Protein Synthesis during Germination: Shedding New Light on a Classical Question

Abstract

Bacterial spores were discovered by Cohn and Koch in the species Bacillus subtilis and Bacillus anthracis and described in two jointly published papers in the year 1876 (1). Among the most striking of their observations was that spores are metabolically dormant and highly resistant. In spite of these features, however, spores can break dormancy extremely rapidly; within minutes of sensing the appropriate stimulus (typically, a small molecule such as an amino acid or a sugar) the spore converts to a vegetative cell and resumes growth and division (2). During the first step in this revival process, known as germination, the dormant spore sheds its protective outer layers, takes up water, and swells. In the second step, called outgrowth, the spore begins active production of new cellular macromolecules. Outgrowth is complete when the cell fully converts to a rod shape; the now fully restored vegetative cell is poised to begin division. This rapid reawakening from the dead (or the near dead) raises a number of intriguing and very deep questions, including (i) how can metabolism be reactivated so quickly after the completion of germination and (ii) do spores really lack all metabolic activity? Or, to phrase it another way, just how dead are spores, anyway?Elucidating the mechanistic basis of germination and outgrowth is one of the longest-running challenges in microbiology; despite nearly 150 years of research, there are still major gaps in our understanding of these processes. Nonetheless, since germination was first observed, a large body of evidence has accumulated documenting that, throughout the course of germination, up until the start of outgrowth, spores are, indeed, quite dormant; no metabolic or biosynthetic processes (until quite recently) have been detected. While these data appeared to exclude significant levels of metabolic activity during germination, an outstanding question has always lingered: could germinating spores possess some degree of physiologically important metabolic activity that is simply too low to detect or for some other reason inaccessible to measurement by the methods used so far?This issue was addressed in a landmark study in 2015 by Sinai et al. (3). These authors showed, first, that protein synthesis occurs prior to the completion of germination in two ways: (i) by a novel biochemical approach (bio-orthogonal noncanonical amino acid tagging [BONCAT]) that specifically identifies newly synthesized proteins and (ii) by monitoring the timing, during germination, of the appearance of fluorescently tagged versions of some of the proteins discovered by BONCAT. Second, and more directly relevant to our discussion here, these authors argued that protein synthesis is required for successful germination by demonstrating that (i) protein synthesis inhibitors (specifically, the antibiotics tetracycline and lincomycin) prevent germination (cleverly overcoming the limitations of previous experiments attempting to use antibiotics in a similar manner, by increasing spore permeability during antib...

Towards the minimal proteome of life: Proteome profiles ofBacillus subtilisvegetative cells and spores

“…To jump start the germination, the macromolecules needed are pre-synthesized during sporulation 4 . Even though the spores are metabolically dormant 5 researchers are still in pursuit of finding the exact molecular, biochemical and biophysical mechanisms behind the germination process [6][7][8] . Despite numerous indications from genetic studies, the existing hypotheses on the molecular basis of spore revival remains, at the mechanistic level, unanswered 8 .…”

“…Even though the spores are metabolically dormant 5 researchers are still in pursuit of finding the exact molecular, biochemical and biophysical mechanisms behind the germination process [6][7][8] . Despite numerous indications from genetic studies, the existing hypotheses on the molecular basis of spore revival remains, at the mechanistic level, unanswered 8 . Processes such as water uptake, dipicolinic acid (DPA) release, cortex hydrolysis and signal transduction therein are still not completely understood [5][6][7] .…”

Cationic Amphipathic Antimicrobial Peptides Perturb the Inner Membrane of Germinated Spores Thus Inhibiting Their Outgrowth

“…Germination of spores leads to the release of CaDPA, cortex hydrolysis, and rehydration of the spore core causing swelling, followed by the increase in mobility of the macromolecular components and that of the inner membrane ( Setlow, 2003 ; Setlow et al, 2017 ). Protein synthesis has also been reported to occur during germination, but it still remains a subject of debate ( Sinai et al, 2015 ; Boone and Driks, 2016 ; Korza et al, 2016 ). The spore inner membrane is remodeled, expands, and become permeable ( Setlow et al, 2017 ).…”

Integrative analysis of proteome and transcriptome dynamics duringBacillus subtilisspore revival

“…A study monitoring the temporal expression of mRNA during spore germination and outgrowth has demonstrated the systematic onset of transcription 1,4 whereas a detailed analysis of reviving spores has provided information on newly synthesized proteins of Bacillus subtilis spores including a putative set of enzymes that are crucial for the onset of protein synthesis 3,10 . One particular aspect of spore revival, whether protein synthesis is required for germination to occur or not, is topic of much recent debate 9,11,12 .…”