The Molecular Timeline of a Reviving Bacterial Spore

Sinai, Lior; Rosenberg, Alex; Smith, Yoav; Segev, Einat; Ben-Yehuda, Sigal

doi:10.1016/j.molcel.2014.12.019

Cited by 116 publications

(183 citation statements)

References 44 publications

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“…We previously reported that the doubling time of the ⌬hpf mutant does not significantly differ from that of the WT (43 and 40 min, respectively) (21), and time-lapse microscopy studies failed to provide evidence that the ⌬hpf mutant continues to divide during stationary phase (data not shown). Previous work has demonstrated that bacteria can remain metabolically active without dividing (42,61,62). Taken together, these data suggest that hibernating ribosomes do not influence cell division but probably do have effects on other cellular activities.…”

Section: Discussionsupporting

confidence: 61%

Ribosome Hibernation Facilitates Tolerance of Stationary-Phase Bacteria to Aminoglycosides

McKay

Portnoy

2015

Antimicrob Agents Chemother

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Upon entry into stationary phase, bacteria dimerize 70S ribosomes into translationally inactive 100S particles by a process called ribosome hibernation. Previously, we reported that the hibernation-promoting factor (HPF) of Listeria monocytogenes is required for 100S particle formation and facilitates adaptation to a number of stresses. Here, we demonstrate that HPF is required for the high tolerance of stationary-phase cultures to aminoglycosides but not to beta-lactam or quinolone antibiotics. The sensitivity of a ⌬hpf mutant to gentamicin was suppressed by the bacteriostatic antibiotics chloramphenicol and rifampin, which inhibit translation and transcription, respectively. Disruption of the proton motive force by the ionophore carbonyl cyanide m-chlorophenylhydrazone or mutation of genes involved in respiration also suppressed the sensitivity of the ⌬hpf mutant. Accordingly, ⌬hpf mutants had aberrantly high levels of ATP and reducing equivalents during prolonged stationary phase. Analysis of bacterial uptake of fluorescently labeled gentamicin demonstrated that the ⌬hpf mutant harbored increased intracellular levels of the drug. Finally, deletion of the main ribosome hibernation factor of Escherichia coli, ribosome modulation factor (rmf), rendered these bacteria susceptible to gentamicin. Taken together, these data suggest that HPF-mediated ribosome hibernation results in repression of the metabolic activity that underlies aminoglycoside tolerance. HPF is conserved in nearly every bacterial pathogen, and the role of ribosome hibernation in antibiotic tolerance may have clinical implications. Listeria monocytogenes is a Gram-positive, facultative intracellular pathogen that poses serious medical risks for pregnant women, immunocompromised individuals, and infants (1, 2). The significance of L. monocytogenes as a foodborne pathogen was recently highlighted when contaminated cantaloupes caused the deadliest U.S. foodborne disease outbreak since 1924 (3). This bacterium is hardy and able to survive in a variety of environments, including soil, groundwater, and processed meats and cheeses, and within mammalian cells (2, 4-6). L. monocytogenes can also form biofilms that can persist for years in food processing plants, despite repeated exposure to disinfectants (7,8). Persistent L. monocytogenes contamination of food processing plants remains a significant source of foodborne illness (9-11). Understanding the molecular mechanisms that allow L. monocytogenes to survive and persist in harsh environments is crucial for developing strategies to ensure food safety.Ribosome dimerization is a highly conserved yet relatively underappreciated arm of the classic ribosome cycle. During stress, bacteria are able to dimerize 70S ribosomes into inactive hibernating 100S ribosome dimers. These dimers lack mRNA and tRNA and have been shown to be translationally silent in vitro (12, 13). Upon introduction of fresh nutrients, 100S ribosomes are immediately dissociated into 70S ribosomes and available for translation (14-16). Thus,...

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Section: Discussionsupporting

confidence: 61%

Ribosome Hibernation Facilitates Tolerance of Stationary-Phase Bacteria to Aminoglycosides

McKay

Portnoy

2015

Antimicrob Agents Chemother

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“…While wild-type spores treated for ϳ20 h at 75 to 80°C retained almost normal viability even though almost all of their rRNA was lost, obviously rRNA must be synthesized in order for spores to grow out, since there is much protein synthesis in this period of spore development (13,(31)(32)(33)(34). To compare these wildtype spores' outgrowth kinetics, spores left untreated and spores treated at 75 or 80°C were incubated in medium that promoted spore germination, outgrowth, and subsequent vegetative growth and these processes were monitored by measuring the OD 600 of the incubated spores (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In spite of the evidence noted above, most of which is from work published 30 to 40 years ago, there are recent reports suggesting that metabolic activity does take place in dormant spores, including RNA synthesis and degradation, and that some protein synthesis takes place in and is required for the process of spore germination (12,13). Equally importantly, one of these reports suggested that these events in dormant spores could modify their germination properties significantly, in particular when spores were incubated for extended times at 37 to 50°C, leading to degradation of much of spores' rRNA (12).…”

mentioning

confidence: 99%

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

et al. 2016

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ABSTRACTrRNAs of dormant spores of Bacillus subtilis were >95% degraded during extended incubation at 50°C, as reported previously (E. Segev, Y. Smith, and S. Ben-Yehuda, Cell 148:139 -114, 2012, doi:http://dx.doi.org/10.1016/j.cell.2011.11.059), and this was also true of spores of Bacillus megaterium. Incubation of spores of these two species for ϳ20 h at 75 to 80°C also resulted in the degradation of all or the great majority of the 23S and 16S rRNAs, although this rRNA degradation was slower than nonenzymatic hydrolysis of purified rRNAs at these temperatures. This rRNA degradation at high temperature generated almost exclusively oligonucleotides with minimal levels of mononucleotides. RNase Y, suggested to be involved in rRNA hydrolysis during B. subtilis spore incubation at 50°C, did not play a role in B. subtilis spore rRNA breakdown at 80°C. Twenty hours of incubation of Bacillus spores at 70°C also decreased the already minimal levels of ATP in dormant spores 10-to 30-fold, to <0.01% of the total free adenine nucleotide levels. Spores depleted of rRNA were viable and germinated relatively normally, often even faster than starting spores. Their return to vegetative growth was also similar to that of untreated spores for B. megaterium spores and slower for heat-treated B. subtilis spores; accumulation of rRNA took place only after completion of spore germination. These findings thus strongly suggest that protein synthesis is not essential for Bacillus spore germination. Spores of Bacillus species formed in sporulation are metabolically dormant and extremely resistant to many severe treatments, including heat, radiation, and toxic chemicals (1-3). Although these spores can survive in this dormant resistant state for many years, if given the appropriate stimulus, generally small molecules that are found in environments that are appropriate for the growth of these bacteria, the spores can rapidly break dormancy in the process of spore germination and then outgrowth (1, 4, 5). Notably, spores' extreme resistance properties are lost in the process of germination and early outgrowth, and thus, germinated spores are much easier to kill than dormant spores. This property has significant applied implications, as dormant spores are the vectors for much food spoilage, as well as some foodborne and other human diseases. Consequently, there is significant interest in mechanisms that determine rates of spore germination and how sporulation conditions, as well as storage conditions, modify spores' resistance properties and their rates of germination.In addition to the applied interest in spore resistance and germination, there has also long been much basic interest in the mechanism of spore germination, in particular, the signal transduction pathways operating during the process (4, 5). The major proteins involved in Bacillus spore germination are as follows. (i) Germinant receptors (GRs) in spores' inner membrane (IM) recognize and respond to the physiological germinants specific for spores of each species/strain. (ii)...

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“…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.…”

mentioning

confidence: 99%

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

Boone

Driks

2016

J Bacteriol

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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...

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The Molecular Timeline of a Reviving Bacterial Spore

Cited by 116 publications

References 44 publications

Ribosome Hibernation Facilitates Tolerance of Stationary-Phase Bacteria to Aminoglycosides

Ribosome Hibernation Facilitates Tolerance of Stationary-Phase Bacteria to Aminoglycosides

Changes in Bacillus Spore Small Molecules, rRNA, Germination, and Outgrowth after Extended Sublethal Exposure to Various Temperatures: Evidence that Protein Synthesis Is Not Essential for Spore Germination

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

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