Crossing design shapes patterns of genetic variation in synthetic recombinant populations of Saccharomyces cerevisiae

Abstract“Synthetic recombinant” populations have emerged as a useful tool for dissecting the genetics of complex traits. They can be used to derive inbred lines for fine QTL mapping, or the populations themselves can be sampled for experimental evolution. In the latter application, investigators generally value maximizing genetic variation in constructed populations. This is because in evolution experiments initiated from such populations, adaptation is primarily fueled by standing genetic variation. Despite this reality, little has been done to systematically evaluate how different methods of constructing synthetic populations shape initial patterns of variation. Here we seek to address this issue by comparing outcomes in synthetic recombinant Saccharomyces cerevisiae populations created using one of two strategies: pairwise crossing of isogenic strains or simple mixing of strains in equal proportion. We also explore the impact of the varying the number of parental strains. We find that more genetic variation is initially present and maintained when population construction includes a round of pairwise crossing. As perhaps expected, we also observe that increasing the number of parental strains typically increases genetic diversity. In summary, we suggest that when constructing populations for use in evolution experiments, simply mixing founder strains in equal proportion may limit the adaptive potential.

Synergy Between Beta-Lactams and Lipo-, Glyco-, and Lipoglycopeptides, Is Independent of the Seesaw Effect in Methicillin-Resistant Staphylococcus aureus

Methicillin-resistant S. aureus (MRSA) are resistant to beta-lactams, but synergistic activity between beta-lactams and glycopeptides/lipopeptides is common. Many have attributed this synergy to the beta-lactam-glycopeptide seesaw effect; however, this association has not been rigorously tested. The objective of this study was to determine whether the seesaw effect is necessary for synergy and to measure the impact of beta-lactam exposure on lipid metabolism. We selected for three isogenic strains with reduced susceptibility to vancomycin, daptomycin, and dalbavancin by serial passaging the MRSA strain N315. We used whole genome sequencing to identify genetic variants that emerged and tested for synergy between vancomycin, daptomycin, or dalbavancin in combination with 6 beta-lactams with variable affinity for staphylococcal penicillin binding proteins (PBPs), including nafcillin, meropenem, ceftriaxone, ceftaroline, cephalexin, and cefoxitin, using time-kills. We observed that the seesaw effect with each beta-lactam was variable and the emergence of the seesaw effect for a particular beta-lactam was not necessary for synergy between that beta-lactam and vancomycin, daptomycin, or dalbavancin. Synergy was more commonly observed with vancomycin and daptomycin based combinations than dalbavancin in time-kills. Among the beta-lactams, cefoxitin and nafcillin were the most likely to exhibit synergy using the concentrations tested, while cephalexin was the least likely to exhibit synergy. Synergy was more common among the resistant mutants than the parent strain. Interestingly N315-D1 and N315-DAL0.5 both had mutations in vraTSR and walKR despite their differences in the seesaw effect. Lipidomic analysis of all strains exposed to individual beta-lactams at subinhibitory concentrations suggested that in general, the abundance of cardiolipins (CLs) and most free fatty acids (FFAs) positively correlated with the presence of synergistic effects while abundance of phosphatidylglycerols (PGs) and lysylPGs mostly negatively correlated with synergistic effects. In conclusion, the beta-lactam-glycopeptide seesaw effect and beta-lactam-glycopeptide synergy are distinct phenomena. This suggests that the emergence of the seesaw effect may not have clinical importance in terms of predicting synergy. Further work is warranted to characterize strains that don’t exhibit beta-lactam synergy to identify which strains should be targeted with combination therapy and which ones cannot and to further investigate the potential role of CLs in mediating synergy.

Crossing design shapes patterns of genetic variation in synthetic recombinant populations of Saccharomyces cerevisiae

Multiparent or synthetic recombinant populations those created by combining distinct isogenic founders to establish a single recombinant background have emerged as a useful tool for dissecting the genetics of complex traits. Synthetic recombinant populations can be used to derive inbred lines in which quantitative traits can be mapped, or the recombinant populations themselves can be sampled for experimental evolution. Especially for the latter application, investigators generally value maximizing genetic variation in a recombinant population; in other words, a population harboring relatively equal contributions of the genetic backgrounds of each isogenic founder strain is a desirable resource. It is well-documented that in evolution experiments initiated from recombinant or outbred ancestral populations, the subsequent adaptation that occurs in evolved populations is driven by standing genetic variation, rather than de novo mutations. Despite the demonstrated importance of initial genetic variation to the adaptive process, little has been done to systematically evaluate methods of constructing a synthetic recombinant population, for creating resources for evolution experiments. Here we seek to address this issue by comparing patterns of genetic variation in different synthetic recombinant populations of Saccharomyces cerevisiae created using one of two combination strategies: pairwise crossing of isogenic strains or simple mixing of strains in equal proportion. We also explore the impact of the varying the number of parental strains used in each strategy. We find that more genetic variation is initially present and subsequently maintained over generations when population construction includes a round of pairwise crossing. We also observe that when using a given crossing strategy, increasing the number of parental strains typically increases genetic diversity. In summary, we suggest that when creating recombinant populations for use in experimental evolution studies, simply mixing founder strains in equal proportion may limit the adaptive potential of that population.

Old data and friends improve with age: Advancements with the updated tools of GeneNetwork

Understanding gene-by-environment interactions is important across biology, particularly behaviour. Families of isogenic strains are excellently placed, as the same genome can be tested in multiple environments. The BXD's recent expansion to 140 strains makes them the largest family of murine isogenic genomes, and therefore give great power to detect QTL. Indefinite reproducible genometypes can be leveraged; old data can be reanalysed with emerging tools to produce novel biological insights. To highlight the importance of reanalyses, we obtained drug- and behavioural-phenotypes from Philip et al. 2010, and reanalysed their data with new genotypes from sequencing, and new models (GEMMA and R/qtl2). We discover QTL on chromosomes 3, 5, 9, 11, and 14, not found in the original study. We narrowed down the candidate genes based on their ability to alter gene expression and/or protein function, using cis-eQTL analysis, and variants predicted to be deleterious. Co-expression analysis ('gene friends') and human PheWAS were used to further narrow candidates. Prominent candidate genes include: Slitrk6 in a Chr 14 QTL for locomotion in the center of an open field, we show to be part of a coexpression network involved in voluntary movement, and association with neuropsychiatric phenotypes in PheWAS; and Cdk14, one of only 3 genes in a Chr 5 QTL for handling induced convulsions after ethanol treatment, that is regulated by the anticonvulsant drug valproic acid. By using families of isogenic strains, we can reuse and reanalyse data to discover novel and highly plausible candidate genes involved in response to the environment.

Disbalancing Envelope Stress Responses as a Strategy for Sensitization of Escherichia coli to Antimicrobial Agents

Disbalancing envelope stress responses was investigated as a strategy for sensitization of Escherichia coli to antimicrobial agents. Seventeen isogenic strains were selected from the KEIO collection with deletions in genes corresponding to the σE, Cpx, Rcs, Bae, and Psp responses. Antimicrobial activity against 20 drugs with different targets was evaluated by disk diffusion and gradient strip tests. Growth curves and time-kill curves were also determined for selected mutant-antimicrobial combinations. An increase in susceptibility to ampicillin, ceftazidime, cefepime, aztreonam, ertapenem, and fosfomycin was detected. Growth curves for Psp response mutants showed a decrease in optical density (OD) using sub-MIC concentrations of ceftazidime and aztreonam (ΔpspA and ΔpspB mutants), cefepime (ΔpspB and ΔpspC mutants) and ertapenem (ΔpspB mutant). Time-kill curves were also performed using 1xMIC concentrations of these antimicrobials. For ceftazidime, 2.9 log10 (ΔpspA mutant) and 0.9 log10 (ΔpspB mutant) decreases were observed at 24 and 8 h, respectively. For aztreonam, a decrease of 3.1 log10 (ΔpspA mutant) and 4 log1010 (ΔpspB mutant) was shown after 4–6 h. For cefepime, 4.2 log10 (ΔpspB mutant) and 2.6 log10 (ΔpspC mutant) decreases were observed at 8 and 4 h, respectively. For ertapenem, a decrease of up to 6 log10 (ΔpspB mutant) was observed at 24 h. A deficient Psp envelope stress response increased E. coli susceptibility to beta-lactam agents such as cefepime, ceftazidime, aztreonam and ertapenem. Its role in repairing extensive inner membrane disruptions makes this pathway essential to bacterial survival, so that disbalancing the Psp response could be an appropriate target for sensitization strategies.

Causes and consequences of pattern diversification in a spatially self-organizing microbial community

Surface-attached microbial communities constitute a vast amount of life on our planet. They contribute to all major biogeochemical cycles, provide essential services to our society and environment, and have important effects on human health and disease. They typically consist of different interacting genotypes that arrange themselves non-randomly across space (referred to hereafter as spatial self-organization). While spatial self-organization is important for the functioning, ecology, and evolution of these communities, the underlying determinants of spatial self-organization remain unclear. Here, we performed a combination of experiments, statistical modeling, and mathematical simulations with a synthetic cross-feeding microbial community consisting of two isogenic strains. We found that two different patterns of spatial self-organization emerged at the same length and time scales, thus demonstrating pattern diversification. This pattern diversification was not caused by initial environmental heterogeneity or by genetic heterogeneity within populations. Instead, it was caused by nongenetic heterogeneity within populations, and we provide evidence that the source of this nongenetic heterogeneity is local differences in the initial spatial positionings of individuals. We further demonstrate that the different patterns exhibit different community-level properties; namely, they have different expansion speeds. Together, our results demonstrate that pattern diversification can emerge in the absence of initial environmental heterogeneity or genetic heterogeneity within populations and can affect community-level properties, thus providing novel insights into the causes and consequences of microbial spatial self-organization.

The serine protease HtrA plays a key role in heat-induced dispersal of pneumococcal biofilms

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx by forming multicellular biofilms. Due to the high level of asymptomatic carriage, transition to infections, such as otitis media, pneumonia, sepsis, and meningitis, occurs often enough that the pneumococcus remains a major cause of disease and death globally. Virus infection and virus-induced responses, such as increased temperature (fever), trigger release of virulent bacteria from colonizing biofilms. The exact mechanisms involved in pneumococcal egress during biofilm dispersal remain unknown, although we hypothesize that disruption of the biofilm matrix encasing the bacteria is necessary. Here, we utilized established in vitro biofilm dispersal models to investigate the involvement of proteases in bacterial egress from pneumococcal biofilms. We demonstrate the importance of protease activity, both through increased bacterial release following addition of proteases and reduced heat-induced biofilm dispersal in the presence of protease inhibitors. We identify a key role for the surface-exposed serine protease HtrA, but not PrtA, in heat-induced biofilm dispersal. Bacterial release from htrA-negative biofilms was significantly reduced compared to wild-type isogenic strains but was restored and increased above wild-type levels following addition of recombinant HtrA. Understanding the specific mechanisms involved in bacterial egress may provide novel targets for future strategies aimed to specifically interfere with disease progression without disturbing nasopharyngeal biofilm colonization.

Dimercaptosuccinic acid in combination with carbapenems against isogenic strains of Escherichia coli producing or not producing a metallo-β-lactamase in vitro and in murine peritonitis

Background Carbapenemase-producing Enterobacterales represent a major therapeutic challenge. MBLs, requiring zinc at their catalytic site, could be inhibited by meso-dimercaptosuccinic acid (DMSA), a heavy metal chelator already widely used for treating lead intoxication. Objectives To evaluate the activity of carbapenems alone or combined with DMSA against MBL-producing Escherichia coli in a severe murine peritonitis model. Methods Isogenic strains of wild-type E. coli CFT073 producing the MBLs NDM-1, VIM-2 and IMP-1, and the control serine carbapenemases OXA-48 and KPC-3 were constructed. MIC determinations and time–kill assays were performed for imipenem, meropenem and ertapenem alone or in combination with DMSA. Infected mice were treated intraperitoneally for 24 h with imipenem, DMSA or their combination. Bacterial counts in peritoneal fluid and spleen were assessed at 24 h. Results DMSA in combination with each carbapenem caused a significant decrease in the MICs for all MBL-producing strains, in a concentration-dependent manner, but did not provide benefit against non-MBL strains. In mice infected with the NDM-1-producing strain, the combination of imipenem and DMSA significantly reduced bacterial counts in peritoneal fluid (P = 0.0006) and spleen (P < 0.0001), as compared with imipenem alone, with no benefit against the KPC-3-producing and CFT073 strains. DMSA concentrations in plasma of mice were comparable to those obtained in humans with a standard oral dose. Conclusions DMSA restores the activity of carbapenems against MBL-producing strains, and its combination with carbapenems appears to be a promising strategy for the treatment of NDM-producing E. coli infections.

Sequential Time-Kill, a Simple Experimental Trick To Discriminate between Pharmacokinetics/Pharmacodynamics Models with Distinct Heterogeneous Subpopulations versus Homogenous Population with Adaptive Resistance

ABSTRACT Experiments were conducted with polymyxin B and two Klebsiella pneumonia isogenic strains (the wild type, KP_WT, and its transconjugant carrying the mobile colistin resistance gene, KP_MCR-1) to demonstrate that conducting two consecutive time-kill experiments (sequential TK) represents a simple approach to discriminate between pharmacokinetics/pharmacodynamics models with two heterogeneous subpopulations or adaptive resistance.

Interaction-dependent effects of surface structure on microbial spatial self-organization

Surface-attached microbial communities consist of different cell types that, at least to some degree, organize themselves non-randomly across space (referred to as spatial self-organization). While spatial self-organization can have important effects on the functioning, ecology and evolution of communities, the underlying determinants of spatial self-organization remain unclear. Here, we hypothesize that the presence of physical objects across a surface can have important effects on spatial self-organization. Using pairs of isogenic strains of Pseudomonas stutzeri , we performed range expansion experiments in the absence or presence of physical objects and quantified the effects on spatial self-organization. We demonstrate that physical objects create local deformities along the expansion frontier, and these deformities increase in magnitude during range expansion. The deformities affect the densities of interspecific boundaries and diversity along the expansion frontier, and thus affect spatial self-organization, but the effects are interaction-dependent. For competitive interactions that promote sectorized patterns of spatial self-organization, physical objects increase the density of interspecific boundaries and diversity. By contrast, for cross-feeding interactions that promote dendritic patterns, they decrease the density of interspecific boundaries and diversity. These qualitatively different outcomes are probably caused by fundamental differences in the orientations of the interspecific boundaries. Thus, in order to predict the effects of physical objects on spatial self-organization, information is needed regarding the interactions present within a community and the general geometric shapes of spatial self-organization that emerge from those interactions. This article is part of the theme issue ‘Conceptual challenges in microbial community ecology’.