Mycobacterial chaperonins in cellular proteostasis: Evidence for chaperone function of Cpn60.1 and Cpn60.2-mediated protein folding.

Mycobacterium tuberculosis encodes two chaperonin proteins, MtbCpn60.1 and MtbCpn60.2, that share substantial sequence similarity with the Escherichia coli chaperonin, GroEL. However, unlike GroEL, MtbCpn60.1 and MtbCpn60.2 purify as lower-order oligomers. Previous studies have shown that MtbCpn60.2 can functionally replace GroEL in E. coli, while the function of MtbCpn60.1 remained an enigma. Here, we demonstrate the molecular chaperone function of MtbCpn60.1 and MtbCpn60.2, by probing their ability to assist the folding of obligate chaperonin clients, DapA, FtsE and MetK, in an E. coli strain depleted of endogenous GroEL. We show that both MtbCpn60.1 and MtbCpn60.2 support cell survival and cell division by assisting the folding of DapA and FtsE, but only MtbCpn60.2 completely rescues GroEL-depleted E. coli cells. We also show that, unlike MtbCpn60.2, MtbCpn60.1 has limited ability to support cell growth and proliferation and assist the folding of MetK. Our findings suggest that the client pools of GroEL and MtbCpn60.2 overlap substantially, while MtbCpn60.1 folds only a small subset of GroEL clients. We conclude that the differences between MtbCpn60.1 and MtbCpn60.2 may be a consequence of their intrinsic sequence features, which affect their thermostability, efficiency, clientomes and modes of action.

A Bayesian non-parametric mixed-effects model of microbial growth curves.

Substantive changes in gene expression, metabolism, and the proteome are manifested in overall changes in microbial population growth. Quantifying how microbes grow is therefore fundamental to areas such as genetics, bioengineering, and food safety. Traditional parametric growth curve models capture the population growth behavior through a set of summarizing parameters. However, estimation of these parameters from data is confounded by random effects such as experimental variability, batch effects or differences in experimental material. A systematic statistical method to identify and correct for such confounding effects in population growth data is not currently available. Further, our previous work has demonstrated that parametric models are insufficient to explain and predict microbial response under non-standard growth conditions. Here we develop a hierarchical Bayesian non-parametric model of population growth that identifies the latent growth behavior and response to perturbation, while simultaneously correcting for random effects in the data. This model enables more accurate estimates of the biological effect of interest, while better accounting for the uncertainty due to technical variation. Additionally, modeling hierarchical variation provides estimates of the relative impact of various confounding effects on measured population growth.

Minichaperone (GroEL191-345) mediated folding of MalZ proceeds by binding and release of native and functional intermediates.

The isolated apical domain of GroEL consisting of residues 191-345 (known as "minichaperone") binds and assists the folding of a wide variety of client proteins without GroES and ATP, but the mechanism of its action is still unknown. In order to probe into the matter, we have examined minichaperone-mediated folding of a large aggregation prone protein Maltodextrin-glucosidase (MalZ). The key objective was to identify whether MalZ exists free in solution, or remains bound to, or cycling on and off the minichaperone during the refolding process. When GroES was introduced during refolding process, production of the native MalZ was inhibited. We also observed the same findings with a trap mutant of GroEL, which stably captures a predominantly non-native MalZ released from minichaperone during refolding process, but does not release it. Tryptophan and ANS fluorescence measurements indicated that refolded MalZ has the same structure as the native MalZ, but that its structure when bound to minichaperone is different. Surface plasmon resonance measurements provide an estimate for the equilibrium dissociation constant KD for the MalZ-minichaperone complex of 0.21 ±â€¯0.04 µM, which are significantly higher than for most GroEL clients. This showed that minichaperone interacts loosely with MalZ to allow the protein to change its conformation and fold while bound during the refolding process. These observations suggest that the minichaperone works by carrying out repeated cycles of binding aggregation-prone protein MalZ in a relatively compact conformation and in a partially folded but active state, and releasing them to attempt to fold in solution.

Reconstructing promoter activity from Lux bioluminescent reporters.

The bacterial Lux system is used as a gene expression reporter. It is fast, sensitive and non-destructive, enabling high frequency measurements. Originally developed for bacterial cells, it has also been adapted for eukaryotic cells, and can be used for whole cell biosensors, or in real time with live animals without the need for euthanasia. However, correct interpretation of bioluminescent data is limited: the bioluminescence is different from gene expression because of nonlinear molecular and enzyme dynamics of the Lux system. We have developed a computational approach that, for the first time, allows users of Lux assays to infer gene transcription levels from the light output. This approach is based upon a new mathematical model for Lux activity, that includes the actions of LuxAB, LuxEC and Fre, with improved mechanisms for all reactions, as well as synthesis and turn-over of Lux proteins. The model is calibrated with new experimental data for the LuxAB and Fre reactions from Photorhabdus luminescens-the source of modern Lux reporters-while literature data has been used for LuxEC. Importantly, the data show clear evidence for previously unreported product inhibition for the LuxAB reaction. Model simulations show that predicted bioluminescent profiles can be very different from changes in gene expression, with transient peaks of light output, very similar to light output seen in some experimental data sets. By incorporating the calibrated model into a Bayesian inference scheme, we can reverse engineer promoter activity from the bioluminescence. We show examples where a decrease in bioluminescence would be better interpreted as a switching off of the promoter, or where an increase in bioluminescence would be better interpreted as a longer period of gene expression. This approach could benefit all users of Lux technology.

Structural and Functional Analysis of the Escherichia coli Acid-Sensing Histidine Kinase EvgS.

The EvgS/EvgA two-component system of Escherichia coli is activated in response to low pH and alkali metals and regulates many genes, including those for the glutamate-dependent acid resistance system and a number of efflux pumps. EvgS, the sensor kinase, is one of five unconventional histidine kinases (HKs) in E. coli and has a large periplasmic domain and a cytoplasmic PAS domain in addition to phospho-acceptor, HK and dimerization, internal receiver, and phosphotransfer domains. Mutations that constitutively activate the protein at pH 7 map to the PAS domain. Here, we built a homology model of the periplasmic region of EvgS, based on the structure of the equivalent region of the BvgS homologue, to guide mutagenesis of potential key residues in this region. We show that histidine 226 is required for induction and that it is structurally colocated with a proline residue (P522) at the top of the predicted transmembrane helix that is expected to play a key role in passing information to the cytoplasmic domains. We also show that the constitutive mutations in the PAS domain can be further activated by low external pH. Expression of the cytoplasmic part of the protein alone also gives constitutive activation, which is lost if the constitutive PAS mutations are present. These findings are consistent with a model in which EvgS senses both external and internal pH and is activated by a shift from a tight inactive to a weak active dimer, and we present an analysis of the purified cytoplasmic portion of EvgS that supports this.IMPORTANCE One of the ways bacteria sense their environment is through two-component systems, which have one membrane-bound protein to do the sensing and another inside the cell to turn genes on or off in response to what the membrane-bound protein has detected. The membrane-bound protein must thus be able to detect the stress and signal this detection event to the protein inside the cell. To understand this process, we studied a protein that helps E. coli to survive exposure to low pH, which it must do before taking up residence in the gastrointestinal tract. We describe a predicted structure for the main sensing part of the protein and identify some key residues within it that are involved in the sensing and signaling processes. We propose a mechanism for how the protein may become activated and present some evidence to support our proposal.

Replacement of GroEL in Escherichia coli by the Group II Chaperonin from the Archaeon Methanococcus maripaludis.

UNLABELLED: Chaperonins are required for correct folding of many proteins. They exist in two phylogenetic groups: group I, found in bacteria and eukaryotic organelles, and group II, found in archaea and eukaryotic cytoplasm. The two groups, while homologous, differ significantly in structure and mechanism. The evolution of group II chaperonins has been proposed to have been crucial in enabling the expansion of the proteome required for eukaryotic evolution. In an archaeal species that expresses both groups of chaperonins, client selection is determined by structural and biochemical properties rather than phylogenetic origin. It is thus predicted that group II chaperonins will be poor at replacing group I chaperonins. We have tested this hypothesis and report here that the group II chaperonin from Methanococcus maripaludis (Mm-cpn) can partially functionally replace GroEL, the group I chaperonin of Escherichia coli Furthermore, we identify and characterize two single point mutations in Mm-cpn that have an enhanced ability to replace GroEL function, including one that allows E. coli growth after deletion of the groEL gene. The biochemical properties of the wild-type and mutant Mm-cpn proteins are reported. These data show that the two groups are not as functionally diverse as has been thought and provide a novel platform for genetic dissection of group II chaperonins. IMPORTANCE: The two phylogenetic groups of the essential and ubiquitous chaperonins diverged approximately 3.7 billion years ago. They have similar structures, with two rings of multiple subunits, and their major role is to assist protein folding. However, they differ with regard to the details of their structure, their cofactor requirements, and their reaction cycles. Despite this, we show here that a group II chaperonin from a methanogenic archaeon can partially substitute for the essential group I chaperonin GroEL in E. coli and that we can easily isolate mutant forms of this chaperonin with further improved functionality. This is the first demonstration that these two groups, despite the long time since they diverged, still overlap significantly in their functional properties.

Characterization of mutations in the PAS domain of the EvgS sensor kinase selected by laboratory evolution for acid resistance in Escherichia coli.

Laboratory-based evolution and whole-genome sequencing can link genotype and phenotype. We used evolution of acid resistance in exponential phase Escherichia coli to study resistance to a lethal stress. Iterative selection at pH 2.5 generated five populations that were resistant to low pH in early exponential phase. Genome sequencing revealed multiple mutations, but the only gene mutated in all strains was evgS, part of a two-component system that has already been implicated in acid resistance. All these mutations were in the cytoplasmic PAS domain of EvgS, and were shown to be solely responsible for the resistant phenotype, causing strong upregulation at neutral pH of genes normally induced by low pH. Resistance to pH 2.5 in these strains did not require the transporter GadC, or the sigma factor RpoS. We found that EvgS-dependent constitutive acid resistance to pH 2.5 was retained in the absence of the regulators GadE or YdeO, but was lost if the oxidoreductase YdeP was also absent. A deletion in the periplasmic domain of EvgS abolished the response to low pH, but not the activity of the constitutive mutants. On the basis of these results we propose a model for how EvgS may become activated by low pH.

The Escherichia coli Acid Stress Response and Its Significance for Pathogenesis.

Escherichia coli has a remarkable ability to survive low pH and possesses a number of different genetic systems that enable it to do this. These may be expressed constitutively, typically in stationary phase, or induced by growth under a variety of conditions. The activities of these systems have been implicated in the ability of E. coli to pass the acidic barrier of the stomach and to become established in the gastrointestinal tract, something causing serious infections. However, much of the work characterizing these systems has been done on standard laboratory strains of E. coli and under conditions which do not closely resemble those found in the human gut. Here we review what is known about acid resistance in E. coli as a model laboratory organism and in the context of its lifestyle as an inhabitant-sometimes an unwelcome one-of the human gut.

Structural basis of inhibition of Mycobacterium tuberculosis DprE1 by benzothiazinone inhibitors.

Resistance against currently used antitubercular therapeutics increasingly undermines efforts to contain the worldwide tuberculosis (TB) epidemic. Recently, benzothiazinone (BTZ) inhibitors have shown nanomolar potency against both drug-susceptible and multidrug-resistant strains of the tubercle bacillus. However, their proposed mode of action is lacking structural evidence. We report here the crystal structure of the BTZ target, FAD-containing oxidoreductase Mycobacterium tuberculosis DprE1, which is essential for viability. Different crystal forms of ligand-free DprE1 reveal considerable levels of structural flexibility of two surface loops that seem to govern accessibility of the active site. Structures of complexes with the BTZ-derived nitroso derivative CT325 reveal the mode of inhibitor binding, which includes a covalent link to conserved Cys387, and reveal a trifluoromethyl group as a second key determinant of interaction with the enzyme. Surprisingly, we find that a noncovalent complex was formed between DprE1 and CT319, which is structurally identical to CT325 except for an inert nitro group replacing the reactive nitroso group. This demonstrates that binding of BTZ-class inhibitors to DprE1 is not strictly dependent on formation of the covalent link to Cys387. On the basis of the structural and activity data, we propose that the complex of DrpE1 bound to CT325 is a representative of the BTZ-target complex. These results mark a significant step forward in the characterization of a key TB drug target.

Laboratory adapted Escherichia coli K-12 becomes a pathogen of Caenorhabditis elegans upon restoration of O antigen biosynthesis.

Escherichia coli has been the leading model organism for many decades. It is a fundamental player in modern biology, facilitating the molecular biology revolution of the last century. The acceptance of E. coli as model organism is predicated primarily on the study of one E. coli lineage; E. coli K-12. However, the antecedents of today's laboratory strains have undergone extensive mutagenesis to create genetically tractable offspring but which resulted in loss of several genetic traits such as O antigen expression. Here we have repaired the wbbL locus, restoring the ability of E. coli K-12 strain MG1655 to express the O antigen. We demonstrate that O antigen production results in drastic alterations of many phenotypes and the density of the O antigen is critical for the observed phenotypes. Importantly, O antigen production enables laboratory strains of E. coli to enter the gut of the Caenorhabditis elegans worm and to kill C. elegans at rates similar to pathogenic bacterial species. We demonstrate C. elegans killing is a feature of other commensal E. coli. We show killing is associated with bacterial resistance to mechanical shear and persistence in the C. elegans gut. These results suggest C. elegans is not an effective model of human-pathogenic E. coli infectious disease.

Exploitation of microbial activities at low pH to enhance planetary health.

Awareness is growing that human health cannot be considered in isolation but is inextricably woven with the health of the environment in which we live. It is, however, under-recognized that the sustainability of human activities strongly relies on preserving the equilibrium of the microbial communities living in/on/around us. Microbial metabolic activities are instrumental for production, functionalization, processing, and preservation of food. For circular economy, microbial metabolism would be exploited to produce building blocks for the chemical industry, to achieve effective crop protection, agri-food waste revalorization, or biofuel production, as well as in bioremediation and bioaugmentation of contaminated areas. Low pH is undoubtedly a key physical-chemical parameter that needs to be considered for exploiting the powerful microbial metabolic arsenal. Deviation from optimal pH conditions has profound effects on shaping the microbial communities responsible for carrying out essential processes. Furthermore, novel strategies to combat contaminations and infections by pathogens rely on microbial-derived acidic molecules that suppress/inhibit their growth. Herein, we present the state-of-the-art of the knowledge on the impact of acidic pH in many applied areas and how this knowledge can guide us to use the immense arsenal of microbial metabolic activities for their more impactful exploitation in a Planetary Health perspective.

On the potential role of naturally occurring carboxylic organic acids as anti-infective agents: opportunities and challenges.

Carboxylic organic acids are intermediates of central carbon metabolic pathways (e.g. acetic, propionic, citric, and lactic acid) long known to have potent antimicrobial potential, mainly at acidic pHs. The food industry has been leveraging those properties for years, using many of these acids as preservatives to inhibit the growth of pathogenic and/or spoilage fungal and bacterial species. A few of these molecules (the most prominent being acetic acid) have been used as antiseptics since Hippocratic medicine, mainly to treat infected wounds in patients with burns. With the growth of antibiotic therapy, the use of carboxylic acids (and other chemical antiseptics) in clinical settings lost relevance; however, with the continuous emergence of multi-antibiotic/antifungal resistant strains, the search for alternatives has intensified. This prospective article raises awareness of the potential of carboxylic acids to control infections in clinical settings, considering not only their previous exploitation in this context (which we overview) but also the positive experience of their safe use in food preservation. At a time of great concern with antimicrobial resistance and the slow arrival of new antimicrobial therapeutics to the market, further exploration of organic acids as anti-infective molecules may pave the way to more sustainable prophylactic and therapeutic approaches.

The unusual mycobacterial chaperonins: evidence for in vivo oligomerization and specialization of function.

The pathogen Mycobacterium tuberculosis expresses two chaperonins, one (Cpn60.1) dispensable and one (Cpn60.2) essential. These proteins have been reported not to form oligomers despite the fact that oligomerization of chaperonins is regarded as essential for their function. We show here that the Cpn60.2 homologue from Mycobacterium smegmatis also fails to oligomerize under standard conditions. However, we also show that the Cpn60.2 proteins from both organisms can replace the essential groEL gene of Escherichia coli, and that they can function with E. coli GroES cochaperonin, as well as with their cognate cochaperonin proteins, strongly implying that they form oligomers in vivo. We show that the Cpn60.1 proteins, but not the Cpn60.2 proteins, can complement for loss of the M. smegmatis cpn60.1 gene. We investigated the oligomerization of the Cpn60.2 proteins using analytical ultracentrifugation and mass spectroscopy. Both form monomers under standard conditions, but they form higher order oligomers in the presence of kosmotropes and ADP or ATP. Under these conditions, their ATPase activity is significantly enhanced. We conclude that the essential mycobacterial chaperonins, while unstable compared to many other bacterial chaperonins, do act as oligomers in vivo, and that there has been specialization of function of the mycobacterial chaperonins following gene duplication.

A COST Action on microbial responses to low pH: Developing links and sharing resources across the academic-industrial divide.

We present work of our COST Action on "Understanding and exploiting the impacts of low pH on micro-organisms". First, we summarise a workshop held at the European Federation of Biotechnology meeting on Microbial Stress Responses (online in 2020) on "Industrial applications of low pH stress on microbial bio-based production", as an example of an initiative fostering links between pure and applied research. We report the outcomes of a small survey on the challenging topic of developing links between researchers working in academia and industry that show that, while people in different sectors strongly support such links, barriers remain that obstruct this process. We present the thoughts of an expert panel held as part of the workshop above, where people with experience of collaboration between academia and industry shared ideas on how to develop and maintain links. Access to relevant information is essential for research in all sectors, and because of this we have developed, as part of our COST Action goals, two resources for the free use of all researchers with interests in any aspects of microbial responses to low pH. These are (1) a comprehensive database of references in the literature on different aspects of acid stress responses in different bacterial and fungal species, and (2) a database of research expertise across our network. We invite the community of researchers working in this field to take advantage of these resources to identify relevant literature and opportunities for establishing collaborations.

RcsB is required for inducible acid resistance in Escherichia coli and acts at gadE-dependent and -independent promoters.

RcsB interacts with GadE to mediate acid resistance in stationary-phase Escherichia coli K-12. We show here that RcsB is also required for inducible acid resistance in exponential phase and that it acts on promoters that are not GadE regulated. It is also required for acid resistance in E. coli O157:H7.

Use of Transposon Directed Insertion-Site Sequencing to Probe the Antibacterial Mechanism of a Model Honey on E. coli K-12.

Antimicrobial resistance is an ever-growing health concern worldwide that has created renewed interest in the use of traditional anti-microbial treatments, including honey. However, understanding the underlying mechanism of the anti-microbial action of honey has been hampered due to the complexity of its composition. High throughput genetic tools could assist in understanding this mechanism. In this study, the anti-bacterial mechanism of a model honey, made of sugars, hydrogen peroxide, and gluconic acid, was investigated using genome-wide transposon mutagenesis combined with high-throughput sequencing (TraDIS), with the strain Escherichia coli K-12 MG1655 as the target organism. We identified a number of genes which when mutated caused a severe loss of fitness when cells were exposed to the model honey. These genes encode membrane proteins including those involved in uptake of essential molecules, and components of the electron transport chain. They are enriched for pathways involved in intracellular homeostasis and redox activity. Genes involved in assembly and activity of formate dehydrogenase O (FDH-O) were of particular note. The phenotypes of mutants in a subset of the genes identified were confirmed by phenotypic screening of deletion strains. We also found some genes which when mutated led to enhanced resistance to treatment with the model honey. This study identifies potential synergies between the main honey stressors and provides insights into the global antibacterial mechanism of this natural product.

Chaperonin Abundance Enhances Bacterial Fitness.

The ability of chaperonins to buffer mutations that affect protein folding pathways suggests that their abundance should be evolutionarily advantageous. Here, we investigate the effect of chaperonin overproduction on cellular fitness in Escherichia coli. We demonstrate that chaperonin abundance confers 1) an ability to tolerate higher temperatures, 2) improved cellular fitness, and 3) enhanced folding of metabolic enzymes, which is expected to lead to enhanced energy harvesting potential.

The hrcA and hspR regulons of Campylobacter jejuni.

The human pathogen Campylobacter jejuni has a classic heat shock response, showing induction of chaperones and proteases plus several unidentified proteins in response to a small increase in growth temperature. The genome contains two homologues to known heat shock response regulators, HrcA and HspR. Previous work has shown that HspR controls several heat-shock genes, but the hrcA regulon has not been defined. We have constructed single and double deletions of C. jejuni hrcA and hspR and analysed gene expression using microarrays. Only a small number of genes are controlled by these two regulators, and the two regulons overlap. Strains mutated in hspR, but not those mutated in hrcA, showed enhanced thermotolerance. Some genes previously identified as being downregulated in a strain lacking hspR showed no change in expression in our experiments.

Mapping the Transcriptional and Fitness Landscapes of a Pathogenic E. coli Strain: The Effects of Organic Acid Stress under Aerobic and Anaerobic Conditions.

Several methods are available to probe cellular responses to external stresses at the whole genome level. RNAseq can be used to measure changes in expression of all genes following exposure to stress, but gives no information about the contribution of these genes to an organism's ability to survive the stress. The relative contribution of each non-essential gene in the genome to the fitness of the organism under stress can be obtained using methods that use sequencing to estimate the frequencies of members of a dense transposon library grown under different conditions, for example by transposon-directed insertion sequencing (TraDIS). These two methods thus probe different aspects of the underlying biology of the organism. We were interested to determine the extent to which the data from these two methods converge on related genes and pathways. To do this, we looked at a combination of biologically meaningful stresses. The human gut contains different organic short-chain fatty acids (SCFAs) produced by fermentation of carbon compounds, and Escherichia coli is exposed to these in its passage through the gut. Their effect is likely to depend on both the ambient pH and the level of oxygen present. We, therefore, generated RNAseq and TraDIS data on a uropathogenic E. coli strain grown at either pH 7 or pH 5.5 in the presence or absence of three SCFAs (acetic, propionic and butyric), either aerobically or anaerobically. Our analysis identifies both known and novel pathways as being likely to be important under these conditions. There is no simple correlation between gene expression and fitness, but we found a significant overlap in KEGG pathways that are predicted to be enriched following analysis of the data from the two methods, and the majority of these showed a fitness signature that would be predicted from the gene expression data, assuming expression to be adaptive. Genes which are not in the E. coli core genome were found to be particularly likely to show a positive correlation between level of expression and contribution to fitness.

The Signaling Molecule Indole Inhibits Induction of the AR2 Acid Resistance System in Escherichia coli.

Induction of the AR2 acid response system of Escherichia coli occurs at a moderately low pH (pH 5.5) and leads to high levels of resistance to pH levels below 2.5 in the presence of glutamate. Induction is mediated in part by the EvgAS two component system. Here, we show that the bacterial signaling molecule indole inhibits the induction of key promoters in the AR2 system and blocks the development of glutamate-dependent acid resistance. The addition of tryptophan, the precursor for indole biosynthesis, had the same effects, and this block was relieved in a tnaA mutant, which is unable to synthesize indole. Expression of a constitutively active EvgS protein was able to relieve the inhibition caused by indole, consistent with EvgS being inhibited directly or indirectly by indole. Indole had no effect on autophosphorylation of the isolated cytoplasmic domain of EvgS. This is consistent with a model where indole directly or indirectly affects the ability of EvgS to detect its inducing signal or to transduce this information across the cytoplasmic membrane. The inhibitory activity of indole on the AR2 system is not related to its ability to act as an ionophore, and, conversely, the ionophore CCCP had no effect on acid-induced AR2 promoter activity, showing that the proton motive force is unlikely to be a signal for induction of the AR2 system.