excluded wetlands, for which coarser estimates are available3. The resulting carbon transfer from terrestrial systems to inland waters there-fore amounts to 5.7 Pg per year2,3, substantially higher than was previously thought (Fig. 1).
Where is all this carbon coming from? There are three main possibilities: soil res-piration, which increases inorganic carbon concentrations in groundwater; soil erosion, which transports organic-rich particles into streams; and the entry of a substantial amount of dead biomass into water courses in forested and wetland systems7. At present, it is not clear which source contributes most to car-bon transfer from the land to surface waters. Researchers are therefore using advanced methods of organic and isotope geochemistry to disentangle these terrestrial carbon sources and to discriminate between aquatic and terrestrial organic matter8.
The CO2 emissions predicted by Raymond and co-workers’ analysis are largest from tropical rivers and lakes in southeast Asia and Amazonia. Because tropical regions are seri-ously under-represented in global data sets, additional studies of carbon concentrations in the predicted hotspot areas in the tropics are urgently needed. Efforts to constrain data for global emissions of methane — a potent greenhouse gas — from inland waters are also a high priority9.
Two other fundamental issues need to be addressed in future work. First, gas transfer along river networks can be dominated by high emission rates at local discontinuities, such as weirs, rapids, waterfalls or turbine releases in hydropower plants9. It is therefore
questionable whether a continuous model for gas-transfer velocities based on large-scale geographical parameters, such as that used by Raymond et al., represents the most adequate description of the gas-transfer process.
The other issue concerns the heavy modifi-cations that have been made to surface water systems during the past two centuries by chan-nelization and damming. For example, cutting off and draining the wetlands of the lower Danube has reduced the seasonally flooded area of the river corridor by 72%, resulting in an artificial river morphology that cannot be predicted by geographical scaling laws10. Raymond and colleagues’ surprising results call for more specific investigations of how hydraulic constructions in river systems affect global biogeochemical cycles. ■
Bernhard Wehrli is at the Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, 8092 Zurich, Switzerland, and at the Swiss Federal Institute of Aquatic Science and Technology, Kastanienbaum. e-mail: [email protected]
1. Raymond, P. A. et al. Nature 503, 355–359 (2013).2. Battin, T. J. et al. Nature Geosci. 2, 598–600 (2009). 3. Aufdenkampe, A. K. et al. Front. Ecol. Environ. 9,
53–60 (2011).4. Stumm, W. & Morgan, J. J. Aquatic Chemistry:
Chemical Equilibria and Rates in Natural Waters 3rd edn (Wiley, 1995).
5. Raymond, P. A. et al. Limnol. Oceanogr. Fluids Environ. 2, 41–53 (2012).
6. Downing, J. A. et al. Limnol. Oceanogr. 51, 2388–2397 (2006).
7. Zurbrügg, R., Suter, S., Lehmann, M. F., Wehrli, B. & Senn, D. B. Biogeosciences 10, 23–38 (2013).
8. Feng, X. et al. Proc. Natl Acad. Sci. USA 110, 14168–14173 (2013).
9. Abril, G. et al. Glob. Biogeochem. Cycles 19, GB4007 (2005).
10. Tockner, K., Uehlinger, U. & Robinson, C. T. Rivers of Europe (Elsevier, 2009).
A N T I B I O T I C S
Killing the survivors Antibiotic-tolerant, dormant variants of otherwise antibiotic-sensitive bacteria underlie many chronic and relapsing infections. A small molecule has been identified that can efficiently eradicate these persister cells. See Article p.365
K E N N G E R D E S & H A N N E I N G M E R
It is well documented that the incidence of pathogenic antibiotic-resistant bac-teria is increasing at an alarming pace. But
it is less well known that almost all bacteria, including major pathogens, generate persisters — slow-growing or hibernating cells that are tolerant to multiple antibiotics. Importantly, these variants form at frequencies higher than mutation rates and, consistent with this, seem to be genetically identical to the antibiotic- sensitive organism. Persister cells are a primary source of chronic and relapsing bacterial infec-tions1 because they are difficult or impossible to eradicate using conventional antibiotics. There is therefore a pressing need to develop treatments that can kill persisters. On page 365
of this issue, Conlon et al.2 present evidence that acyldepsipeptides, an emerging class of antibiotic, efficiently kill persisters of certain types of bacterium. Remarkably, when used in conjunction with conventional antibiotics, one of these agents completely eliminated the bacterium Staphylo coccus aureus growing in culture, and also cured an S. aureus infection in mice.
The phenomenon of persistence was discov-ered almost 70 years ago, but only recently3,4 was strong support obtained for the hypothesis that persisters are rare, slow-growing, bacter-ial cells genetically identical to the rest of the population (typically occurring at a frequency of 1 in 10,000 to 1 in 1 million cells in a rapidly growing population). Phenotypic heterogen-eity (in which genetically identical cells exhibit
Figure 1 | Inland waters in the global carbon cycle. The numbers above the rising arrows indicate carbon dioxide emissions from inland waters in petagrams of carbon per year (1 Pg is 109 tonnes), including data for rivers and lakes now reported by Raymond et al.1 and for emissions from wetlands3. Also shown are data for the export of dissolved carbon by rivers to the ocean2 (dark blue arrow) and the amount of organic carbon stored in sediments and wetland soils2 (descending arrows). The numbers indicate that a substantial fraction of the carbon fixed by terrestrial vegetation must be laterally exported from land into surface waters (green arrow), which affects regional carbon budgets on land. (Figure adapted from ref. 3.)
Land
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different traits) among bacteria can be viewed as a bet-hedging strategy that has evolved to increase survival under environmental insults that could otherwise eradicate the entire population5.
The common signalling molecule guano-sine tetraphosphate (ppGpp; also called ‘magic spot’) can render bacterial cells persistent, and is synthesized either by stochastic switching (as in the bet-hedging strategy)4 or in response to environmental stress6. But because most exist-ing antibiotics target actively growing bacteria, they do not kill these magic-spot-protected persisters.
Acyldepsipeptides (ADEPs) efficiently kill Gram-positive bacteria (a broad bacterial group that includes several human patho-gens, including S. aureus) at astonishingly low concentrations, and are effective against cells that are resistant to many other antibiot-ics7. Bacterial killing by ADEPs is the result of uncontrolled activation of a protein called ClpP (ref. 7), which is a subunit of the protease enzyme Clp. This enzyme is found in all bac-teria and it functions in protein-quality control and in the regulated degradation of specific proteins. The protein-degrading subunit of Clp consists of two heptameric ClpP rings with small central pores through which peptide
chains are threaded into the central proteo-lytic chamber, after being unfolded by ClpP- associated ATPase enzymes8,9. (The small size of the pore explains why ClpP is largely inactive without these protein-unfolding enzymes.) Structural studies have revealed that ADEPs bind directly to ClpP, independently of the ATPases, and dramatically increase the size of the central pore in ClpP (Fig. 1a). This provides unregulated access for peptides and proteins to the proteolytic chamber10,11, and the resulting increase in protein degradation leads to cell death.
Conlon et al. studied non-growing S. aureus cells reminiscent of the persisters formed dur-ing infection, and found that application of the compound ADEP4 caused the degradation of more than 400 bacterial proteins. This obser-vation led to the straightforward and easily tested hypothesis that ADEP4 would kill per-sisters, and the authors found that the drug efficiently killed non-growing, stationary-phase bacteria. Remarkably, in combination with a conventional antibiotic (rifampicin, linezolid or ciprofloxacin) that does not kill persisters when administered alone, ADEP4 totally eradicated persisters from a flask cul-ture (Fig. 1b), from a biofilm (a distinct form of aggregated bacteria growing on a surface)
and from an in vitro hollow-fibre model used for assessing the efficacy of antibiotics. Even more strikingly, the authors found that ADEP4 eradicated severe, deep-seated S. aureus infections in the thighs of mice.
These results raise several key questions. First, will bacteria develop resistance to ADEPs and other compounds that kill bacteria by acti-vating bacterial proteases? Bacteria almost always rapidly become resistant to new anti-biotics, so it is to be expected that this will also be the case with ADEPs. But general proteases such as Clp are typically either essential for bacterial survival or are required for bacterial virulence. Therefore, if spontaneous S. aureus mutants arise that lack ClpP and are resistant to ADEPs, they will display greatly reduced virulence9. Second, will the breakthrough pre-sented by Conlon and colleagues lead to the development of more-effective antibiotics for treating relapsing and chronic infections? We believe that this is probable. Most antibiotics that actively kill bacteria do so by corrupting a cellular target that is particularly active dur-ing bacterial growth, whereas ADEPs activate their cellular target whether the bacteria are growing or not. Unsurprisingly, the research group is now testing a second class of antibiotic that activates ClpP (ref. 12). This growing body of results generates hope that antibiotics for the treatment of persistent infections will be available in the future. ■
Kenn Gerdes is at the Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Newcastle upon Tyne NE2 4AX, UK, and in the Department of Biology, University of Copenhagen, Denmark. Hanne Ingmer is in the Department of Veterinary Disease Biology, University of Copenhagen, 1870 Frederiksberg, Denmark.e-mails: [email protected]; [email protected]
1. Lewis, K. Annu. Rev. Microbiol. 64, 357–372 (2010).2. Conlon, B. P. et al. Nature 503, 365–370 (2013).3. Balaban, N. Q., Merrin, J., Chait, R., Kowalik, L. &
Leibler, S. Science 305, 1622–1625 (2004).4. Maisonneuve, E., Castro-Camargo, M. & Gerdes, K.
Cell 154, 1140–1150 (2013).5. Veening, J. W., Smits, W. K. & Kuipers, O. P. Annu.
Rev. Microbiol. 62, 193–210 (2008).6. Dalebroux, Z. D. & Swanson, M. S. Nature Rev.
Microbiol. 10, 203–212 (2012).7. Brötz-Oesterhelt, H. et al. Nature Med. 11,
1082–1087 (2005).8. Baker, T. A. & Sauer, R. T. Biochim. Biophys. Acta
1823, 15–28 (2012).9. Frees, D., Savijoki, K., Varmanen, P. & Ingmer, H. Mol.
Microbiol. 63, 1285–1295 (2007).10. Li, D. H. et al. Chem. Biol. 17, 959–969 (2010).11. Lee, B. G. et al. Nature Struct. Mol. Biol. 17, 471–478
(2010).12. Leung, E. et al. Chem. Biol. 18, 1167–1178
(2011).13. Sowole, M. A., Alexopoulos, J. A., Cheng, Y. Q.,
Ortega, J. & Konermann, L. J. Mol. Biol. 425, 4508–4519 (2013).
This article was published online on 13 November 2013.
Antibiotic A Antibiotics A + B Antibiotic A + ADEP4
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Figure 1 | ADEPs activate ClpP and kill persister bacteria. a, Clp is a bacterial protein-degrading enzyme. Its proteolytic subunit ClpP forms a ring-shaped barrel containing a small pore (amino acids lining the pore are shown in red). The pore is normally gated by ClpP-associated ATPase enzymes, which control the entry of protein substrates into the ClpP chamber. Acyldepsipeptide (ADEP) molecules (purple) that bind to the ATPase docking sites on ClpP cause a conformational change that widens the pore, leading to deregulated protein degradation. (Structures provided by Lars Konermann13.) b, Conlon et al.2 show that the combination of ADEP4 and a conventional antibiotic kills non-growing persister cells — the small, slow-growing population of cells that persist during treatment with conventional antibiotics (alone or in combination).
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