Genetic Network Architecture and Environmental Cues Drive Spatial Organization of Phenotypic Division of Labor in Streptomyces coelicolor

ABSTRACT A number of bacteria are known to differentiate into cells with distinct phenotypic traits during processes such as biofilm formation or the development of reproductive structures. These cell types, by virtue of their specialized functions, embody a division of labor. However, how bacteria build spatial patterns of differentiated cells is not well understood. Here, we examine the factors that drive spatial patterns in divisions of labor in colonies of Streptomyces coelicolor, a multicellular bacterium capable of synthesizing an array of antibiotics and forming complex reproductive structures (e.g., aerial hyphae and spores). Using fluorescent reporters, we demonstrate that the pathways for antibiotic biosynthesis and aerial hypha formation are activated in distinct waves of gene expression that radiate outwards in S. coelicolor colonies. We also show that the spatiotemporal separation of these cell types depends on a key activator in the developmental pathway, AdpA. Importantly, when we manipulated local gradients by growing competing microbes nearby, or through physical disruption, expression in these pathways could be decoupled and/or disordered, respectively. Finally, the normal spatial organization of these cell types was partially restored with the addition of a siderophore, a public good made by these organisms, to the growth medium. Together, these results indicate that spatial divisions of labor in S. coelicolor colonies are determined by a combination of physiological gradients and regulatory network architecture, key factors that also drive patterns of cellular differentiation in multicellular eukaryotic organisms. IMPORTANCE Streptomyces coelicolor is a multicellular bacterium that differentiates into specialized cell types and produces a diverse array of natural products. While much is known about the genetic networks that regulate development and antibiotic biosynthesis in S. coelicolor, what drives the spatial organization of these activities within a colony remains to be explored. By using time-lapse microscopy to monitor gene expression in developmental and antibiotic biosynthesis pathways, we found that expression in these pathways occurs in spatiotemporally separated waves. Normally, expression of the antibiotic biosynthesis pathway preceded expression in the developmental pathway; however, this order was compromised in a mutant lacking a key developmental regulator. Furthermore, when we disrupted the local gradients during S. coelicolor growth, we observed disordered patterns of gene expression within colonies. Together, these results indicate that spatial divisions of labor in S. coelicolor colonies are determined by a combination of regulatory network architecture and physiological gradients.

High Resolution Imaging Mass Spectrometry of Bacterial Microcolonies at Ecological Scales

Microbes interact with the world around them at the chemical level. However, directly examining the chemical exchange between microbes, and microbes and their environment, at ecological scales, i.e. the scale of a single bacterial cell or small groups of cells, remains a key challenge. Here we address this obstacle by presenting a methodology that enables Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) of bacterial microcolonies. By combining optimized sample preparation with sub-atmospheric pressure MALDI, we demonstrate that chemical output from groups of as few as ~50 cells can be visualized with MALDI-IMS. Application of this methodology to Bacillus subtilis and Streptomyces coelicolor revealed heterogeneity in chemical output across microcolonies, and asymmetrical metabolite production when cells grew within physiological gradients produced by Medicago sativa roots. Taken together, these results indicate that MALDI-IMS can readily visualize metabolites made by very small assemblages of bacterial cells, and that even these small groups of cells can differentially produce metabolites in response to local chemical gradients.

Regulatory interaction of ATP Na+ and Cl- in the turnover cycle of the NaK2Cl cotransporter.

To probe the mechanism by which intracellular ATP, Na+, and Cl- influence the activity of the NaK2Cl cotransporter, we measured bumetanide-sensitive (BS) 86Rb fluxes in the osteosarcoma cell line UMR-106-01. Under physiological gradients of Na+, K+, and Cl-, depleting cellular ATP by incubation with deoxyglucose and antimycin A (DOG/AA) for 20 min at 37 degrees C reduced BS 86Rb uptake from 6 to 1 nmol/mg protein per min. Similar incubation with 0.5 mM ouabain to inhibit the Na+ pump had no effect on the uptake, excluding the possibility that DOG/AA inhibited the uptake by modifying the cellular Na+ and K+ gradients. Loading the cells with Na+ and depleting them of K+ by a 2-3-h incubation with ouabain or DOG/AA increased the rate of BS 86Rb uptake to approximately 12 nmol/mg protein per min. The unidirectional BS 86Rb influx into control cells was approximately 10 times faster than the unidirectional BS 86Rb efflux. On the other hand, at steady state the unidirectional BS 86Rb influx and efflux in ouabain-treated cells were similar, suggesting that most of the BS 86Rb uptake into the ouabain-treated cells is due to K+/K+ exchange. The entire BS 86Rb uptake into ouabain-treated cells was insensitive to depletion of cellular ATP. However, the influx could be converted to ATP-sensitive influx by reducing cellular Cl- and/or Na+ in ouabain-treated cells to impose conditions for net uptake of the ions. The BS 86Rb uptake in ouabain-treated cells required the presence of Na+, K+, and Cl- in the extracellular medium. Thus, loading the cells with Na+ induced rapid 86Rb (K+) influx and efflux which, unlike net uptake, were insensitive to cellular ATP. Therefore, we suggest that ATP regulates a step in the turnover cycle of the cotransporter that is required for net but not K+/K+ exchange fluxes. Depleting control cells of Cl- increased BS 86Rb uptake from medium-containing physiological Na+ and K+ concentrations from 6 to approximately 15 nmol/mg protein per min. The uptake was blocked by depletion of cellular ATP with DOG/AA and required the presence of all three ions in the external medium. Thus, intracellular Cl- appears to influence net uptake by the cotransporter. Depletion of intracellular Na+ was as effective as depletion of Cl- in stimulating BS 86Rb uptake.(ABSTRACT TRUNCATED AT 400 WORDS)

Ionophore-mediated coupling between ion fluxes and amino acid absorption in mouse ascites-tumour cells. Restoration of the physiological gradients of methionine by valinomycin in the absence of adenosine triphosphate

1. Preparations of mouse ascites-tumour cells depleted of ATP and Na+ ions accumulated l-methionine, in the presence of cyanide and deoxyglucose, from a 1mm solution containing 80mequiv. of Na+/l and about 5mequiv. of K+/l. Valinomycin increased, from about 4 to 16, the maximum value of the ratio of the cellular to extracellular concentrations of methionine formed under these conditions without markedly affecting the distributions of Na+ and of K+. Similar observations were made with 2-aminoisobutyrate, glycine and l-leucine. Increasing the extracellular concentration of K+ progressively decreased the accumulation of methionine in the presence of valinomycin. Over the physiological range of ionic gradients, the system behaved as though the absorption of methionine with Na+ was closely coupled to the electrogenic efflux of K+ through the ionophore. The process was insensitive to ouabain and so the sodium pump was probably not involved. 2. The amount of methionine accumulated during energy metabolism was similar to the optimal accumulation in the presence of valinomycin when ATP was lacking. It was also similarly affected by increasing the methionine concentration. 3. A mixture of nigericin and tetrachlorosalicylanilide mimicked the action of valinomycin. The anilide derivative inhibited the absorption of 2-aminoisobutyrate in the presence of valinomycin, but not in its absence. 4. Gramicidin inhibited methionine absorption and caused the preparations to absorb Na+ and lose K+. 5. The observations appear to verify the principle underlying the gradient hypothesis by showing that the tumour cells can efficiently couple the electrochemical gradient of Na+ to the amino acid gradient.

Transplantations de gonades indifférenciées chez l'hermaphrodite simultané Eisenia foetida (Oligochète, Lumbricidae)

Transplantations of undifferentiated gonads in the simultaneous hermaphrodite Eisenia foetida (Oligochaeta, Lumbricidae) leading to evidence of local factors (inductors?) of sexual differentiation The hypothesis of a ♀ sex capable of autodifferentiation and of a ♂ sex depending on an androgen hormone has been verified in several gonochoric or hermaphrodite invertebrates. In order to test the validity of this hypothesis in the hermaphrodite Eisenia foetida we have investigated the influence of the gonads' environment on their differentiation by transplanting undifferentiated gonads, dorsally and ventrally, on different parts of the body. The gonads, taken with the adjacent wall of the body from new-born worms, are grafted on worms of the same age; some grafts do, others do not, retain their nervous system. The control transplantation in which a graft of a given presumptive sex is transplanted in a region of the same sex can lead to inversions. These inversions have a rare and short-lived character in the gonads coming from presumptive testes. They are more frequent and more or less durable in the gonads coming from presumptive ovaries. These results can only be explained by the existence, within the grafts of the two presumptive sexes, of a dominant relationship between one and the other sexual potentiality which can be momentarily inverted by trophic disturbances caused by the transplantation. The heterosexual transplantations, in which a graft of a given presumptive sex is transplanted in the region of the opposite sex, show that the frequency of the inversions is doubled in relation to the frequency obtained in the control transplantations. The inversions which occur in the presumptive testes can be lasting and even permanent. These results lead to the opinion that to the effects of transplantation is added an inductive action from the host, leading to masculinity in the ♂ region and femininity in the ♀ region. The transplantations outside of the sexual ventral regions lead to inversions of which the character and frequency recall those obtained in the control transplantations. They suggest that these regions are devoid of inductive sexualizing power. The very slight variations of frequency of the inversions observed along the antero-posterior and dorso-ventral axes can be explained by the existence of physiological gradients acting on the balance of the graft's own potentialities. The removal of the nervous system in the grafts shows that this system plays a part, probably trophic, in favour of the masculine potentialities of the graft. In conclusion, our experiments lead to the rejection of the hypothesis of the ♀ sex capable of autodifferentiation in Eisenia, in order to adopt that of a sexual balance between the ♂ and ♀ potentialities determining, according to their dominant relationship, the synthesis of ♂ or ♀ ‘inductors’ respectively in the ♂ and ♀ ventral regions. These inductors act in turn on the bipotential sexual cells.