Critical knowledge gaps and research needs related to the environmental dimensions of antibiotic resistance

There is growing understanding that the environment plays an important role both in the transmission of antibiotic resistant pathogens and in their evolution. Accordingly, researchers and stakeholders world-wide seek to further explore the mechanisms and drivers involved, quantify risks and identify suitable interventions. There is a clear value in establishing research needs and coordinating efforts within and across nations in order to best tackle this global challenge. At an international workshop in late September 2017, scientists from 14 countries with expertise on the environmental dimensions of antibiotic resistance gathered to define critical knowledge gaps. Four key areas were identified where research is urgently needed: 1) the relative contributions of different sources of antibiotics and antibiotic resistant bacteria into the environment; 2) the role of the environment, and particularly anthropogenic inputs, in the evolution of resistance; 3) the overall human and animal health impacts caused by exposure to environmental resistant bacteria; and 4) the efficacy and feasibility of different technological, social, economic and behavioral interventions to mitigate environmental antibiotic resistance.(1)Peer reviewe

Nystrom T: Regulation of or factor competition by the alarmone ppGpp

. Similarly, an in vivo competition assay showed that the ability of both 32 and S to compete with 70 is diminished in cells lacking ppGpp. Consistently, the fraction of S and 32 bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of factors in accordance with cellular demands during physiological stress

Regulation of σ factor competition by the alarmone ppGpp

Many regulons controlled by alternative σ factors, including σ(S) and σ(32), are poorly induced in cells lacking the alarmone ppGpp. We show that ppGpp is not absolutely required for the activity of σ(S)-dependent promoters because underproduction of σ(70), specific mutations in rpoD (rpoD40 and rpoD35), or overproduction of Rsd (anti-σ(70)) restored expression from σ(S)-dependent promoters in vivo in the absence of ppGpp accumulation. An in vitro transcription/competition assay with reconstituted RNA polymerase showed that addition of ppGpp reduces the ability of wild-type σ(70) to compete with σ(32) for core binding and the mutant σ(70) proteins, encoded by rpoD40 and rpoD35, compete less efficiently than wild-type σ(70). Similarly, an in vivo competition assay showed that the ability of both σ(32) and σ(S) to compete with σ(70) is diminished in cells lacking ppGpp. Consistently, the fraction of σ(S) and σ(32) bound to core was drastically reduced in ppGpp-deficient cells. Thus, the stringent response encompasses a mechanism that alters the relative competitiveness of σ factors in accordance with cellular demands during physiological stress

Differential role of cytosolic Hsp70s in longevity assurance and protein quality control

70 kDa heat shock proteins (Hsp70) are essential chaperones of the protein quality control network; vital for cellular fitness and longevity. The four cytosolic Hsp70's in yeast, Ssa1-4, are thought to be functionally redundant but the absence of Ssa1 and Ssa2 causes a severe reduction in cellular reproduction and accelerates replicative aging. In our efforts to identify which Hsp70 activities are most important for longevity assurance, we systematically investigated the capacity of Ssa4 to carry out the different activities performed by Ssa1/2 by overproducing Ssa4 in cells lacking these Hsp70 chaperones. We found that Ssa4, when overproduced in cells lacking Ssa1/2, rescued growth, mitigated aggregate formation, restored spatial deposition of aggregates into protein inclusions, and promoted protein degradation. In contrast, Ssa4 overproduction in the Hsp70 deficient cells failed to restore the recruitment of the disaggregase Hsp104 to misfolded/aggregated proteins, to fully restore clearance of protein aggregates, and to bring back the formation of the nucleolus-associated aggregation compartment. Exchanging the nucleotide-binding domain of Ssa4 with that of Ssa1 suppressed this 'defect' of Ssa4. Interestingly, Ssa4 overproduction extended the short lifespan of ssa1 Delta ssa2 Delta mutant cells to a lifespan comparable to, or even longer than, wild type cells, demonstrating that Hsp104-dependent aggregate clearance is not a prerequisite for longevity assurance in yeast. Author summary: All organisms have proteins that network together to stabilize and protect the cell throughout its lifetime. One of these types of proteins are the Hsp70s (heat shock protein 70). Hsp70 proteins take part in folding other proteins to their functional form, untangling proteins from aggregates, organize aggregates inside the cell and ensure that damaged proteins are destroyed. In this study, we investigated three closely related Hsp70 proteins in yeast; Ssa1, 2 and 4, in an effort to describe the functional difference of Ssa4 compared to Ssa1 and 2 and to answer the question: What types of cellular stress protection are necessary to reach a normal lifespan? We show that Ssa4 can perform many of the same tasks as Ssa1 and 2, but Ssa4 doesn't interact in the same manner as Ssa1 and 2 with other types of proteins. This leads to a delay in removing protein aggregates created after heat stress. Ssa4 also cannot ensure that misfolded proteins aggregate correctly inside the nucleus of the cell. However, this turns out not to be necessary for yeast cells to achieve a full lifespan, which shows us that as long as cells can prevent aggregates from forming in the first place, they can reach a full lifespan

Increased RNA polymerase availability directs resources towards growth at the expense of maintenance

Nutritionally induced changes in RNA polymerase availability have been hypothesized to be an evolutionary primeval mechanism for regulation of gene expression and several contrasting models have been proposed to explain how such ‘passive' regulation might occur. We demonstrate here that ectopically elevating Escherichia coli RNA polymerase (Eσ70) levels causes an increased expression and promoter occupancy of ribosomal genes at the expense of stress-defense genes and amino acid biosynthetic operons. Phenotypically, cells overproducing Eσ70 favours growth and reproduction at the expense of motility and damage protection; a response reminiscent of cells with no or diminished levels of the alarmone guanosine tetraphosphate (ppGpp). Consistently, we show that cells lacking ppGpp displayed markedly elevated levels of free Eσ70 compared with wild-type cells and that the repression of ribosomal RNA expression and reduced growth rate of mutants with constitutively elevated levels of ppGpp can be suppressed by overproducing Eσ70. We conclude that ppGpp modulates the levels of free Eσ70 and that this is an integral part of the alarmone's means of regulating a trade-off between growth and maintenance