation rate compared to intra-rhombomeric cells. However, little is known regarding their functions and the mechanisms that regulate their formation.     

Cell accumulation in the junctional region of denervated muscle

If skeletal muscles are denervated, the number of mononucleated cells in the connective tissue between muscle fibers increases. Since interstitial cells might remodel extracellular matrix, and since extracellular matrix in nerve and muscle plays a direct role in reinnervation of the sites of the original neuromuscular junctions, we sought to determine whether interstitial cell accumulation differs between junctional and extrajunctional regions of denervated muscle. We found in muscles from frog and rat that the increase in interstitial cell number was severalfold (14-fold for frog, sevenfold for rat) greater in the vicinity of junctional sites than in extrajunctional regions. Characteristics of the response at the junctional sites of frog muscles are as follows. During chronic denervation, the accumulation of interstitial cells begins within 1 wk and it is maximal by 3 wk. Reinnervation 1-2 wk after nerve damage prevents the maximal accumulation. Processes of the cells form a multilayered veil around muscle fibers but make little, if any, contact with the muscle cell or its basal lamina sheath. The results of additional experiments indicate that the accumulated cells do not originate from terminal Schwann cells or from muscle satellite cells. Most likely the cells are derived from fibroblasts that normally occupy the space between muscle fibers and are known to make and degrade extracellular matrix components.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Protein-mediated adhesion of Lactobacillus acidophilus BG2FO4 on human enterocyte and mucus-secreting cell lines in culture.

The adhesion of Lactobacillus acidophilus BG2FO4, a human stool isolate, to two human enterocytelike cell lines (Caco-2 and HT-29) and to the mucus secreted by a subpopulation of mucus-secreting HT29-MTX cells was investigated. Scanning electron microscopy revealed that the bacteria interacted with the well-defined apical microvilli of Caco-2 cells without cell damage and with the mucus secreted by the subpopulation of HT29-MTX cells. The adhesion to Caco-2 cells did not require calcium and involved an adhesion-promoting factor that was present in the spent supernatant of L. acidophilus cultures. This factor promoted adhesion of poorly adhering human Lactobacillus casei GG but did not promote adhesion of L. casei CNRZ 387, a strain of dairy origin. The adherence components on the bacterial cells and in the spent supernatant were partially characterized. Carbohydrates on the bacterial cell wall appeared to be partly responsible for the interaction between the bacteria and the extracellular adhesion-promoting factor. The adhesion-promoting factor was proteinaceous, since trypsin treatment dramatically decreased the adhesion of the L. acidophilus strain. The adhesion-promoting factor may be an important component of Lactobacillus species that colonize the gastrointestinal tract.     

Cannibalism enhances biofilm development in Bacillus subtilis

Cannibalism is a mechanism to delay sporulation in Bacillus subtilis. Cannibal cells express the skf and sdp toxin systems to lyse a fraction of their sensitive siblings. The lysed cells release nutrients that serve to feed the community, effectively delaying spore formation. Here we provide evidence that the subpopulation of cells that differentiates into cannibals is the same subpopulation that produces the extracellular matrix that holds cells together in biofilms. Cannibalism and matrix formation are both triggered in response to the signalling molecule surfactin. Nutrients released by the cannibalized cells are preferentially used by matrix-producing cells, as they are the only cells expressing resistance to the Skf and Sdp toxins. As a result this subpopulation increases in number and matrix production is enhanced when cannibalism toxins are produced. The cannibal/matrix-producing subpopulation is also generated in response to antimicrobials produced by other microorganisms and may thus constitute a defense mechanism to protect B. subtilis from the action of antibiotics in natural settings.     

Leukemia Inhibitory Factor Is an Autocrine Survival Factor for Schwann Cells

Abstract: Schwann cells play a major role in promoting nerve survival and regeneration after injury. Their activities include providing neurotrophic factors and increasing the production of extracellular matrix components and cell surface adhesion molecules to promote axon regeneration. Following nerve transection, leukemia inhibitory factor (LIF) is up-regulated by Schwann cells at the injury site. LIF receptors are also up-regulated at the nerve injury site, but their cellular localization and function have not been fully characterized. We demonstrate that Schwann cells express mRNAs for LIF and the LIF receptor components LIF receptor subunit β and glycoprotein 130 in vitro. We also show that although LIF is not required for the genesis of Schwann cells, it can potentiate the survival of differentiated Schwann cells in the context of neuregulin support. Not only does exogenous LIF promote survival under these conditions, but addition of the soluble LIF receptor (LIF binding protein) and anti-LIF antibodies significantly reduced cell survival, suggesting that LIF exerts autocrine effects. These results suggest that Schwann cell survival following nerve injury is potentially modulated by LIF.     

Characteristics of electrical activity in different sections of nerve endings in the frog

In experiments on frog sartorius neuromuscular preparations, the evoked electrical responses of nerve endings were recorded by extracellular microelectrodes. It was shown that in proximal parts of the nerve ending, the three-phase response (+ - +) with a high amplitude negative phase occurred due to motor nerve stimulation. With movement of the extracellular electrode in distal direction a certain increase of the initial positive phase and a significant decrease of the negative one were observed. At the end of the terminal that response transformed to the monophasic one (+). On local iontophoretic application of tetrodotoxin (TTX) to the recording site two components of the nerve ending response were revealed: TTX-insensitive and TTX-sensitive. A significant decrease of the TTX-sensitive component occurred along the course of the nerve ending. That component was absent from the distal synaptic areas. It is concluded that in frog nerve ending, the action potential propogates with decrement while depolarization of the end parts of the terminal is passive in nature.     

Characterization of NADPH-diaphorase-positive glial cells of the tench optic nerve after axotomy

NADPH-diaphorase (ND) positive cell types were characterized throughout the optic nerve of the tench in normal conditions and after optic nerve transection with survival periods of 1, 3, 7, 14, 30, 60, 120 and 180 days. Astrocytic markers (S100 and glutamine synthetase) and the microglial marker tomato lectin were employed. In the control prechiasmatic optic nerve two types (types I and II) of ND-positive glial cells appeared. All type I cells showed S100 immunoreactivity, whereas only a subpopulation of them were positive to glutamine synthetase. Type II cells only presented S100 immunoreactivity. In the control anterior optic tract, all ND-positive glial cells (type III) presented immunolabeling to S100 and glutamine synthetase. After transection, types I and II did not show any changes in the staining patterns for the glial markers when observed. Two new types of ND-positive glial cells (types IV and V) were observed after axotomy. All type IV cells were S100-immunopositive, and a subpopulation presented glutamine synthetase immunolabeling. Only a subpopulation of type V cells showed glutamine synthetase immunostaining. The presence of type IV or V cells in the lesioned optic nerve occurred simultaneously with significant decreases or absence of type I cells. These data suggest that type I and III cells are astrocytes and type II cells are oligodendrocytes. Types IV and V cells are the result of the activation of type I cells after optic nerve section. The polymorphism observed in ND-positive cells may reflect different cell functions during degenerative and regenerative processes.     

A two-dimensional ERK-AKT signaling code for an NGF-triggered cell-fate decision

Growth factors activate Ras, PI3K, and other signaling pathways. It is not well understood how these signals are translated by individual cells into a decision to proliferate or differentiate. Here, using single-cell image analysis of nerve growth factor (NGF)-stimulated PC12 cells, we identified a two-dimensional phospho-ERK (pERK)-phospho-AKT (pAKT) response map with a curved boundary that separates differentiating from proliferating cells. The boundary position remained invariant when different stimuli were used or upstream signaling components perturbed. We further identified Rasa2 as a negative feedback regulator that links PI3K to Ras, placing the stochastically distributed pERK-pAKT signals close to the decision boundary. This allows for uniform NGF stimuli to create a subpopulation of cells that differentiates with each cycle of proliferation. Thus, by linking a complex signaling system to a simpler intermediate response map, cells gain unique integration and control capabilities to balance cell number expansion with differentiation.     

Brain lipid binding protein in axon-Schwann cell interactions and peripheral nerve tumorigenesis

Loss of axonal contact characterizes Schwann cells in benign and malignant peripheral nerve sheath tumors (MPNST) from neurofibromatosis type 1 (NF1) patients. Tumor Schwann cells demonstrate NF1 mutations, elevated Ras activity, and aberrant epidermal growth factor receptor (EGFR) expression. Using cDNA microarrays, we found that brain lipid binding protein (BLBP) is elevated in an EGFR-positive subpopulation of Nf1 mutant mouse Schwann cells (Nf1(-/-) TXF) that grows away from axons; BLBP expression was not affected by farnesyltransferase inhibitor, an inhibitor of H-Ras. BLBP was also detected in EGFR-positive cell lines derived from Nf1:p53 double mutant mice and human MPNST. BLBP expression was induced in normal Schwann cells following transfection with EGFR but not H-Ras12V. Furthermore, EGFR-mediated BLBP expression was not inhibited by dominant-negative H-Ras, indicating that BLBP expression is downstream of Ras-independent EGFR signaling. BLBP-blocking antibodies enabled process outgrowth from Nf1(-/-) TXF cells and restored interaction with axons, without affecting cell proliferation or migration. Following injury, BLBP expression was induced in normal sciatic nerves when nonmyelinating Schwann cells remodeled their processes. These data suggest that BLBP, stimulated by Ras-independent pathways, regulates Schwann cell-axon interactions in normal peripheral nerve and peripheral nerve tumors.     

Formation of junctional complexes at sites of contact of hemocytes with tissue elements in degenerating nerves of the crayfish, Orconectes virilis

Hemocytes, which contain large cytoplasmic granules, invade the multilamellate glial sheath of ventral ganglion nerve roots of the crayfish following surgical interruption of these nerves. Electron microscopic examination of sections of plasticembedded tissues and replicas of freeze-cleaved ganglion roots reveals numerous slender cytoplasmic extensions of the hemocytes present in damaged nerve sheaths. Many of these microvillous extensions contact glial cells and filamentous extracellular masses. At sites of contact, the microvilli are flattened and occasionally electron-dense material is present in the hemocyte cytoplasm subjacent to the plasma membrane that is closely apposed to a glial cell or connective tissue. Intramembranous surfaces of hemocyte plasmalemmae exposed by freeze-fracture, exhibit particle aggregates 700–2500 Å in diameter. Individual particles are 95–105 Å in diameter. Since the particle aggregates correspond in overall dimension and position in the cell to the sites of contact of hemocyte processes with other sheath components, it is assumed that the two structures are equivalent and represent a junctional complex very similar in structure to some hemidesmosomes. Results from this study strongly suggest that granulated crustacean hemocytes, in response to surgical injury of nerves, invade the damaged nerve sheath and identify damaged glial cells and connective tissue by forming slender cytoplasmic processes which contact elements of the sheath. Tissue components contacted by the hemocytes may subsequently be phagocytosed by them. This is the first report of an invertebrate hemocyte-mediated response to tissue damage in which evidence is presented that the hemocyte may identify necrotic cells and extracellular matrix by forming junctional complexes with them. Crustacean hemocytes, therefore, are likely much more complex functionally than has been previously estimated.     

Pituitary adenylate cyclase-activating polypeptide in intrinsic and extrinsic nerves of the rat pancreas

Pituitary adenylate cyclase-activating polypeptide (PACAP) is the latest member of the vasoactive intestinal polypeptide (VIP) family of neuropeptides present in nerve fibres in many peripheral organs. Using double immunohistochemistry, with VIP as a marker for intrinsic innervation and calcitonin-gene related peptide (CGRP) as a marker for mainly extrinsic innervation, the distribution and localization of PACAP were studied in the rat pancreas. PACAP was demonstrated in nerve fibres in all compartments of the pancreas and in a subpopulation of intrapancreatic VIP-containing ganglion cells. PACAP and VIP were co-stored in intra- and interlobular nerve fibres innervating acini, blood vessels, and in nerve fibres within the islets of Langerhans. No PACAP immunoreactivity was observed in the islet cells. Another population of PACAP-immunoreactive nerve fibres co-localized with CGRP innervated ducts, blood vessels and acini. PACAP/CGRP-positive nerve fibres were also demonstrated within the islets. Neonatal capsaicin reduced the PACAP-38 concentration by approximately 50%, and accordingly a marked reduction in PACAP/CGRP-immunoreactive nerve fibres in the exocrine and endocrine pancreas was observed. Bilateral subdiaphragmatic vagotomy caused a slight but significant decrease in the PACAP-38 concentration compared with controls. In conclusion, PACAP-immunoreactive nerve fibres in the rat pancreas seem to have dual origin: extrinsic, most probably sensory fibres co-storing CGRP; and intrinsic, constituting a subpopulation of VIP-containing nerve cell bodies and fibres innervating acinar cells and islet cells. Our data provide a morphological basis for the reported effects of PACAP in the pancreas and suggest that PACAP-containing nerves in the rat pancreas may have both efferent and sensory functions.     

Patterning of olfactory sensory connections is mediated by extracellular matrix proteins in the nerve layer of the olfactory bulb

In early rat embryos when axons from sensory neurons first contact the olfactory bulb primordium, lactosamine-containing glycans (LCG) are detected on neurons that are broadly distributed within the olfactory epithelium, but that project axons to a very restricted region of the ventromedial olfactory bulb. LCG(+) axons extend through channels defined by the coexpression of galectin-1 and beta2-laminin. These two extracellular matrix molecules are differentially expressed, along with semaphorin 3A, by subsets of ensheathing cells in the ventral nerve layer of the olfactory bulb. The overlapping expression of these molecules creates an axon-sorting domain that is capable of promoting and repelling subsets of olfactory axons. Specifically, LCG(+) axons preferentially grow into the region of the nerve layer that expresses high amounts of galectin-1, beta2-laminin, and semaphorin 3A, whereas neuropilin-1(+) axons grow in a complementary pattern, avoiding the ventral nerve layer and projecting medially and laterally. These studies suggest that initial patterning of olfactory epithelium to olfactory bulb connections is, in part, dependent on extracellular components of the embryonic nerve layer that mediate convergence and divergence of specific axon subsets.     

Transmitter release increases intracellular calcium in perisynaptic Schwann cells in situ.

Glial cells isolated from the nervous system are sensitive to neurotransmitters and may therefore be involved in synaptic transmission. The sensitivity of individual perisynaptic Schwann cells to activity of a single synapse was investigated, in situ, at the frog neuromuscular junction by monitoring changes in intracellular Ca2+ in the Schwann cells. Motor nerve stimulation induced an increase in intracellular Ca2+ in these Schwann cells; this increase was greatly reduced when transmitter release was blocked. Furthermore, local application of the cotransmitters acetylcholine and ATP evoked Ca2+ responses even in the absence of extracellular Ca2+. Successive trains of nerve stimuli or applications of transmitters resulted in progressively smaller Ca2+ responses. We conclude that transmitter released during synaptic activity can evoke release of intracellular Ca2+ in perisynaptic Schwann cells. This Ca2+ signal may play a role in the maintenance or modulation of a synapse. These data show that synaptic transmission involves three cellular components with both postsynaptic and glial components responding to transmitter secretion.     

Brief exposure to neurotrophins produces a calcium-dependent increase in choline acetyltransferase activity in cultured rat septal neurons

We demonstrate that brief (30-min) exposure of cultured embryonic rat septal neurons to neurotrophins (NTs) increases choline acetyltransferase (ChAT) activity by 20-50% for all tested NTs (nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4, each at 100 ng/ml). The increase in ChAT activity was first detected 12 h after NT exposure, persisted at least 48 h, and was not mediated by increased neuronal survival or action-potential activity. Under some conditions, the response to brief NT exposure was as great as that produced by continuous exposure. NT stimulation of ChAT activity was inhibited by inhibitors of p75- and Trk kinase-mediated signaling, by removal of extracellular Ca2+ during the period of NT exposure, and by buffering intracellular Ca2+ with BAPTA. Application of nerve growth factor and brain-derived neurotrophic factor transiently increased [Ca2+] within a subpopulation of neurons. NT stimulation of ChAT activity was not affected significantly by cyclic AMP agonists or antagonists. These findings suggest that brief exposure to NTs can have a long-lasting effect on cholinergic transmission, and that this effect requires Ca2+, but not cyclic AMP.     

Fibrillin-2 is dispensable for peripheral nerve development,myelination and regeneration

The extracellular matrix of peripheral nerve is formed from a diverse set of macromolecules, including glycoproteins, collagens and proteoglycans. Recent studies using knockout animal models have demonstrated that individual components of the extracellular matrix play a vital role in peripheral nerve development and regeneration. In this study we identified fibrillin-1 and fibrillin-2, large modular structural glycoproteins, as components of the extracellular matrix of peripheral nerve. Previously it was found that fibrillin-2 null mice display joint contractures, suggesting a possible defect of the peripheral nervous system in these animals. Close examination of the peripheral nerves of fibrillin-2 deficient animals described here revealed some structural abnormalities in the perineurium, while general structure of the nerve and molecular composition of nerve extracellular matrix remained unchanged. We also found that in spite of the obvious motor function impairment, fibrillin-2 null mice failed to display changes of nerve conduction properties or nerve regeneration capacity. Based on the data obtained we can conclude that peripheral neuropathy should be excluded as the cause of the impairment of locomotory function and joint contractures observed in fibrillin-2 deficient animals.