Search

The emergence of new resistant bacterial strains is a worldwide challenge. A resistant bacterial population can emerge from a single cell that acquires resistance or persistence. Hence, new ways of tackling the mechanism of antibiotic response, such as single cell studies are required. It is necessary to see what happens at the single cell level, in order to understand what happens at the population level. To date, neither bulk nor single-cell methods are able to link the heterogeneity of single-cell susceptibility to the population-scale response to antibiotics. Here, we present a platform that measures the ability of individual E.coli cells to form small colonies at different ciprofloxacin concentrations, by using anchored microfluidic drops and an image and data analysis pipelines. The microfluidic results are benchmarked against classical microbiology measurements of antibiotic susceptibility, showing an agreement between the pooled microfluidicchip and replated bulk measurements. Further, the experimental likelihood of a single cell to form a colony is used to provide a probabilistic antibiotic susceptibility curve. In addition to the probabilistic viewpoint, the microfluidic format enables the characterization of morphological features over time for a large number of individual cells. This pipeline can be used to compare the response of different bacterial strains to antibiotics with different action mechanisms.

The integron is a bacterial recombination system that allows acquisition, stockpiling and expression of promoterless genes embedded in cassettes. Some integrons, like the one found in the second chromosome ofVibrio cholerae, can be particularly massive, gathering hundreds of non-expressed cassettes. It is unclear how such genetic structures can be stabilized in bacterial genomes. Here, we reveal that the orientation of integrons toward replication within bacterial chromosomes is essential to their stability. Indeed, we show that upon inversion of theV. choleraechromosomal integron, the cassette excision rate of its cassettes is dramatically increased. This correlates with a strong growth defect which we show is mostly due to the excision of a remarkable type of cassettes bearing their own promoter and encoding toxin– antitoxin systems. This “abortive excision” of toxin–antitoxin systems can prevent the inversion of chromosomal integrons and the associated extensive loss of cassettes. Our analysis of the available sedentary chromosomal integrons in genome database show an over-representation of toxin–antitoxin cassettes in large integrons. This study thus provides a striking example of the relationship between genome organization, genome stability and an emerging property of toxin–antitoxin systems.

The repABC replication/partitioning loci are widespread in extrachromosomal replicons of Alphaproteobacteria . They are evolutionarily diverse, subject to multi-layer self-regulation, and are responsible for the maintenance of different types of replicons, such as plasmids (e.g., Agrobacterium pTi and pRi tumorigenic and rhizogenic plasmids), megaplasmids (e.g., Sinorhizobium pSymA and pSymB) and essential chromids (e.g., secondary chromosomes of Agrobacterium , Brucella and Rhodobacter ).

A realistic numerical model is used to study the circulation and mixing of the Salish Sea, a large, complex estuarine system on the United States and Canadian west coast. The Salish Sea is biologically productive and supports many important fisheries but is threatened by recurrent hypoxia and ocean acidification, so a clear understanding of its circulation patterns and residence times is of value. The estuarine exchange flow is quantified at 39 sections over 3 years (2017–2019) using the Total Exchange Flow method. Vertical mixing in the 37 segments between sections is quantified as opposing vertical transports: the efflux and reflux. Efflux refers to the rate at which deep, landward-flowing water is mixed up to become part of the shallow, seaward-flowing layer. Similarly, reflux refers to the rate at which upper layer water is mixed down to form part of the landward inflow. These horizontal and vertical transports are used to create a box model to explore residence times in a number of different sub-volumes, seasons, and years. Residence times from the box model are generally found to be longer than those based on simpler calculations of flushing time. The longer residence times are partly due to reflux, and partly due to incomplete tracer homogenization in sub-volumes. The methods presented here are broadly applicable to other estuaries.

The Washington State Department of Ecology conducted a large-scale ocean acidification (OA) study in greater Puget Sound to: (1) produce a marine carbon dioxide (CO2) system dataset capable of distinguishing between long-term anthropogenic changes and natural variability, (2) characterize how rivers and freshwater drive OA conditions in the region, and (3) understand the relative influence of cumulative anthropogenic forcing on regional OA conditions. Marine CO2 system data were collected monthly at 20 stations between October 2018 and February 2020. While additional data are still needed, the climate-level data collected thus far have uncovered novel insights into spatiotemporal distributions of and variability in the regional marine CO2 system, especially at low salinities in shallow, river-forced shelf regions. The data provide a strong foundation with which to continue monitoring OA conditions across the region. More importantly, this work represents the first successful long-term OA monitoring program undertaken at the state-level by a regulatory agency. Therefore, we offer the work described herein as a blueprint to help state and local scientists and environmental and natural resource managers develop, implement, and conduct long-term OA monitoring programs and studies in their own contexts and jurisdictions.

Metastatic cancer is incurable because tumors evolve resistance to all classes of therapy, resulting the in the deaths of 10 million people each year. Therapy resistance is typically attributed to the presence of a cancer stem cell or genetic tumor cell heterogeneity. However, therapeutic resistance can also emerge through a transient cell state: the Polyaneuploid Cancer Cell (PACC) state. The Polyaneuploid transition (PAT) is an evolutionary conserved polyploid program accessed by cancer cells in response to an external stress such as chemotherapy. Once the stress subsides, cells exit the PACC state and repopulate a non-polyploid population that is resistant to multiple classes of therapy. We hypothesize that the PAT facilitates inherited universal therapeutic resistance. We utilized the bacteria E. coli as a model system of the evolutionary conserved polyploid mechanism of resistance. The E. coli strain MG1655 was engineered to express an hupA::GFP reporter to monitor nucleoid DNA or a ibpA::GFP reporter to monitor the aggregation of misfolded proteins. We treated these cultures with ciprofloxacin at near lethal doses for 12 hours and recorded the dynamics with fluorescent time-lapse microscopy. Individual bacteria underwent a transient phase of polyploidization causing an elongated filamentous phenotype that is at least an order of magnitude bigger compared to the unstressed bacteria. During filamentation, we found that the misfolded protein aggregates are shuttled along the cell body to the tips. Eventually, the filaments bud resulting in a population of “typical” length cells. The buds that are produced by the filaments are filled with an excess of misfolded protein aggregate and, surprisingly, are commonly void of DNA. This response is unexpected in a cell that is already resistant due to the high fitness cost. However, if the cell is not resistant, then each misfolded protein may represent an attempt to solve the antibiotic puzzle and to achieve a resistant phenotype. In turn, the ability to efficiently collect and dispose of the misfolded proteins, i.e., the previous failed attempt, may be advantageous to reduce the need for protein degradation and ensure that future protein production is not impaired. Taken together, this finding suggests that antibiotic resistance might be dependent on the ability to endure repeated futile endeavors to explore the phenotypic landscape rather than selecting for individuals that already exist at an evolutionary optimum. As a result, the PAT may act as a buffer by which cancer cells “buy time” to search within the phenotypic landscape and look for a path to resistance. Future work will specifically test this model in cancer cells. If true, then this finding would suggest that therapeutic resistance can be actively delayed by inhibiting the PAT during first line treatment. Citation Format: George Butler, Julia Bos, Robert H. Austin, Sarah R. Amend, Kenneth J. Pienta. The polyaneuploid transition as a hedge against failures in resistance acquisition [abstract]. In: Proceedings of the AACR Special Conference on the Evolutionary Dynamics in Carcinogenesis and Response to Therapy; 2022 Mar 14-17. Philadelphia (PA): AACR; Cancer Res 2022;82(10 Suppl):Abstract nr B022.

International audience; Bacteria which grow not on the featureless agar plates of the microbiology lab but in the real world mustnavigate topologies which are nontrivially complex, such as mazes or fractals. We show that chemo-sensitive motileE. colican efficiently explore nontrivial mazes in times much shorter than a no-memory(Markovian) walk would predict, and can collectively escape from a fractal topology. The strategies usedby the bacteria include individual power-law probability distribution function exploration, the launching ofchemotactic collective waves with preferential branching at maze nodes and defeating of fractal pumping,and bet hedging in case the more risky attempts to find food fail.

Significance We have discovered an emergent mechanism by which Escherichia coli can escape high-stress regions, such as near-lethal concentrations of antibiotics, by forming long motile helical filaments that are poized at the critical shear buckling point: 2 π twist rotations independent of the length of the filament. All filaments, independent of length, have the same twist, indicating that this is a highly evolved response. The helices do not tumble as nonstressed bacteria do but rather, over a period of tens of seconds, reverse direction. The result is that the persistence length of the filaments’ translational motion is much larger than in unstressed normal-size bacteria, giving filaments an extremely large effective diffusion coefficient, allowing the bacteria to escape high existential stress.

Fingerprinting ocean acidification (OA) in US West Coast waters is extremely challenging due to the large magnitude of natural carbonate chemistry variations common to these regions. Additionally, quantifying a change requires information about the initial conditions, which is not readily available in most coastal systems. In an effort to address this issue, we have collated high-quality publicly available data to characterize the modern seasonal carbonate chemistry variability in marine surface waters of the US Pacific Northwest. Underway ship data from version 4 of the Surface Ocean CO2 Atlas, discrete observations from various sampling platforms, and sustained measurements from regional moorings were incorporated to provide ∼  100 000 inorganic carbon observations from which modern seasonal cycles were estimated. Underway ship and discrete observations were merged and gridded to a 0.1°  ×  0.1° scale. Eight unique regions were identified and seasonal cycles from grid cells within each region were averaged. Data from nine surface moorings were also compiled and used to develop robust estimates of mean seasonal cycles for comparison with the eight regions. This manuscript describes our methodology and the resulting mean seasonal cycles for multiple OA metrics in an effort to provide a large-scale environmental context for ongoing research, adaptation, and management efforts throughout the US Pacific Northwest. Major findings include the identification of unique chemical characteristics across the study domain. There is a clear increase in the ratio of dissolved inorganic carbon (DIC) to total alkalinity (TA) and in the seasonal cycle amplitude of carbonate system parameters when moving from the open ocean North Pacific into the Salish Sea. Due to the logarithmic nature of the pH scale (pH  =  −log10[H+], where [H+] is the hydrogen ion concentration), lower annual mean pH values (associated with elevated DIC  :  TA ratios) coupled with larger magnitude seasonal pH cycles results in seasonal [H+] ranges that are ∼  27 times larger in Hood Canal than in the neighboring North Pacific open ocean. Organisms living in the Salish Sea are thus exposed to much larger seasonal acidity changes than those living in nearby open ocean waters. Additionally, our findings suggest that lower buffering capacities in the Salish Sea make these waters less efficient at absorbing anthropogenic carbon than open ocean waters at the same latitude.All data used in this analysis are publically available at the following websites: Surface Ocean CO2 Atlas version 4 coastal data, https://doi.pangaea.de/10.1594/PANGAEA.866856 (Bakker et al., 2016a);National Oceanic and Atmospheric Administration (NOAA) West Coast Ocean Acidification cruise data, https://doi.org/10.3334/CDIAC/otg.CLIVAR_NACP_West_Coast_Cruise_2007 (Feely and Sabine, 2013); https://doi.org/10.7289/V5JQ0XZ1 (Feely et al., 2015b); https://data.nodc.noaa.gov/cgi-bin/iso?id=gov.noaa.nodc:0157445 (Feely et al., 2016a); https://doi.org/10.7289/V5C53HXP (Feely et al., 2015a);University of Washington (UW) and Washington Ocean Acidification Center cruise data, https://doi.org/10.5281/zenodo.1184657 (Fassbender et al., 2018);Washington State Department of Ecology seaplane data, https://doi.org/10.5281/zenodo.1184657 (Fassbender et al., 2018);NOAA Moored Autonomous pCO2 (MAPCO2) buoy data, https://doi.org/10.3334/CDIAC/OTG.TSM_LAPUSH_125W_48N (Sutton et al., 2012); https://doi.org/10.3334/CDIAC/OTG.TSM_WA_125W_47N (Sutton et al., 2013); https://doi.org/10.3334/CDIAC/OTG.TSM_DABOB_122W_478N (Sutton et al., 2014a); https://doi.org/10.3334/CDIAC/OTG.TSM_TWANOH_123W_47N (Sutton et al., 2016a);UW Oceanic Remote Chemical/Optical Analyzer buoy data, https://doi.org/10.5281/zenodo.1184657 (Fassbender et al., 2018);NOAA Pacific Coast Ocean Observing System cruise data, https://doi.org/10.5281/zenodo.1184657 (Fassbender et al., 2018).

We tested the in vivo potential of a MHC class I-restricted blocking peptide to sufficiently lower an anti-viral CTL response for preventing virus-induced CTL-mediated autoimmune diabetes (insulin-dependent diabetes mellitus (IDDM)) in vivo without affecting systemic viral clearance. By designing and screening several peptides with high binding affinities to MHC class I H-2Db for best efficiency in blocking killing of target cells by lymphocytic choriomeningitis virus (LCMV) and other viral CTL, we identified the peptide for this study. In vitro, it selectively lowered CTL killing restricted to the Db allele, which correlated directly with the affinity of the respective epitopes. Expression of the blocking peptide in the target cell lowered recognition of all Db-restricted LCMV epitopes. In addition, in vitro expansion of LCMV memory CTL was prevented, resulting in decreased IFN-γ secretion. In vivo, a 2-wk treatment with this peptide lowered the LCMV Db-restricted CTL response by over threefold without affecting viral clearance. However, the CTL reduction by the peptide treatment was sufficient to prevent LCMV-induced IDDM in rat insulin promoter-LCMV-glycoprotein transgenic mice. Following LCMV infection, these mice develop IDDM, which depends on Db-restricted anti-self (viral) CTL. Precursor numbers of splenic LCMV-CTL in peptide-treated mice were reduced, but their cytokine profile was not altered, indicating that the peptide did not induce regulatory cells. Further, non-LCMV-CTL recognizing the blocking peptide secreted IFN-γ and did not protect from IDDM. This study demonstrates that in vivo treatment with a MHC class I blocking peptide can prevent autoimmune disease by directly affecting expansion of autoreactive CTL.

Bacteria under external stress can reveal unexpected emergent phenotypes. We show that the intensely studied bacterium Escherichia coli can transform into long, highly motile helical filaments poized at a torsional buckling criticality when exposed to minimum inhibitory concentrations of several antibiotics. While the highly motile helices are physically either right- or left-handed, the motile helices always rotate with a right-handed angular velocity [Formula: see text] , which points in the same direction as the translational velocity [Formula: see text] of the helix. Furthermore, these helical cells do not swim by a “run and tumble” but rather synchronously flip their spin [Formula: see text] and thus translational velocity—backing up rather than tumbling. By increasing the translational persistence length, these dynamics give rise to an effective diffusion coefficient up to 20 times that of a normal E. coli cell. Finally, we propose an evolutionary mechanism for this phenotype’s emergence whereby the increased effective diffusivity provides a fitness advantage in allowing filamentous cells to more readily escape regions of high external stress.

International audience; The reactive species of oxygen and chlorine damage cellular components, potentially leading to cell death. In proteins, the sulfur-containing amino acid methionine is converted to methionine sulfoxide, which can cause a loss of biological activity. To rescue proteins with methionine sulfoxide residues, living cells express methionine sulfoxide reductases (Msrs) in most subcellular compartments, including the cytosol, mitochondria and chloroplasts(1-3). Here we report the identification of an enzymatic system, MsrPQ, repairing proteins containing methionine sulfoxide in the bacterial cell envelope, a compartment particularly exposed to the reactive species of oxygen and chlorine generated by the host defence mechanisms. MsrP, a molybdo-enzyme, and MsrQ, a haem-binding membrane protein, are widely conserved throughout Gram-negative bacteria, including major human pathogens. MsrPQ synthesis is induced by hypochlorous acid, a powerful antimicrobial released by neutrophils. Consistently, MsrPQ is essential for the maintenance of envelope integrity under bleach stress, rescuing a wide series of structurally unrelated periplasmic proteins from methionine oxidation, including the primary periplasmic chaperone SurA. For this activity, MsrPQ uses electrons from the respiratory chain, which represents a novel mechanism to import reducing equivalents into the bacterial cell envelope. A remarkable feature of MsrPQ is its capacity to reduce both rectus (R-) and sinister (S-) diastereoisomers of methionine sulfoxide, making this oxidoreductase complex functionally different from previously identified Msrs. The discovery that a large class of bacteria contain a single, non-stereospecific enzymatic complex fully protecting methionine residues from oxidation should prompt a search for similar systems in eukaryotic subcellular oxidizing compartments, including the endoplasmic reticulum.

The systematic emergence of drug resistance remains a major problem in the treatment of infectious diseases (antibiotics) and cancer (chemotherapy), with possible common fundamental origins linking bacterial antibiotic resistance and emergence of chemotherapy resistance. The common link may be evolution in a complex fitness landscape with connected small population niches. We report a detailed method for observing bacterial adaptive behavior in heterogeneous microfluidic environment designed to mimic the environmental heterogeneity found in natural microbial niches. First, the device is structured with multiple connected micro-chambers that allow the cell population to communicate and organize into smaller populations. Second, bacteria evolve within an antibiotic gradient generated throughout the micro-chambers that creates a wide range of fitness landscapes. High-resolution images of the adaptive response to the antibiotic stress are captured by epifluorescence microscopy at various levels of the bacterial organization for quantitative analysis. Thus, the experimental setup we have developed provides a powerful frame for visualizing evolution at work: bacterial movement, survival and death. It also presents a basis for exploring the rates at which drug resistance arises in bacteria and other biological contexts such as cancer.

Fingerprinting ocean acidification (OA) in U.S. West Coast waters is extremely challenging due to the large magnitude of natural carbonate chemistry variations common to these regions. Additionally, quantifying a change requires information about the initial conditions, which is not readily available in most coastal systems. In an effort to address this issue, we have collated high-quality, publicly-available data to characterize the modern seasonal carbonate chemistry variability in marine surface waters of the Pacific Northwest. Underway ship data from Version 4 of the Surface Ocean CO2 Atlas, discrete observations from various sampling platforms, and sustained measurements from regional moorings were incorporated to provide ~ 100,000 inorganic carbon observations from which modern seasonal cycles were estimated. Underway ship and discrete observations were merged and gridded to a 0.1° × 0.1° scale. Eight unique regions were identified and seasonal cycles from grid cells within each region were averaged. Data from nine surface moorings were also compiled and used to develop robust estimates of mean seasonal cycles for comparison with the eight regions. This manuscript describes our methodology and the resulting mean seasonal cycles for multiple OA metrics in an effort to provide large-scale, environmental context for ongoing research, adaptation, and management efforts throughout the Pacific Northwest. Major findings include the identification of unique chemical characteristics across the study domain. There is a clear increase in the ratio of dissolved inorganic carbon (DIC) to total alkalinity (TA) and in the seasonal cycle amplitude of carbonate system parameters when moving from the open ocean North Pacific into the Salish Sea. Due to the logarithmic nature of the pH scale (pH = −log10[H+], where [H+] is the hydrogen ion concentration), lower annual mean pH values (associated with elevated DIC : TA) coupled with larger magnitude seasonal pH cycles results in seasonal [H+] ranges that are ~ 27 times larger in Hood Canal than in the neighboring North Pacific open ocean. Organisms living in the Salish Sea are thus exposed to much larger seasonal acidity changes than those living in nearby open ocean waters. Additionally, our findings suggest that lower buffering capacities in the Salish Sea make these waters less efficient at absorbing anthropogenic carbon than open ocean waters at the same latitude. All data used in this analysis are publically available at the following websites: • Surface Ocean CO2 Atlas Version 4 coastal data, doi:10.1594/PANGAEA.866856; • National Oceanic and Atmospheric Administration (NOAA) West Coast Ocean Acidification cruise data, doi:10.3334/CDIAC/otg.CLIVAR_NACP_West_Coast_Cruise_2007; doi:10.3334/CDIAC/OTG.COAST_WCOA2011; doi:10.3334/CDIAC/OTG.COAST_WCOA2012; doi:10.7289/V5C53HXP; • University of Washington (UW) and Washington Ocean Acidification Center cruise data, doi:10.5281/zenodo.1184657; • Washington State Department of Ecology seaplane data, 10.5281/zenodo.1184657; • NOAA Moored Autonomous pCO2 (MAPCO2) Buoy data, doi:10.3334/CDIAC/OTG.TSM_LAPUSH_125W_48N; doi:10.3334/CDIAC/OTG.TSM_WA_125W_47N; doi:10.3334/CDIAC/OTG.TSM_DABOB_122W_478N; doi:10.3334/CDIAC/OTG.TSM_TWANOH_123W_47N; • UW Oceanic Remote Chemical/Optical Analyzer Buoy data, doi:10.5281/zenodo.1184657; • NOAA Pacific Coast Ocean Observing System cruise data, doi:10.5281/zenodo.1184657.

The systematic emergence of drug resistance remains a major problem in the treatment of infectious diseases (antibiotics) and cancer (chemotherapy), with possible common fundamental origins linking bacterial antibiotic resistance and emergence of chemotherapy resistance. The common link may be evolution in a complex fitness landscape with connected small population niches. We report a detailed method for observing bacterial adaptive behavior in heterogeneous microfluidic environment designed to mimic the environmental heterogeneity found in natural microbial niches. First, the device is structured with multiple connected micro-chambers that allow the cell population to communicate and organize into smaller populations. Second, bacteria evolve within an antibiotic gradient generated throughout the micro-chambers that creates a wide range of fitness landscapes. High-resolution images of the adaptive response to the antibiotic stress are captured by epifluorescence microscopy at various levels of the bacterial organization for quantitative analysis. Thus, the experimental setup we have developed provides a powerful frame for visualizing evolution at work: bacterial movement, survival and death. It also presents a basis for exploring the rates at which drug resistance arises in bacteria and other biological contexts such as cancer.

In the presence of extensive DNA damage, eukaryotes activate endonucleases to fragment their chromosomes and induce apoptotic cell death. Apoptotic-like responses have recently been described in bacteria, but primarily in specialized mutant backgrounds, and the factors responsible for DNA damage-induced chromosome fragmentation and death have not been identified. Here we find that wild-type Caulobacter cells induce apoptotic-like cell death in response to extensive DNA damage. The bacterial apoptosis endonuclease (BapE) protein is induced by damage but not involved in DNA repair itself, and mediates this cell fate decision. BapE fragments chromosomes by cleaving supercoiled DNA in a sequence-nonspecific manner, thereby perturbing chromosome integrity both in vivo and in vitro. This damage-induced chromosome fragmentation pathway resembles that of eukaryotic apoptosis. We propose that damage-induced programmed cell death can be a primary stress response for some bacterial species, providing isogenic bacterial communities with advantages similar to those that apoptosis provides to multicellular organisms.

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

Bacteria can rapidly evolve resistance to antibiotics via the SOS response, a state of high-activity DNA repair and mutagenesis. We explore here the first steps of this evolution in the bacterium Escherichia coli. Induction of the SOS response by the genotoxic antibiotic ciprofloxacin changes the E. coli rod shape into multichromosome-containing filaments. We show that at subminimal inhibitory concentrations of ciprofloxacin the bacterial filament divides asymmetrically repeatedly at the tip. Chromosome-containing buds are made that, if resistant, propagate nonfilamenting progeny with enhanced resistance to ciprofloxacin as the parent filament dies. We propose that the multinucleated filament creates an environmental niche where evolution can proceed via generation of improved mutant chromosomes due to the mutagenic SOS response and possible recombination of the new alleles between chromosomes. Our data provide a better understanding of the processes underlying the origin of resistance at the single-cell level and suggest an analogous role to the eukaryotic aneuploidy condition in cancer.

We have constructed a microfabricated circular corral for bacteria made of rings of concentric funnels which channel motile bacteria outwards via non-hydrodynamic interactions with the funnel walls. Initially bacteria do move rapidly outwards to the periphery of the corral. At the edge, nano-slits allow for the transport of nutrients into the device while keeping the bacteria from escaping. After a period of time in which the bacteria increase their cell density in this perimeter region, they are then able to defeat the physical constrains of the funnels by launching back-propagating collective waves. We present the basic data and some nonlinear modeling which can explain how bacterial population waves propagate through a physical funnel, and discuss possible biological implications.

In Saccharomyces cerevisiae, the telomerase complex binds to chromosome ends and is activated in late S-phase through a process coupled to the progression of the replication fork. Here, we show that the single-stranded DNA-binding protein RPA (replication protein A) binds to the two daughter telomeres during telomere replication but only its binding to the leading-strand telomere depends on the Mre11/Rad50/Xrs2 (MRX) complex. We further demonstrate that RPA specifically co-precipitates with yKu, Cdc13 and telomerase. The interaction of RPA with telomerase appears to be mediated by both yKu and the telomerase subunit Est1. Moreover, a mutation in Rfa1 that affects both the interaction with yKu and telomerase reduces the dramatic increase in telomere length of a rif1Δ, rif2Δ double mutant. Finally, we show that the RPA/telomerase association and function are conserved in Schizosaccharomyces pombe. Our results indicate that in both yeasts, RPA directly facilitates telomerase activity at chromosome ends.

The Washington State Department of Ecology (Ecology) maintains moorings in Puget Sound and Willapa Bay (Figure 1) as part of a more extensive monitoring program.

Methionine ranks among the amino acids most sensitive to oxidation, which converts it to a racemic mixture of methionine- S -sulfoxide (Met-S-SO) and methionine- R -sulfoxide (Met-R-SO). The methionine sulfoxide reductases MsrA and MsrB reduce free and protein-bound MetSO, MsrA being specific for Met-S-SO and MsrB for Met-R-SO. In the present study, we report that an Escherichia coli metB 1 auxotroph lacking both msrA and msrB is still able to use either of the two MetSO enantiomers. This indicates that additional methionine sulfoxide reductase activities occur in E. coli . BisC, a poorly characterized biotin sulfoxide reductase, was identified as one of these new methionine sulfoxide reductases. BisC was purified and found to exhibit reductase activity with free Met-S-SO but not with free Met-R-SO as a substrate. Moreover, a metB 1 msrA msrB bisC strain of E. coli was unable to use Met-S-SO for growth, but it retained the ability to use Met-R-SO. Mass spectrometric analyses indicated that BisC is unable to reduce protein-bound Met-S-SO. Hence, this study shows that BisC has an essential role in assimilation of oxidized methionines. Moreover, this work provides the first example of an enzyme that reduces free MetSO while having no activity on peptide-bound MetSO residues.