Psychrophilic (cold-active) organisms have developed enzymes that facilitate sufficient metabolic activity at low temperatures to sustain life. This occurs through molecular adaptations that tend to increase protein flexibility at the expense of stability. However, psychrophiles also vary in their growth conditions. Eurypsychrophiles thrive over a wide temperature range and often prefer temperatures above 20 °C, while stenopsychrophiles grow optimally below 15 °C and are more narrowly adapted to cold temperatures. To elucidate differences between these two classes of enzymes, we here compare the stability and unfolding kinetics of two orthologues of the basal household enzyme triose phosphate isomerase, one from the stenopsychrophilic Antarctic permafrost bacterium Rhodonellum psychrophilum (sTPI) and the other from the eurypsychrophilic Greenland ikaite column bacterium Rhodococcus sp. JG-3 (eTPI). Remarkably, sTPI proved significantly more thermostable and resistant to chemical denaturation than its eurypsychrophilic counterpart, eTPI, in the absence of ionic components in solution, whereas inclusion of electrostatic screening agents in the form of sodium chloride or the charged denaturant guanidinium chloride largely cancelled out this difference. Thus, electrostatics play a prominent role in stabilizing the stenopsychrophilic sTPI, and a mandatory low-temperature growth environment does not preclude the development of considerable thermotolerance for individual enzymes. We were able to increase the thermostability of sTPI using an evolutionary machine learning model, which transferred several sTPI residues into the eTPI active site. While the stabilizing effect was modest, the combination of individual mutations was additive, underscoring the potential of combining multiple beneficial mutations to achieve enhanced enzyme properties.

Cold-active enzymes hold promise for energy-efficient processes. Amylases are widely used in household and industrial applications, but only a few are cold-active. Here we describe three novel secreted amylases, Rho13, Ika2 and I3C6, all from bacteria growing in the cold and alkaline ikaite columns in Greenland. They all hydrolyzed starch to smaller malto-oligomers, but only Rho13 and Ika2 hydrolyzed cyclodextrins, and only Ika2 displayed transglycosylation activity. Ika2 forms a stable dimer, while both Rho13 and I3C6 are mainly monomeric. They all have optimal active temperatures around 30–35 °C and significant enzymatic activity below 20 °C, but Rho13 and I3C6 had an alkaline optimal pH, while Ika2 was markedly acidophilic. They showed complex dependence on Ca2+ concentration, with the activity of Rho13 and I3C6 following a bell-shaped curve and Ika2 being unaffected; however, removal of Ca2+ reduced the stability of all three enzymes. Loss of structure occurred well above the temperature of optimal activity, showing the characteristic psychrophilic divorce between activity and stability. MD simulations showed that Ika2 did not have a well-defined Ca2+ binding site, while Rho13 and I3C6 both maintained one stably bound Ca2+ ion. We identified psychrophilic features as higher levels of backbone fluctuations compared to mesophilic counterparts, based on a lower number of internal hydrogen bonds and salt bridges. This increased fluctuation was also found in regions outside the active site and may provide easier substrate access and accommodation, as well as faster barrier transitions. Our work sheds further light on the many ways in which psychrophilic enzymes adapt to increased catalysis at lower temperatures.

HipA-like kinases are widespread bacterial serine-threonine kinases, yet their regulatory mechanisms remain poorly understood. Here, we characterise two novel HipA-like systems, the monocistronic hipL and bicistronic hipIN, also encoding HipS-like and HIRAN domains. We show that the hipL gene contains an internal translation initiation site producing a smaller variant, HipLS, which counteracts HipL-mediated toxicity via its HipS-like domain. Contrary to this, HipN requires both the HipS-like and the HIRAN domains to neutralise HipI-mediated toxicity. Neither system forms stable toxin-antitoxin (TA) complexes in vitro, distinguishing them from classical type II systems. Finally, we show that autophosphorylation affects HipL but not HipI-mediated toxicity. These findings reveal diverse regulatory architectures in HipA-like TA systems, shaped by domain composition and operon structure.Impact statement Kinases are increasingly recognised as key regulators in bacteria. Here, we show how complex operon and domain structures can contribute to kinase function and regulation, revealing increasingly complex regulatory networks in microbes.

Insights into bacterial metabolic adaptation during stress is crucial for understanding early mechanisms of antibiotic resistance. In the Gram-negative bacterium Escherichia coli, the universal stringent response produces the alarmones (p)ppGpp that target many cellular proteins. The cellular nucleosidase PpnN is regulated by (p)ppGpp and was shown to balance bacterial fitness and persistence during fluoroquinolone exposure. pppGpp and ppGpp both activate PpnN, but differentially regulate its cooperativity via an unknown mechanism; furthermore, the catalytic mechanism of PpnN has remained unclear. Here, we provide mechanistic insights into the interaction of PpnN with a substrate analogue, reaction products, and alarmone molecules, which allows us to understand the catalytic mechanism of this family of nucleosidases and the differential modes of regulation by ppGpp and pppGpp, respectively. Comparison to the homologous plant cytokinin-producing LOG proteins reveals that PpnN utilizes an evolutionarily conserved purine hydrolysis mechanism, which in bacteria is regulated by alarmones during stress.

Bacteria encode a wide range of survival and immunity systems, including CRISPR-Cas, restriction-modification systems, and toxin-antitoxin systems involved in defence against bacteriophages, as well as survival during challenging growth conditions or exposure to antibiotics. Toxin-antitoxin (TA) systems are small two- or three-gene cassettes consisting of a metabolic regulator (the "toxin") and its associated antidote (the "antitoxin"), which also often functions as a transcriptional regulator. TA systems are widespread in the genomes of pathogens but are also present in commensal bacterial species and on plasmids. For mobile elements such as plasmids, TA systems play a role in maintenance, and increasing evidence now points to roles of chromosomal toxin-antitoxin systems in anti-phage defence. Moreover, the widespread occurrence of toxin-antitoxin systems in the genomes of pathogens has been suggested to relate to survival during host infection as well as in persistence during antibiotic treatment. Upon repeated exposure to antibiotics, TA systems have been shown to acquire point mutations as well as more dramatic rearrangements such as in-frame deletions with potential relevance for bacterial survival and pathogenesis. In this review, we present an overview of the known functional and structural consequences of mutations and rearrangements arising in bacterial toxin-antitoxin systems and discuss their relevance for survival and persistence of pathogenic species.

ABSTRACT Transcriptional regulation is a fundamental mechanism in bacteria and is often mediated by repressor proteins. In the type II toxin-antitoxin (TA) system. xre-res , the Xre antitoxin contains a putative helix-turn-helix (HTH) DNA-binding domain and could thus potentially bind to and repress transcription from the xre-res promoter. The structure of the Pseudomonas putida Xre-RES TA complex revealed an unusual 4:2 stoichiometry with two potential DNA-binding sites, suggesting a non-canonical mechanism of transcriptional autoregulation. Here, we show that the activity of the xre-res promoter requires both an intact σ 70 element and the transcriptional start site, and that the Xre–RES complex represses transcription via binding to an imperfect inverted repeat region downstream of the σ 70 element. We furthermore confirm the presence of the unusual 4:2 TA complex in solution and show that it preferably binds the imperfect inverted repeat in a 1:1 ratio. In addition, we show that the isolated Xre antitoxin is an aggregation-prone monomer in vitro and a weak repressor in vivo. We find that the Xre dimer can dissociates within the 4:2 complex and result in a 2:2 TA complex that still neutralizes the RES toxin but cannot bind DNA. Together, our data suggests that the asymmetry of the promoter is important for both transcription and repression, and we propose a model in which the Xre-RES complex regulates transcription through a dynamic and concentration-dependent equilibrium between a non-binding (2:2) and a DNA-binding (4:2) form. IMPORTANCE Transcriptional regulation is fundamental for bacteria to survive in a constantly changing environment and requires the RNA polymerase, which recognizes and binds to a DNA element (the promoter) located upstream of the gene and initiates transcription. Transcriptional repressors can bind DNA and inhibit transcription by the RNA polymerase, but the exact recognition of DNA binding sites remains unclear. The Xre antitoxin from the Pseudomonas putida toxin-antitoxin system Xre-RES contains a putative DNA-binding domain, and the unusual ratio of antitoxin to toxin within the protein complex suggests two potential DNA-binding sites. In this work, we show that Xre-RES auto-regulates its own transcription through an asymmetrical DNA binding-site, optimized to bind both the RNA polymerase and the Xre-RES complex, as well as a concentration-dependent ratio of toxin to antitoxin within the complex. Our work provides insight on how promoters can evolve to fine-tune the regulation of bacterial transcription.

Guanosine tetra- and pentaphosphate nucleotides, (p)ppGpp, function as central secondary messengers and alarmones in bacterial cell biology, signalling a range of stress conditions, including nutrient starvation and exposure to cell-wall-targeting antibiotics, and are critical for survival. While activation of the stringent response and alarmone synthesis on starved ribosomes by members of the RSH (Rel) class of proteins is well understood, much less is known about how single-domain small alarmone synthetases (SASs) and their corresponding alarmone hydrolases, the small alarmone hydrolases (SAHs), are regulated and contribute to (p)ppGpp homeostasis. The substrate spectrum of these enzymes has recently been expanded to include hyperphosphorylated adenosine nucleotides, suggesting that they take part in a highly complex and interconnected signalling network. In this review, we provide an overview of our understanding of the SAHs and discuss their structure, function, regulation, and phylogeny.

ABSTRACT Toxin:antitoxin (TA) systems are widespread in bacteria and were first identified as plasmid addiction systems that kill bacteria lacking a TA-encoding plasmid following cell division. TA systems have also been implicated in bacterial persistence and antibiotic tolerance, which can be precursors of antibiotic resistance. Here, we identified a clinical isolate of Shigella sonnei (CS14) with a remarkably stable pINV virulence plasmid; pINV is usually frequently lost from S. sonnei , but plasmid loss was not detected from CS14. We found that the plasmid in CS14 is stabilized by a single nucleotide polymorphism (SNP) in its vapBC TA system. VapBC TA systems are the most common Type II TA system in bacteria, and consist of a VapB antitoxin and VapC PIN domain-containing toxin. The plasmid stabilizing SNP leads to a Q12L substitution in the DNA-binding domain of VapB, which reduces VapBC binding to its own promoter, impairing vapBC autorepression. However, VapB L12 C mediates high-level plasmid stabilization because VapB L12 is more prone to degradation by Lon than wild-type VapB; this liberates VapC to efficiently kill bacteria that no longer contain a plasmid. Of note, mutations that confer tolerance to antibiotics in Escherichia coli also map to the DNA-binding domain of VapBC encoded by the chromosomally integrated F plasmid. We demonstrate that the tolerance mutations also enhance plasmid stabilization by the same mechanism as VapB L12 . Our findings highlight the links between plasmid maintenance and antibiotic tolerance, both of which can promote the development of antimicrobial resistance. IMPORTANCE Our work addresses two processes, the maintenance of plasmids and antibiotic tolerance; both contribute to the development of antimicrobial resistance in bacteria that cause human disease. Here, we found a single nucleotide change in the vapBC toxin:antitoxin system that stabilizes the large virulence plasmid of Shigella sonnei . The mutation is in the vapB antitoxin gene and makes the antitoxin more likely to be degraded, releasing the VapC toxin to efficiently kill cells without the plasmid (and thus unable to produce more antitoxin as an antidote). We found that vapBC mutations in E. coli that lead to antibiotic tolerance (a precursor to resistance) also operate by the same mechanism ( i.e. , generating VapB that is prone to cleavage); free VapC during tolerance will arrest bacterial growth and prevent susceptibility to antibiotics. This work shows the mechanistic links between plasmid maintenance and tolerance, and has applications in biotech and in the design and evaluation of vaccines against shigellosis.

In Escherichia coli , the 14-cistron phn operon encoding carbon-phosphorus lyase allows for utilisation of phosphorus from a wide range of stable phosphonate compounds containing a C-P bond. As part of a complex, multi-step pathway, the PhnJ subunit was shown to cleave the C-P bond via a radical mechanism, however, the details of the reaction could not immediately be reconciled with the crystal structure of a 220 kDa PhnGHIJ C-P lyase core complex, leaving a significant gap in our understanding of phosphonate breakdown in bacteria. Here, we show using single-particle cryogenic electron microscopy that PhnJ mediates binding of a double dimer of the ATP-binding cassette proteins, PhnK and PhnL, to the core complex. ATP hydrolysis induces drastic structural remodelling leading to opening of the core complex and reconfiguration of a metal-binding and putative active site located at the interface between the PhnI and PhnJ subunits.

Many bacteria encode multiple toxin–antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of the Escherichia coli hipBA system, HipA, is a kinase that inhibits translation via phosphorylation of glutamyl-tRNA synthetase. Enteropathogenic E. coli O127:H6 encodes the hipBA-like, tripartite TA system; hipBST, in which the HipT toxin specifically targets the tryptophanyl-tRNA synthetase, TrpS. Notably, in the tripartite system, the function as antitoxin has been taken over by the third protein, HipS, but the molecular details of how activity of HipT is inhibited remain poorly understood. Here, we show that HipBST is structurally different from E. coli HipBA and that the unique HipS protein, which is homologous to the N-terminal subdomain of HipA, inhibits the kinase through insertion of a conserved Trp residue into the active site. We also show how auto-phosphorylation at two conserved sites in the kinase toxin serve different roles and affect the ability of HipS to neutralize HipT. Finally, solution structural studies show how phosphorylation affects overall TA complex flexibility.

ABSTRACT Structural biology describes biological processes at the molecular level and is an integral part of undergraduate study programs in molecular biosciences. Students are often fascinated by the visualizations created by molecular graphics software, which allow them to see the molecular world for the first time. Today, molecular visualization and structural analysis do not require expensive high-end computers but can be performed on the students' own laptops and are therefore highly suited for active learning approaches. We have designed a semester-long learning path that integrates molecular graphics and structural analysis using PyMOL into an undergraduate course in biomolecular structure and function. Compared to stand-alone PyMOL introductions, the semester-long learning path allows for an improved pedagogical design. The path progressively introduces more advanced functions in relevant scientific contexts and allows for spaced repetition. Advanced analysis functions in PyMOL are available only via the command line, so the learning path also teaches basic scripting and serves as an accessible introduction to computational thinking because a few lines of code can produce stunning results. Student surveys carried out at the end of the course suggest that the learning path supported the ability to perform structural analysis to a high degree. Moreover, a simulated exam showed that almost all students were able to carry out basic visualization tasks using PyMOL scripts, while three-quarters could undertake advanced structural analysis after following the course. In summary, integration of molecular graphics software with teaching of structural biochemistry allows a hands-on approach to analyzing molecular mechanisms and introduces biologically oriented students to computational thinking.

Phosphorus (P) is an essential macronutrient that can be extracted from the environment by several metabolic pathways. In E. coli, phosphate deprivation activates the the 14-cistron phn operon that encodes carbon-phosphorus (C-P) lyase. C-P lyase confers the ability to extract phosphorus from a wide range of phosphonate compounds containing the chemically highly stable C-P bond. Phosphonates are widespread in nature and due to their similarity to phosphate esters, have important applications in agriculture and biomedicine, as pesticides (glyphosate, RoundUp) and antibiotics, respectively. C-P lyase catalyses a complex multi-step pathway that directly depends on 10 of the encoded proteins (PhnGHIJKLMNOP). Of these, the PhnJ subunit was shown to be responsible for cleavage of the C-P bond via a strict anaerobic glycyl radical mechanism that requires an iron-sulphur (Fe S ) cluster and S-adenosyl methionine (SAM) for radical activation. Surprisingly, however, this mechanism was not immediately compatible with the crystal structure of a PhnGHIJ C-P lyase core complex, which placed key residues at a significant distance, thus leaving a large gap in our understanding of the mechanism of phosphonate breakdown. The phn operon also encodes two ATP-binding cassette (ABC) proteins with homology to the nucleotide-binding domains of ABC transporters, PhnK and PhnL, for which no function has been assigned. Here, we present four high-resolution (~2 Å), single-particle cryo-electron microscopy structures of 300+ kDa C-P lyase complexes in several key, functional states. Together, these structures reveal how the PhnGHIJ core complex interacts with a unique double dimer of PhnK and PhnL subunits, in which PhnK, in a tight ATP-bound conformation, bridges the core complex and PhnL. We also show using additional structures determined under ATP turnover conditions, that ATP hydrolysis in PhnK induces a dramatic remodelling of the core complex, leading to opening and large-scale movement of several subunits. Moreover, we show that ATP hydrolysis and binding of both ABC subunits is required for growth of E. coli on phosphonate. Finally, we demonstrate that C-P lyase in the closed state binds substrate at a metal-binding site located at the interface between the PhnI and PhnJ subunits, and that ATP hydrolysis leads to a reorganisation of this active site in a way likely contributes to substrate/product exchange. In summary, we provide several novel insights into the elusive process of phosphonate breakdown by C-P lyase in microorganisms and represent a solid structural basis for understanding the catalytic process. Our structural data also uncover a hitherto unknown configuration of ABCs in which two ABC dimers directly interact with each other that have broad implications for our understanding of the role of this module in biological systems. 1. Metcalf, W. W. & Wanner, B. L. Gene 129, 27-32, (1993). 2. Kamat, S. S., Williams, H. J., Dangott, L. J., Chakrabarti, M. & Raushel, F. M. Nature 497, 132-136, (2013). 3. Seweryn, P. et al. Nature 525, 68-72, (2015).

Many bacteria encode multiple toxin-antitoxin (TA) systems targeting separate, but closely related, cellular functions. The toxin of the E. coli hipBA system, HipA, is a kinase that inhibits inhibits translation via phosphorylation of glutamyl-tRNA synthetase. Enteropathogenic E. coli (EPEC) O127:H6 encodes an additional, tripartite TA module, hipBST, for which the HipT toxin was shown to specifically target tryptophanyl-tRNA synthetase, TrpS. Surprisingly, the function as antitoxin has been taken over by the third protein, HipS, but the molecular details of how activity of HipT is controlled remain poorly understood. Here, we show that HipBST is markedly different from HipBA and that the unique HipS protein, which is homologous to the N-terminal subdomain of HipA, has evolved to function as antitoxin by breaking the kinase active site. We also show how auto-phosphorylation at two conserved sites in the kinase toxin serve to dually regulate binding of HipS and kinase activity. Finally, we demonstrate that the HipBST complex is dynamic and present a cohesive model for the regulation and activation of this type of three-component system.