For calculating Wald test p-values, the coefficients were scaled by their standard errors and then compared to a standard Normal distribution. rise to the macroscopic fruiting bodies composed of at least two cell types, myxospores and peripheral rods [2]. Within the fruiting bodies, rod-shaped cells differentiate into spherical myxospores that are metabolically inactive and resistant to harsh environments [2]. Peripheral rods are ostensibly less drastic as the cells do not enter dormancy or appear to change strikingly morphologically [2,5]. Peripheral rods remain metabolically active outside of the fruiting bodies [5C7]. When nutrients become readily available, both cell types respond to the stimuli by returning to a vegetative state, albeit, peripheral rods respond more quickly than myxospores, which must undergo germination [7]. In the multicellular development of [2]. However, stationary cells exhibit similar characteristics to TC-E 5006 peripheral rods. During the transition from exponential growth to the stationary phase, a number of morphological and physiological changes take place. The composition of the cellular envelope is altered and a series of stress-related genes is upregulated prior to or upon entering stasis [8,11,12]. As with stationary phase cells, there have been limited analyses of peripheral rods. However, there are perceivable similarities between the two cell types. Peripheral rod cells have been shown to alter their cell wall, and sigma factors (e.g. SigD) are upregulated in a manner vital to development [11C14]. Peripheral rods also possess a single chromosome and Hapln1 maintain a rod-shaped morphology, characteristics found in stationary cells. TC-E 5006 Due to the similarities, we address the distinction of peripheral rods as a differentiated cell type through a comparative analysis [15]. The study focuses on cell structure and response signaling induced by environmental stresses. Moreover, the use of Next Generation Sequencing (NGS) provides an in-depth look at the transcriptomic profile of cell types. We demonstrate that the expression patterns of the peripheral rods are different from any other cell type observed. This study also gives insight into the possible origin and developmental pathway of peripheral rods. 2.?Materials and methods 2.1. Bacterial strains, growth, and media All strains used are derivatives of the wild-type strain DK1622. strains were grown in CTTYE 1% casitone (Difco, Franklin Lakes, NJ), 10 mM Tris-HCl (pH 7.6), 1 mM KH2PO4, 8 mM MgSO4) broth or on CTTYE plates containing 1% agar. Stationary cells were passaged three times before being collected at a Klett value of 230. Low nutrient cells were grown in 0.08% CTTYE following an established protocol [16]. 2.2. Microscopy Phase contrast microscopy was used to visualize and photograph cells. Nikon Eclipse 80i light microscope with 100 oil immersion objective and 10X ocular along with a Q-Imaging MicroPublisher 3.3 RTV camera were used to image cells. 2.3. Development Development was induced either TC-E 5006 with a submerged liquid culture buffer system [1,16] or on TPM agar plates (10 mM Tris [pH 7.6], 8 mM MgSO4, and 1 mM KH2PO4 containing 1.5% agar). Cells developed in a humidity chamber at 33C. Cells were harvested and quick-frozen in liquid nitrogen [16]. 2.4. Purification of peripheral rods Peripheral rods were purified from myxospores in the fruiting body by using an adaptation of previous protocols [5,15]. Fruiting bodies were removed from developmental plates after four days. Cells were scraped from TPM agar with a spatula and suspended in 1 ml of 10 mM sodium phosphate, pH 7.2. This resuspension was then applied to a sucrose step gradient with levels of 60%, 30%, 15%, and 5% sucrose in 10 mM sodium phosphate, pH 7.2. Samples were subjected to centrifugation at 400 for 15 min in an HB-4 rotor. The 5% sucrose fraction contains rods, and the 30C60% sucrose fractions contain myxospores. The purity of the peripheral rod samples was verified using microscopy. 2.5. RNA isolation, integrity, and quality assessment Total RNA was extracted from N2 snap-frozen cells.