Kristian Kvint

The bacterial universal stress protein: function and regulation.

The universal stress protein A (UspA) superfamily encompasses an ancient and conserved group of proteins that are found in bacteria, Archea, fungi, flies and plants. The Escherichia coli UspA is produced in response to a large number of different environmental onslaughts and UspA is one of the most abundant proteins in growth-arrested cells. Although insights into the regulation of the E. coli uspA gene have been gained, the exact roles of the Usp proteins and Usp domains remain enigmatic; they appear, in some cases, to be linked to resistance to DNA-damaging agents and to respiratory uncouplers.

Negative regulation by RpoS: a case of sigma factor competition

A mutation in the Escherichia coli gene encoding the stationary phase‐inducible sigma factor (σs, RpoS) not only abolishes transcription of some genes in stationary phase, but also causes superinduction of other stationary phase‐induced genes. We have examined this phenomenon of repression by σs using as a model system the divergently transcribed stationary phase‐inducible genes, uspA and uspB. uspA is transcribed by σ70‐programmed RNA polymerase and is superinduced in an rpoS mutant, while uspB induction is σs dependent. The data suggest that the superinduction of uspA is caused by an increased amount of σ70 bound to RNA polymerase in the absence of the competing σs. Increasing the ability of σ70 to compete against σs by overproducing σ70 mimics the effect of an rpoS mutation by causing superinduction of σ70‐dependent stationary phase‐inducible genes (uspA and fadD), silencing of σs‐dependent genes (uspB, bolAp1 and fadL) and inhibiting the development of σs‐dependent phenotypes, such as hydrogen peroxide resistance in stationary phase. In addition, overproduction of σs markedly reduced stationary phase expression of a σ70‐dependent promoter. Thus, we conclude that sigma factors compete for a limiting amount of RNA polymerase during stationary phase. The implications of this competition in the passive control of promoter activity is discussed.

Emergency derepression: stringency allows RNA polymerase to override negative control by an active repressor

The uspA promoter, driving production of the universal stress protein A in response to diverse stresses, is demonstrated to be under dual control. One regulatory pathway involves activation of the promoter by the alarmone guanosine 3′,5′‐bisphosphate, via the β‐subunit of RNA polymerase, whereas the other consists of negative control by the FadR repressor. In contrast to canonical dual control by activation and repression circuits, which depends on concomitant activation and derepression for induction to occur, the ppGpp‐dependent activation of the uspA promoter overrides repression by an active FadR under conditions of severe cellular stress (starvation). The ability of RNA polymerase to overcome repression during stringency depends, in part, on the strength of the FadR operator. This emergency derepression is operative on other FadR‐regulated genes induced by starvation and is argued to be an essential regulatory mechanism operating during severe stress.

Absence of Mitochondrial Translation Control Proteins Extends Life Span by Activating Sirtuin-Dependent Silencing

Altered mitochondrial functionality can extend organism life span, but the underlying mechanisms are obscure. Here we report that inactivating SOV1, a member of the yeast mitochondrial translation control (MTC) module, causes a robust Sir2-dependent extension of replicative life span in the absence of respiration and without affecting oxidative damage. We found that SOV1 interacts genetically with the cAMP-PKA pathway and the chromatin remodeling apparatus. Consistently, Sov1p-deficient cells displayed reduced cAMP-PKA signaling and an elevated, Sir2p-dependent, genomic silencing. Both increased silencing and life span extension in sov1Δ cells require the PKA/Msn2/4p target Pnc1p, which scavenges nicotinamide, a Sir2p inhibitor. Inactivating other members of the MTC module also resulted in Sir2p-dependent life span extension. The data demonstrate that the nuclear silencing apparatus senses and responds to the absence of MTC proteins and that this response converges with a pathway for life span extension elicited by reducing TOR signaling.

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 alarmones means of regulating a trade‐off between growth and maintenance.

Opposing roles of Ubp3‐dependent deubiquitination regulate replicative life span and heat resistance

The interplay between molecular chaperones, ubiquitin/deubiquitinating enzymes, and proteasomes is a critical element in protein homeostasis. Among these factors, the conserved deubiquitinase, Ubp3, has the interesting ability, when overproduced, to suppress the requirement for the major cytosolic Hsp70 chaperones. Here, we show that Ubp3 overproduction counteracts deficiency of Hsp70s by the removal of damaged proteins deposited in inclusion bodies (JUNQ) during both aging and heat stress. Consistent with this, Ubp3 destabilized, deubiquitinated, and diminished the toxicity of the JUNQ‐associated misfolded protein Ubc9ts in a proteasome‐dependent manner. In contrast, another misfolded model protein, ∆ssCPY*, was stabilized by Ubp3‐dependent deubiquitination demonstrating a dual role for Ubp3, saving or destroying aberrant protein species depending on the stage at which the damaged protein is committed for destruction. We present genetic evidence for the former of these activities being key to Ubp3‐dependent suppression of heat sensitivity in Hsp70‐deficient cells, whereas protein destruction suppresses accelerated aging. We discuss the data in view of how heat stress and aging might elicit differential damage and challenges on the protein homeostasis network.

Loss of Ubp3 increases Silencing, decreases Unequal Recombination in rDNA, and shortens the Replicative Life Span in Saccharomyces cerevisiae

Ubp3 is an antisilencing factor. Accordingly, loss of Upb3 leads to lower RNAPII occupancy in heterochromatic regions and suppression of unequal recombination in rDNA. However, ubp3Δ mutants have a shortened replicative life span, suggesting that recombination frequency is not directly correlated with aging.

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.