Two-Component System Sensor Kinases from Asgardian Archaea May Be Witnesses to Eukaryotic Cell Evolution.

The signal transduction paradigm in bacteria involves two-component systems (TCSs). Asgardarchaeota are archaea that may have originated the current eukaryotic lifeforms. Most research on these archaea has focused on eukaryotic-like features, such as genes involved in phagocytosis, cytoskeleton structure, and vesicle trafficking. However, little attention has been given to specific prokaryotic features. Here, the sequence and predicted structural features of TCS sensor kinases analyzed from two metagenome assemblies and a genomic assembly from cultured Asgardian archaea are presented. The homology of the sensor kinases suggests the grouping of Lokiarchaeum closer to bacterial homologs. In contrast, one group from a Lokiarchaeum and a meta-genome assembly from Candidatus Heimdallarchaeum suggest the presence of a set of kinases separated from the typical bacterial TCS sensor kinases. AtoS and ArcB homologs were found in meta-genome assemblies along with defined domains for other well-characterized sensor kinases, suggesting the close link between these organisms and bacteria that may have resulted in the metabolic link to the establishment of symbiosis. Several kinases are predicted to be cytoplasmic; some contain several PAS domains. The data shown here suggest that TCS kinases in Asgardian bacteria are witnesses to the transition from bacteria to eukaryotic organisms.

Plasma membrane damage repair is mediated by an acid sphingomyelinase in Entamoeba histolytica.

Entamoeba histolytica is a pathogen that during its infective process confronts the host defenses, which damages the amoebic plasma membrane (PM), resulting in the loss of viability. However, it is unknown whether amoebic trophozoites are able to repair their PM when it is damaged. Acid sphingomyelinases (aSMases) have been reported in mammalian cells to promote endocytosis and removal of PM lesions. In this work, six predicted amoebic genes encoding for aSMases were found to be transcribed in the HM1:IMSS strain, finding that the EhaSM6 gene is the most transcribed in basal growth conditions and rendered a functional protein. The secreted aSMase activity detected was stimulated by Mg+2 and inhibited by Co+2. Trophozoites that overexpress the EhaSM6 gene (HM1-SM6HA) exhibit an increase of 2-fold in the secreted aSMase activity. This transfectant trophozoites exposed to pore-forming molecules (SLO, Magainin, ß-Defensin 2 and human complement) exhibited an increase from 6 to 25-fold in the secreted aSMase activity which correlated with higher amoebic viability in a Ca+2 dependent process. However, other agents that affect the PM such as hydrogen peroxide also induced an increase of secreted aSMase, but to a lesser extent. The aSMase6 enzyme is N- and C-terminal processed. Confocal and transmission electron microscopy showed that trophozoites treated with SLO presented a migration of lysosomes containing the aSMase towards the PM, inducing the formation of membrane patches and endosomes in the control strain. These cellular structures were increased in the overexpressing strain, indicating the involvement of the aSMase6 in the PM injury repair. The pore-forming molecules induced an increase in the expression of EhaSM1, 2, 5 and 6 genes, meanwhile, hydrogen peroxide induced an increase in all of them. In all the conditions evaluated, the EhaSM6 gene exhibited the highest levels of induction. Overall, these novel findings show that the aSMase6 enzyme from E. histolytica promotes the repair of the PM damaged with pore-forming molecules to prevent losing cell integrity. This novel system could act when encountered with the lytic defense systems of the host.

Possible role played by the SINE2 element in gene regulation, as demonstrated by differential processing and polyadenylation in avirulent strains of E. histolytica.

Entamoeba histolytica represents a useful model in parasitic organisms due to its complex genomic organization and survival mechanisms. To counteract pathogenic organisms, it is necessary to characterize their molecular biology to design new strategies to combat them. In this report, we investigated a less-known genetic element, short interspersed nuclear element 2 (SINE2), that is present in this ameba and is highly transcribed and polyadenylated. In this study, we show that in two different nonvirulent strains of E. histolytica, SINE2 is differentially processed into two transcript fragments, that is, a full-length 560-nt fragment and a shorter 393-nt fragment bearing an approximately 18-nt polyadenylation tail. Sequence analysis of the SINE2 transcript showed that a Musashi-like protein may bind to it. Also, two putative Musashi-like sequences were identified on the transcript. Semiquantitative expression analysis of the two Musashi-like proteins identified in the E. histolytica genome (XP_648918 and XP_649094) showed that XP_64094 is overexpressed in the nonvirulent strains tested. The information available in the literature and the results presented in this report indicate that SINE2 may affect other genes, as observed with the epigenetic silencing of the G3 strain, by an antisense mechanism or via RNA-protein interactions that may ultimately be involved in the phenotype of nonvirulent strains of E. histolytica.

Flotillin homologue is involved in the swimming behavior of Escherichia coli.

Cellular membrane is a key component for maintaining cell shape and integrity. The classical membrane structure and function by Singer and Nicolson groundbreaking model has depicted the membrane as a homogeneous fluid structure. This view has changed by the discovery of discrete domains containing different lipid compositions, called lipid rafts, which play a key role in signal transduction in eukaryotic cells. In the past few years, lipid raft-like structures have been found in bacteria also, constituted by cardiolipin and other modified lipids, perhaps involved in generating a specific site for protein clustering. Here, we report the analysis of a protein termed YqiK from Escherichia coli, a prohibitin homolog that has been implicated in stress sensing by the formation of membrane-associated microdomains. The E. coli yqiK-deficient mutant strain showed an enhanced swimming behavior and was resistant to ampicillin but its response to other stressing conditions was similar to that of the wild-type strain. The abnormal swimming behavior is reversed when the protein is expressed in trans from a plasmid. Also, we demonstrate that YqiK is not redundant with QmcA, another flotillin homolog found in E. coli. Our results, along with the data available in the literature, suggest that YqiK may be involved in the formation of discrete membrane-associated signaling complexes that regulate and agglomerate signaling proteins to generate cell response to chemotaxis.

Development of an oxidative stress sensor in live bacteria using the optimized HyPer2 protein.

Oxidative stress is a key regulator in many cellular processes but also an important burden for living organisms. The source of oxidative damage usually is difficult to measure and assess with analytical tools or chemical indicators. One major limitation is to discriminate the presence of secondary oxidant molecules derived from the cellular metabolism after exposure to the oxidant or the scavenging capacity of reactive oxygen species by cells. Using a whole-cell reporter system based on an optimized HyPer2 protein for Escherichia coli expression, we demonstrate that, as previously shown for eukaryotic organisms, the effect at the transcriptional level of hydrogen peroxide can be monitored in vivo using flow cytometry of bacterial cells without the need of a direct analytical measurement. In this approach, we generated two different HyPer2 expression systems, one that is induced by IPTG and a second one that is induced by oxidative stress responsive promoters to control the expression of the HyPer2 protein and the exposure of higher H2O2 concentrations that has been shown to activate oxidative response genes. Both systems showed that the pathway that leads to the generation of H2O2 in vivo can be traced from H2O2 exposure. Our results indicate that hydrogen peroxide pulses can be readily detected in E. coli cells by a defined fluorescence signature that is H2O2 concentration-dependent. Our findings indicate that although less sensitive than purified protein or expressed in eukaryotic cells, HyPer2 is a good bacterial sensor for H2O2. As proof of concept, this system was used to trace the oxidative capacity of Toluidine Blue O showing that oxidative stress and redox imbalance is generated inside the cell. This system is expanding the repertoire of whole cell probes available for tracing cellular stress in bacteria.

Preparation of Peppermint Oil-Based Nanodevices Loaded with Paclitaxel: Cytotoxic and Apoptosis Studies in HeLa Cells.

In this work, several normal, oil-in-water (o/w) microemulsions (MEs) were prepared using peppermint essential oil, jojoba oil, trans-anethole, and vitamin E as oil phases to test their capacity to load paclitaxel (PTX). Initially, pseudo-ternary partial phase diagrams were constructed in order to find the normal microemulsion region using d-α-tocopherol polyethylene glycol 1000 succinate (TPGS-1000) as surfactant and isobutanol (iso-BuOH) as co-surfactant. Selected ME formulations were loaded with PTX reaching concentrations of 0.6 mg mL-1 for the peppermint oil and trans-anethole MEs, while for the vitamin E and jojoba oil MEs, the maximum concentration was 0.3 mg mL-1. The PTX-loaded MEs were stable according to the results of heating-cooling cycles and mechanical force (centrifugation) test. Particularly, drug release profile for the PTX-loaded peppermint oil ME (MEPP) showed that ∼ 90% of drug was released in the first 48 h. Also, MEPP formulation showed 70% and 90% viability reduction on human cervical cancer (HeLa) cells after 24 and 48 h of exposure, respectively. In addition, HeLa cell apoptosis was confirmed by measuring caspase activity and DNA fragmentation. Results showed that the MEPP sample presented a major pro-apoptotic capability by comparing with the unloaded PTX ME sample.

A protein complex bearing an oxidase with napthalene dihydrodiol dehydrogenase activity is induced in Mucor circinelloides strain YR-1 during growth on polycyclic aromatic compounds.

Fungi are organisms capable of growing in a myriad of conditions and respond to counteract environmental cues. Several locations in the world are polluted with oil and its derivatives, and some microorganisms tolerant to these compounds have been isolated. Some fungi can grow in the presence of molecules such as polycyclic aromatic hydrocarbons as sole carbon sources. In this report, we further characterized the induced enzymes with phenanthrene from Mucor circinelloides YR-1 strain, isolated from a polluted field near a petrochemical facility in México. We identified a putative oxidase that is induced when growth with phenanthrene as sole carbon source at a pH of 8.5 and is NADP+ dependent. We show that this enzyme bears naphthalene dihydrodiol dehydrogenase activity with substrate preference for the cis-naphthalene over the trans-naphthalene, with an optimal pH in the range of 8-10. Mass spectrometry analysis revealed that the induced enzyme belongs to the NADP+ oxidase family enzymes with the typical Rossmann-fold for NADP+ binding. This enzyme seems to form a high molecular weight structure (~ 541 kDa) and with a monomer of 57 kDa, suggesting that the multimer is constituted of 10 subunits. Our findings contribute to understanding of the roles that dihydrodiol dehydrogenases have in organisms exposed to toxic compounds in the environment and can regulate their expression.

Red fluorescent protein (DsRFP) optimization for Entamoeba histolytica expression.

Entamoeba histolytica genetic organization and genome structure is complex and under intense research. The genome is fully sequenced, and several tools have been developed for the molecular study of this organism. Nevertheless, good protein tracking tags that are easy to measure and image, like the fluorescent proteins are lacking. In this report, we codon-optimized the red fluorescent protein from the coral Discosoma striata (DsRFP) for its use in E. histolytica and demonstrated functionality in vivo. We envision that this protein can be widely used for the development of transcriptional reporter systems and protein-tagging applications.

Contribution of neutral sphingomyelinases to in vitro virulence of Entamoeba histolytica.

Amoebiasis is a worldwide health problem caused by the pathogen Entamoeba histolytica. Several virulence factors have been implicated in host invasion, immune evasion, and tissue damage. There are still new factors that remain to be elucidated and characterized. In this work, we obtained amoebic transfectants overexpressing three of the neutral sphingomyelinase enzymes encoded in the E. histolytica genome. The EhnSM3 overexpression induced an increase in hemolytic and cytotoxic activities, besides an increase in gene expression of amoebapore A, B, and C. Meanwhile the EhnSM1 and EhnSM2 overexpression caused an increase in cytopathic activity. In all the neutral sphingomyelinases overexpressing strains, the gene expression levels for cysteine proteinase 5, adhesin 112 and, heavy and light Gal/GalNAc lectin subunits were not affected. We propose that the increase of cytotoxic and lytic effect of EhnSM3 overexpressed strain can be related to the sum of the effect of EhnSM3 plus amoebapores, in a process cell contact-dependent or as mediator by inducing the gene expression of amoebapores enabling a link between EhnSM3 with the virulence phenotype in E. histolytica. Our results suggest a differential role for neutral sphingomyelinases in E. histolytica virulence.

Replacing Standard Reporters from Molecular Cloning Plasmids with Chromoproteins for Positive Clone Selection.

Cloning and expression plasmids are the workhorses of modern molecular biology. Despite the pathway paved by synthetic biology, laboratories around the globe still relay on standard cloning techniques using plasmids with reporter proteins for positive clone selection, such as ß-galactosidase alpha peptide complementation for blue/white screening or ccdB, which encodes for a toxic DNA gyrase. These reporters, when interrupted, serve as a positive clone detection system. In the present report, we show that molecular cloning plasmids bearing the coding sequence for a 25.4 kDa protein, AmilCP, encoded by a 685 bp gene, that is well expressed in Escherichia coli, render blue-purple colonies. Using this reporter protein, we developed and tested a cloning system based on the constitutive expression of the non-toxic AmilCP protein, that once interrupted, the loss of purple color serves to facilitate positive clone selection. The main advantage of this system is that is less expensive than other systems since media do not contain chromogenic markers such as X-gal, which is both expensive and cumbersome to prepare and use, or inductors such as IPTG. We also designed an inducible expression plasmid suitable for recombinant protein expression that also contains AmilCP cloning selection marker, a feature not commonly found in protein expression plasmids. The use of chromogenic reporters opens an important avenue for its application in other organisms besides E. coli for clone selection or even for mutant selection.

Cell damage detection using Escherichia coli reporter plasmids: fluorescent and colorimetric assays.

Bacterial reporter assays are powerful tools used to study the effect of different compounds that affect the physiology of cellular processes. Most bacterial reporters are luciferase based and can be monitored in real time. In the present study we designed and implemented two sets of Escherichia coli bacterial reporter assays, using a multicopy plasmid system. Each reporter strain was constructed using either green fluorescent protein or ß-galactosidase (LacZ) proteins. The designed reporter strains are capable of responding in a specific manner to molecules that either oxidative stress, or membrane, protein, or DNA damage. In order to respond to the desired stimulus, promoter sequences from E. coli were used. These sequences correspond to the promoter of the major catalase (KatG) activated with cellular oxidative damage, the promoter of the ß-hydroxydecanoyl-ACP dehydrase (FabA) which is activated with membrane perturbation, the promoter of DNA recombinase (RecA) which is activated by DNA lesions. For protein misfolding, the promoter of the heat-shock responsive chaperon (DnaK) was used. Our constructs displayed activation to damage from specific stimuli, and low response to nonspecific stimuli was detected. Our results suggest that these types of bacterial reporter strains can be used in semiquantitative (fluorometric) and qualitative (ß-galactosidase activity) studies of different xenobiotic substances and pollutants.

In Vitro Studies of Chromone-Tetrazoles against Pathogenic Protozoa, Bacteria, and Fungi.

In vitro studies to fourteen previously synthesized chromone-tetrazoles and four novel fluorine-containing analogs were conducted against pathogenic protozoan (Entamoeba histolytica), pathogenic bacteria (Pseudomonas aeruginosa, and Staphylococcus aureus), and human fungal pathogens (Sporothrix schenckii, Candida albicans, and Candida tropicalis), which have become in a serious health problem, mainly in tropical countries.

The inorganic pyrophosphatases of microorganisms: a structural and functional review.

Pyrophosphatases (PPases) are enzymes that catalyze the hydrolysis of pyrophosphate (PPi), a byproduct of the synthesis and degradation of diverse biomolecules. The accumulation of PPi in the cell can result in cell death. Although the substrate is the same, there are variations in the catalysis and features of these enzymes. Two enzyme forms have been identified in bacteria: cytoplasmic or soluble pyrophosphatases and membrane-bound pyrophosphatases, which play major roles in cell bioenergetics. In eukaryotic cells, cytoplasmic enzymes are the predominant form of PPases (c-PPases), while membrane enzymes (m-PPases) are found only in protists and plants. The study of bacterial cytoplasmic and membrane-bound pyrophosphatases has slowed in recent years. These enzymes are central to cell metabolism and physiology since phospholipid and nucleic acid synthesis release important amounts of PPi that must be removed to allow biosynthesis to continue. In this review, two aims were pursued: first, to provide insight into the structural features of PPases known to date and that are well characterized, and to provide examples of enzymes with novel features. Second, the scientific community should continue studying these enzymes because they have many biotechnological applications. Additionally, in this review, we provide evidence that there are m-PPases present in fungi; to date, no examples have been characterized. Therefore, the diversity of PPase enzymes is still a fruitful field of research. Additionally, we focused on the roles of H+/Na+ pumps and m-PPases in cell bioenergetics. Finally, we provide some examples of the applications of these enzymes in molecular biology and biotechnology, especially in plants. This review is valuable for professionals in the biochemistry field of protein structure-function relationships and experts in other fields, such as chemistry, nanotechnology, and plant sciences.

High-Throughput Screening Method Using Escherichia coli Keio Mutants for Assessing Primary Damage Mechanism of Antimicrobials.

The Escherichia coli Keio mutant collection has been a tool for assessing the role of specific genes and determining their role in E. coli physiology and uncovering novel functions. In this work, specific mutants in the DNA repair pathways and oxidative stress response were evaluated to identify the primary targets of silver nanoparticles (NPs) and their mechanism of action. The results presented in this work suggest that NPs mainly target DNA via double-strand breaks and base modifications since the recA, uvrC, mutL, and nfo mutants rendered the most susceptible phenotype, rather than involving the oxidative stress response. Concomitantly, during the establishment of the control conditions for each mutant, the katG and sodA mutants showed a hypersensitive phenotype to mitomycin C, an alkylating agent. Thus, we propose that KatG catalase plays a key role as a cellular chaperone, as reported previously for the filamentous fungus Neurospora crassa, a large subunit catalase. The Keio collection mutants may also be a key tool for assessing the resistance mechanism to metallic NPs by using their potential to identify novel pathways involved in the resistance to NPs.

Avirulent UG10 Entamoeba histolytica mutant derived from HM-1:IMSS strain shows limited genome variability and aberrant 5-methyl cytosine genomic distribution.

Entamoeba histolytica, an intestinal parasite of global significance, poses substantial health risks with its associated high morbidity and mortality rates. Despite the current repertoire of molecular tools for the study of gene function in, the regulatory mechanisms governing its pathogenicity remain largely unexplored. This knowledge gap underscores the need to elucidate key genetic determinants orchestrating cellular functions critical to its virulence. Previously, our group generated an avirulent strain, termed UG10, with the same genetic background as the HM1:IMSS strain. UG10 strain, despite showing normal expression levels of well-known virulence factors, was unable to perform in-vitro and in-vivo activities related to amoebic virulence. In this study, we aimed to uncover the genome-wide modifications that rendered the avirulent phenotype of the UG10 strain through whole-genome sequencing. As a complementary approach, we conducted Methylated DNA Immunoprecipitation coupled with sequencing (MeDIP-seq) analysis on both the highly virulent HM1:IMSS strain and the low-virulence UG10 strain to uncover the genome-wide methylation profile. These dual methodologies revealed two aspects of the UG10 avirulent strain. One is the random integration of fragments from the ribosomal gene cluster and tRNA genes, ranging from 120 to 400 bp; and secondly, a clear, enriched methylation profile in the coding and non-coding strand relative to the start codon sequence in genes encoding small GTPases, which is associated with the previously described avirulent phenotype. This study provides the foundation to explore other genetic and epigenetic regulatory circuitries in E. histolytica and novel targets to understand the pathogenic mechanism of this parasite.

Attenuation of In Vitro and In Vivo Virulence Is Associated with Repression of Gene Expression of AIG1 Gene in Entamoeba histolytica.

Entamoeba histolytica virulence results from complex host-parasite interactions implicating multiple amoebic components (e.g., Gal/GalNAc lectin, cysteine proteinases, and amoebapores) and host factors (microbiota and immune response). UG10 is a strain derived from E. histolytica virulent HM-1:IMSS strain that has lost its virulence in vitro and in vivo as determined by a decrease of hemolytic, cytopathic, and cytotoxic activities, increased susceptibility to human complement, and its inability to form liver abscesses in hamsters. We compared the transcriptome of nonvirulent UG10 and its parental HM-1:IMSS strain. No differences in gene expression of the classical virulence factors were observed. Genes downregulated in the UG10 trophozoites encode for proteins that belong to small GTPases, such as Rab and AIG1. Several protein-coding genes, including iron-sulfur flavoproteins and heat shock protein 70, were also upregulated in UG10. Overexpression of the EhAIG1 gene (EHI_180390) in nonvirulent UG10 trophozoites resulted in augmented virulence in vitro and in vivo. Cocultivation of HM-1:IMSS with E. coli O55 bacteria cells reduced virulence in vitro, and the EhAIG1 gene expression was downregulated. In contrast, virulence was increased in the monoxenic strain UG10, and the EhAIG1 gene expression was upregulated. Therefore, the EhAIG1 gene (EHI_180390) represents a novel virulence determinant in E. histolytica.

Theoretical study of ArcB and its dimerization, interaction with anaerobic metabolites, and activation of ArcA.

The complex metabolism of Escherichia coli has been extensively studied, including its response to oxygen availability. The ArcA/B two-component system (TCS) is the key regulator for the transition between these two environmental conditions and has been thoroughly characterized using genetic and biochemical approaches. Still, to date, limited structural data is available. The breakthrough provided by AlphaFold2 in 2021 has brought a reliable tool to the scientific community for assessing the structural features of complex proteins. In this report, we analyzed the structural aspects of the ArcA/B TCS using AlphaFold2 models. The models are consistent with the experimentally determined structures of ArcB kinase. The predicted structure of the dimeric form of ArcB is consistent with the extensive genetic and biochemical data available regarding mechanistic signal perception and regulation. The predicted interaction of the dimeric form of ArcB with its cognate response regulator (ArcA) is also consistent with both the forward and reverse phosphotransfer mechanisms. The ArcB model was used to detect putative binding cavities to anaerobic metabolites, encouraging testing of these predictions experimentally. Finally, the highly accurate models of other ArcB homologs suggest that different experimental approaches are needed to determine signal perception in kinases lacking the PAS domain. Overall, ArcB is a kinase with features that need further testing, especially in determining its crystal structure under different conditions.

Catalases in the pathogenesis of Sporothrix schenckii research.

Pathogenic fungal infection success depends on the ability to escape the immune response. Most strategies for fungal infection control are focused on the inhibition of virulence factors and increasing the effectiveness of antifungal drugs. Nevertheless, little attention has been focused on their physiological resistance to the host immune system. Hints may be found in pathogenic fungi that also inhabit the soil. In nature, the saprophyte lifestyle of fungi is also associated with predators that can induce oxidative stress upon cell damage. The natural sources of nutrients for fungi are linked to cellulose degradation, which in turn generates reactive oxygen species (ROS). Overall, the antioxidant arsenal needed to thrive both in free-living and pathogenic lifestyles in fungi is fundamental for success. In this review, we present recent findings regarding catalases and oxidative stress in fungi and how these can be in close relationship with pathogenesis. Additionally, special focus is placed on catalases of Sporothrix schenckii as a pathogenic model with a dual lifestyle. It is assumed that catalase expression is activated upon exposure to H2O2, but there are reports where this is not always the case. Additionally, it may be relevant to consider the role of catalases in S. schenckii survival in the saprophytic lifestyle and why their study can assess their involvement in the survival and therefore, in the virulence phenotype of different species of Sporothrix and when each of the three catalases are required. Also, studying antioxidant mechanisms in other isolates of pathogenic and free-living fungi may be linked to the virulence phenotype and be potential therapeutic and diagnostic targets. Thus, the rationale for this review to place focus on fungal catalases and their role in pathogenesis in addition to counteracting the effect of immune system reactive oxygen species. Fungi that thrive in soil and have mammal hosts could shed light on the importance of these enzymes in the two types of lifestyles. We look forward to encouraging more research in a myriad of areas on catalase biology with a focus on basic and applied objectives and placing these enzymes as virulence determinants.

Chromogenic Escherichia coli reporter strain for screening DNA damaging agents.

The presence of pollutants in soil and water has given rise to diverse analytical and biological approaches to detect and measure contaminants in the environment. Using bacterial cells as reporter strains represents an advantage for detecting pollutants present in soil or water samples. Here, an Escherichia coli reporter strain expressing a chromoprotein capable of interacting with soil or water samples and responding to DNA damaging compounds is validated. The reporter strain generates a qualitative signal and is based on the expression of the coral chromoprotein AmilCP under the control of the recA promoter. This strain can be used simply by applying soil or water samples directly and rendering activation upon DNA damage. This reporter strain responds to agents that damage DNA (with an apparent detection limit of 1 µg of mitomycin C) without observable response to membrane integrity damage, protein folding or oxidative stress generating agents, in the latter case, DNA damage was observed. The developed reporter strain reported here is effective for the detection of DNA damaging agents present in soils samples. In a proof-of-concept analysis using soil containing chromium, showing activation at 15.56 mg/L of Cr(VI) present in soil and leached samples and is consistent with Cr(III) toxicity at high concentrations (130 µg). Our findings suggest that chromogenic reporter strains can be applied for simple screening, thus reducing the number of samples requiring analytical techniques.

Escherichia coli transcription factors of unknown function: sequence features and possible evolutionary relationships.

Organisms need mechanisms to perceive the environment and respond accordingly to environmental changes or the presence of hazards. Transcription factors (TFs) are required for cells to respond to the environment by controlling the expression of genes needed. Escherichia coli has been the model bacterium for many decades, and still, there are features embedded in its genome that remain unstudied. To date, 58 TFs remain poorly characterized, although their binding sites have been experimentally determined. This study showed that these TFs have sequence variation at the third codon position G+C content but maintain the same Codon Adaptation Index (CAI) trend as annotated functional transcription factors. Most of these transcription factors are in areas of the genome where abundant repetitive and mobile elements are present. Sequence divergence points to groups with distinctive sequence signatures but maintaining the same type of DNA binding domain. Finally, the analysis of the promoter sequences of the 58 TFs showed A+T rich regions that agree with the features of horizontally transferred genes. The findings reported here pave the way for future research of these TFs that may uncover their role as spare factors in case of lose-of-function mutations in core TFs and trace back their evolutionary history.