Energy-dispersive X-ray analysis of the extracellular cadmium sulfide crystallites of Klebsiella aerogenes

Klebsiella aerogenes forms electron-dense partieles on the cell surface in response to the presence of cadmium ions in the growth medium. These particles ranged from 20 to 200 nm in size, and quantitative energy dispersive X-ray analysis established that they comprise cadmium and sulfur in a 1:1 ratio. This observation leads to the conclusion that the particles are cadmium sulfide crystallites. A combination of atomic absorption spectroscopy, inductively coupled plasma mass spectrometry, and acid-labile sulfide analysis revealed that the total intracellular and bound extracellular cadmium:sulfur ratio is also 1:1, which suggests that the bulk of the cadmium is fixed as extracellular cadmium sulfide. The tolerance of K. acrogenes to cadmium ions and the formation of the cadmium sulfide crystallites were dependent on the buffer composition of the growth medium. The addition of cadmium ions to phosphate-buffered media resulted in cadmium phosphate precipitates that remove the potentially toxic cadmium ions from the growth medium. Electrondense particles formed on the surfaces of bacteria grown under these conditions were a combination of cadmium sulfide and cadmium phosphates. The specific bacterial growth rate in the exponential phase of batch cultures was not affected by up to 2mM cadmium in Tricine-buffered medium, but formation of cadmium sulfide crystallites was maximal during the stationary phase of batch culture. Cadmium tolerance was much lower (10 to 150 M) in growth media buffered with Tris, Bistris propane, Bes, Tes, or Hepes. These results illustrate the importance of considering medium composition when comparing levels of bacterial cadmium tolerance.Abbreviations EDXA Energy dispersive X-ray analysis - AAS Atomic absorption spectroscopy - TEM Transmission electron microscopy - SEM Scanning electron microscopy - ICP-MS Inductively coupled plasma mass spectrometry - ALSA Acid-labile sulfide analysis     

Cost,expenditure and vulnerability

The handicap principle (HP) stipulates that signal reliability can be maintained if signals are costly to produce. Yet empirical biologists are typically unable to directly measure evolutionary costs, and instead appeal to expenditure (the time, energy and resources associated with signaling behavior) as a sensible proxy. However the link between expenditure and cost is not always as straightforward as proponents of HP assume. We consider signaling interactions where whether the expenditure associated with signaling is converted into an evolutionary cost is in some sense dependent on the behavior of the intended recipient of the signal. We illustrate this with a few empirical examples and demonstrate that on this alternative expenditure to cost mapping the traditional predictions of HP no longer hold. Instead of full information transfer, a partially informative communication system like those uncovered by Wagner (Games 4(2):163–181, 2013) and Zollman et al. (Proc R Soc B 20121878, 2012) is possible.     

Nucleotide Excision Repair Is a Predominant Mechanism for Processing Nitrofurazone-Induced DNA Damage in Escherichia coli

Nitrofurazone is reduced by cellular nitroreductases to form N²-deoxyguanine (N²-dG) adducts that are associated with mutagenesis and lethality. Much attention recently has been given to the role that the highly conserved polymerase IV (Pol IV) family of polymerases plays in tolerating adducts induced by nitrofurazone and other N²-dG-generating agents, yet little is known about how nitrofurazone-induced DNA damage is processed by the cell. In this study, we characterized the genetic repair pathways that contribute to survival and mutagenesis in Escherichia coli cultures grown in the presence of nitrofurazone. We find that nucleotide excision repair is a primary mechanism for processing damage induced by nitrofurazone. The contribution of translesion synthesis to survival was minor compared to that of nucleotide excision repair and depended upon Pol IV. In addition, survival also depended on both the RecF and RecBCD pathways. We also found that nitrofurazone acts as a direct inhibitor of DNA replication at higher concentrations. We show that the direct inhibition of replication by nitrofurazone occurs independently of DNA damage and is reversible once the nitrofurazone is removed. Previous studies that reported nucleotide excision repair mutants that were fully resistant to nitrofurazone used high concentrations of the drug (200 μM) and short exposure times. We demonstrate here that these conditions inhibit replication but are insufficient in duration to induce significant levels of DNA damage.Replication in the presence of DNA damage is thought to produce most of the mutagenesis, genomic rearrangements, and lethality that occur in all cells. UV-induced photoproducts, X-ray-induced strand breaks, psoralen- or cis-platin-interstrand cross-links, oxidized bases from reactive oxygen species, and base depurination are just a few of the structurally distinct challenges that the replication machinery must overcome. It seems likely that the mechanisms that process these lesions will vary depending on the nature of the impediment.While a number of the lesions described above are known to block replication, the events associated with UV-induced damage have been the most extensively characterized. UV irradiation causes the formation of cyclobutane pyrimidine dimers and 6-4 photoproducts in DNA that block the progression of the replication fork (16, 29, 30, 37). Following the arrest of replication at UV-induced damage, RecA and several RecF pathway proteins are required to process the replication fork such that the blocking lesion is removed or bypassed (2, 5, 6, 8-10). Cells lacking either RecA or any of several RecF pathway proteins are hypersensitive to UV-induced damage and fail to recover replication following disruption by the lesions (2, 6, 10). RecBCD is an exonuclease/helicase complex that is involved in repairing double-strand breaks (38). It also is required for resistance to UV-induced damage, although it is not required to process or restore disrupted replication forks, and the substrates it acts upon after UV irradiation currently remain unclear (3, 10, 19).Survival and the ability to resume DNA synthesis following UV-induced damage depend predominantly on the removal of the lesions by nucleotide excision repair (5, 7, 36). Cells deficient in nucleotide excision repair are unable to remove UV-induced DNA lesions and exhibit elevated levels of mutagenesis, strand exchanges, rearrangements, and cell lethality (16, 33, 34). In cases where replication fork processing or lesion repair is prevented, the recovery of replication and survival become entirely dependent on translesion synthesis by DNA polymerase V (Pol V) (6). However, in repair-proficient cells, the contribution of translesion synthesis to recovery and survival is minor and is detected only following UV doses that exceed the repair capacity of the cell (5, 6).Less is known about how replication recovers from other forms of DNA damage. We chose to characterize nitrofurazone, because a number of studies suggested that N²-deoxyguanine (N²-dG) adducts induced by this and other agents would be processed differently than UV-induced lesions. Nitrofurazone is a topical antibacterial agent that historically has been used for treating burns and skin grafts in patients and animals (14, 15, 32). Nitrofurazone toxicity is known to require activation by cellular nitroreductases (25, 42). However, the mechanism and targets of its antimicrobial properties have yet to be fully elucidated. In addition to its antimicrobial properties, the reduced nitrofurazone metabolites also target DNA and have been shown to induce free radical damage, strand breaks, and N²-dG adducts (26, 40, 42, 45), and they are mutagenic and carcinogenic in rodent models (1, 15, 24, 39).Whereas nucleotide excision repair is the predominant mechanism required for survival after UV-induced damage, a number of studies suggest that translesion synthesis plays a larger role in survival after nitrofurazone-induced DNA damage. dinB mutants lacking Pol IV were shown to be hypersensitive to nitrofurazone compared to cells that constitutively express the polymerase (17). Biochemically, Pol IV and a number of Pol IV homologs from other organisms have been shown to efficiently replicate over a range of N²-dG adducts in vitro (17, 35, 44). In addition, several studies have reported that uvrA mutants, which are defective in nucleotide excision repair, do not exhibit any hypersensitivity to nitrofurazone or other agents that induce similar adducts in vivo (12, 21, 27). Early studies also observed a direct correlation between nitrofurazone-induced mutations and lethality, suggesting that mutagenic lesions persist in the DNA to cause toxicity (21, 23, 27, 43). Consistent with these observations, nitrofuran-induced lesions were found to be poor substrates for nucleotide excision repair in vitro (46).Taken together, these observations suggest to us that the cellular response to nitrofurazone will be distinct from its response to UV irradiation. However, no study has examined the relative contributions that nucleotide excision repair, translesion synthesis, or recombination has in recovering from nitrofurazone-induced damage. In this study, we characterized the mechanism by which nitrofurazone inhibits DNA replication and identified the genes that contribute to the recovery, survival, and mutagenesis of Escherichia coli treated with nitrofurazone. In contrast to previous studies, we found that survival following nitrofurazone-induced damage depends predominantly on nucleotide excision repair. Similarly to UV-induced DNA damage, both the RecF and RecBC pathways contribute to survival following nitrofurazone-induced DNA damage. The contribution of translesion polymerases to survival was minor and was mediated by Pol IV. In addition, we found that nitrofurazone can act to inhibit DNA replication directly when used at higher concentrations. The direct inhibition of replication is reversible and occurs independently of DNA damage, suggesting that DNA is not the primary target of its antimicrobial properties.     

Centrosome to autophagosome signaling: Specific GABARAP regulation by centriolar satellites

Yeast have one Atg8 protein; however, multiple Atg8 orthologs (LC3s and GABARAPs) are found in humans. We discovered that a population of the Atg8 ortholog GABARAP resides on the centrosome and the peri-centrosomal region. This centrosomal pool of GABARAP translocates to forming autophagosomes upon starvation to activate autophagosome formation in a non-hierarchical pathway. How this centrosome-to-phagophore delivery of GABARAP occurs was not understood. To address this, we have shown that the archetypal centriolar satellite protein PCM1 regulates recruitment of GABARAP to the centrosome. PCM1 recruits GABARAP, but not MAP1LC3B, directly to centriolar satellites through a LC3-interacting region (LIR) motif. Furthermore, PCM1, in concert with its interacting centriolar satellite E3 ligase MIB1, controls GABARAP stability, K48-linked ubiquitination and GABARAP-mediated autophagic flux.