Unveiling the importance of the C-terminus in the sugar acid dehydratase of the IlvD/EDD superfamily.

Microbial non-phosphorylative oxidative pathways present promising potential in the biosynthesis of platform chemicals from the hemicellulosic fraction of lignocellulose. An L-arabinonate dehydratase from Rhizobium leguminosarum bv. trifolii catalyzes the rate-limiting step in the non-phosphorylative oxidative pathways, that is, converts sugar acid to 2-dehydro-3-deoxy sugar acid. We have shown earlier that the enzyme forms a dimer of dimers, in which the C-terminal histidine residue from one monomer participates in the formation of the active site of an adjacent monomer. The histidine appears to be conserved across the sequences of sugar acid dehydratases. To study the role of the C-terminus, five variants (H579A, H579F, H579L, H579Q, and H579W) were produced. All variants showed decreased activity for the tested sugar acid substrates, except the variant H579L on D-fuconate, which showed about 20% increase in activity. The reaction kinetic data showed that the substrate preference was slightly modified in H579L compared to the wild-type enzyme, demonstrating that the alternation of the substrate preference of sugar acid dehydratases is possible. In addition, a crystal structure of H579L was determined at 2.4 Å with a product analog 2-oxobutyrate. This is the first enzyme-ligand complex structure from an IlvD/EDD superfamily enzyme. The binding of 2-oxobutyrate suggests how the substrate would bind into the active site in the orientation, which could lead to the dehydration reaction. KEY POINTS: • Mutation of the last histidine at the C-terminus changed the catalytic activity of L-arabinonate dehydratase from R. leguminosarum bv. trifolii against various C5/C6 sugar acids. • The variant H579L of L-arabinonate dehydratase showed an alteration of substrate preferences compared with the wild type. • The first enzyme-ligand complex crystal structure of an IlvD/EDD superfamily enzyme was solved.

Enhancing the amination activity of meso-diaminopimelate dehydrogenase from Symbiobacterium thermophilum by modifying the crucial residue His154 for deamination.

During the deamination and amination processes of meso-diaminopimelate dehydrogenase (meso-DAPDH) from Symbiobacterium thermophilum (StDAPDH), residue R71 was observed to display distinct functions. H154 has been proposed as a basic residue that facilitates water molecules to attack the D-chiral carbon of meso-DAP during deamination. Inspired by the phenomenon of R71, the effects of H154 during deamination and amination were investigated in this study with the goal of enhancing the amination activities of StDAPDH. Single site saturation mutagenesis indicated that almost all of the H154 mutants completely lost their deamination activity towards meso-DAP. However, some H154 variants showed enhanced kcat/Km values towards pyruvic acid and other bulky 2-keto acids, such as 2-oxovaleric acid, 4-methyl-2-oxopentanoic acid, 2-ketobutyric acid, and 3-methyl-2-oxobutanoic acid. When combined with the previously reported W121L/H227I mutant, triple mutants with significantly improved kcat/Km values (2.4-, 2.5-, 2.5-, and 4.0-fold) towards these 2-keto acids were obtained. Despite previous attempts, mutations at the H154 site did not yield the desired results. Moreover, this study not only recognizes the distinctive impact of H154 on both the deamination and amination reactions, but also provides guidance for further high-throughput screening in protein engineering and understanding the catalytic mechanism of StDAPDH.

Heterologous Expression and Purification of Eukaryotic ALA Synthase from E. coli.

This chapter presents a method for the heterologous expression and purification of human ALA synthase from Escherichia coli. Mature ALAS is produced with an N-terminal hexahistidine affinity tag followed by a SUMO fusion tag for solubility and ease of purification. The plasmid is introduced into competent E. coli cells, and robust protein expression is induced with IPTG. The ALAS cofactor, pyridoxal 5'-phosphate, is inserted during protein production to yield an active enzyme upon purification. After cell lysis, the tagged ALAS protein is isolated via a multistep purification that involves an initial nickel-affinity step, affinity tag cleavage and removal, and a final size exclusion chromatography polishing step. Importantly, this protocol is amenable to various ALAS truncations and mutations, opening the door to understanding ALAS biology and its intersections with iron utilization across several organisms.

Genetically encoded Nδ-vinyl histidine for the evolution of enzyme catalytic center.

Genetic code expansion has emerged as a powerful tool for precisely introducing unnatural chemical structures into proteins to improve their catalytic functions. Given the high catalytic propensity of histidine in the enzyme pocket, increasing the chemical diversity of catalytic histidine could result in new characteristics of biocatalysts. Herein, we report the genetically encoded Nδ-Vinyl Histidine (δVin-H) and achieve the wild-type-like incorporation efficiency by the evolution of pyrrolysyl tRNA synthetase. As histidine usually acts as the nucleophile or the metal ligand in the catalytic center, we replace these two types of catalytic histidine to δVin-H to improve the performance of the histidine-involved catalytic center. Additionally, we further demonstrate the improvements of the hydrolysis activity of a previously reported organocatalytic esterase (the OE1.3 variant) in the acidic condition and myoglobin (Mb) catalyzed carbene transfer reactions under the aerobic condition. As histidine is one of the most frequently used residues in the enzyme catalytic center, the derivatization of the catalytic histidine by δVin-H holds a great potential to promote the performance of biocatalysts.

Purification of Recombinant N-terminal Histidine-Tagged Arabidopsis thaliana Phosphoglycolate Phosphatase 1, Glycolate Oxidase 1 and 2, and Hydroxypyruvate Reductase 1.

To measure the kinetic properties of photorespiratory enzymes, it is necessary to work with purified proteins. Protocols to purify photorespiratory enzymes from leaves of various plant species require several time-consuming steps. It is now possible to produce large quantities of recombinant proteins in bacterial cells. They can be rapidly purified as histidine-tagged recombinant proteins by immobilized metal affinity chromatography using Ni2+-NTA-agarose. This chapter describes protocols to purify several Arabidopsis thaliana His-tagged recombinant photorespiratory enzymes (phosphoglycolate phosphatase, glycolate oxidase, and hydroxypyruvate reductase) from Escherichia coli cell cultures using two bacterial strain-plasmid systems: BL21(DE3)-pET and LMG194-pBAD.

C-terminal Poly-histidine Tags Alter Escherichia coli Polyphosphate Kinase Activity and Susceptibility to Inhibition.

In Escherichia coli, many environmental stressors trigger polyphosphate (polyP) synthesis by polyphosphate kinase (PPK1), including heat, nutrient restriction, toxic compounds, and osmotic imbalances. PPK1 is essential for virulence in many pathogens and has been the target of multiple screens for small molecule inhibitors that might serve as new anti-virulence drugs. However, the mechanisms by which PPK1 activity and polyP synthesis are regulated are poorly understood. Our previous attempts to uncover PPK1 regulatory elements resulted in the discovery of PPK1* mutants, which accumulate more polyP in vivo, but do not produce more in vitro. In attempting to further characterize these mutant enzymes, we discovered that the most commonly-used PPK1 purification method - Ni-affinity chromatography using a C-terminal poly-histidine tag - altered intrinsic aspects of the PPK1 enzyme, including specific activity, oligomeric state, and kinetic values. We developed an alternative purification strategy using a C-terminal C-tag which did not have these effects. Using this strategy, we were able to demonstrate major differences in the in vitro response of PPK1 to 5-aminosalicylic acid, a known PPK1 inhibitor, and observed several key differences between the wild-type and PPK1* enzymes, including changes in oligomeric distribution, increased enzymatic activity, and increased resistance to both product (ADP) and substrate (ATP) inhibition, that help to explain their in vivo effects. Importantly, our results indicate that the C-terminal poly-histidine tag is inappropriate for purification of PPK1, and that any in vitro studies or inhibitor screens performed with such tags need to be reconsidered in that light.

Nickel-chelatase activity of SirB variants mimicking the His arrangement in the naturally occurring nickel-chelatase CfbA.

Metal-tetrapyrrole cofactors are involved in multiple cellular functions, and chelatases are key enzymes for the biosynthesis of these cofactors. CfbA is an ancestral, homodimeric-type class II chelatase which is able to use not only Ni2+ as a physiological metal substrate, but also Co2+ as a nonphysiological substrate with higher activity than for Ni2+. The Ni/Co-chelatase function found in CfbA is also observed in SirB, a descendant, monomeric-type class II chelatase. This is despite the distinct active site structure of CfbA and SirB; specifically, CfbA shows a unique four His residue arrangement, unlike other monomeric class II chelatases such as SirB. Herein, we studied the Ni-chelatase activity of SirB variants R134H, L200H, and R134H/L200H, the latter of which mimics the His alignment of CfbA. Our results showed that the SirB R134H variant exhibited the highest Ni-chelatase activity among the SirB enzymes, which in turn suggests that the position of His134 could be more important for the Ni-chelatase activity than that of His200. The SirB R134H/L200H variant showed lower activity than R134H, despite the four His residues found in SirB R134H/L200H. CD spectroscopy showed secondary structure denaturation and a slight difficulty in Ni-binding of SirB R134H/L200H, which may be related to its lower activity. Finally, a docking simulation suggested that the His134 of the SirB R134H variant could function as a base catalyst for the Ni-chelatase reaction in a class II chelatase architecture.

Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO).

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein's surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

Expression and Purification of His-Tagged Variants of Human Hepatitis A Virus 3C Protease.

BACKGROUND: Protease 3C (3Cpro) is the only protease encoded in the human hepatitis A virus genome and is considered as a potential target for antiviral drugs due to its critical role in the viral life cycle. Additionally, 3Cpro has been identified as a potent inducer of ferroptosis, a newly described type of cell death. Therefore, studying the molecular mechanism of 3Cpro functioning can provide new insights into viral-host interaction and the biological role of ferroptosis. However, such studies require a reliable technique for producing the functionally active recombinant enzyme. OBJECTIVE: Here, we expressed different modified forms of 3Cpro with a hexahistidine tag on the N- or C-terminus to investigate the applicability of immobilized metal Ion affinity chromatography (IMAC) for producing 3Cpro. METHODS: We expressed the proteins in Escherichia coli and purified them using IMAC, followed by gel permeation chromatography. The enzymatic activity of the produced proteins was assayed using a specific chromogenic substrate. RESULTS: Our findings showed that the introduction and position of the hexahistidine tag did not affect the activity of the enzyme. However, the yield of the target protein was highest for the variant with seven C-terminal residues replaced by a hexahistidine sequence. CONCLUSION: We demonstrated the applicability of our approach for producing recombinant, enzymatically active 3Cpro.

Hb H disease associated with compound heterozygosity for --SEA deletion and a novel alpha globin chain variant (HBA2:c.175C>A) on the distal histidine in a Chinese family.

OBJECTIVES: In clinical practice, the majority of α-thalassaemia cases arise from deletions of the α-globin genes. However, a subset of cases is attributed to rare haemoglobin variants, which can manifest with borderline or normal screening results, potentially leading to missed diagnoses in clinical practice. METHODS: Blood samples were collected from family members and underwent haematological, DNA and RNA analysis. RESULTS: The five-month-old proband presented a haematological phenotype consistent with Hb H disease. The mother's haematology profile was consistent with an α-thalassaemia carrier, while the father exhibited a borderline reduction in MCV and MCH. MALDI-TOF identified an abnormal α-chain in the proband. DNA analysis revealed a novel α-globin variant (HBA2:c.175C>A, α58His>Asn, Hb DG-Nancheng) affecting the distal histidine in the family. The father and the mother had α-genotype of --SEA/αα and αDG-Nanchengα/αα, respectively; while the proband inherited both mutant alleles (--SEA/αDG-Nanchengα). Sequencing of cDNA from HBA2 gene identified an equal ratio of normal and mutant alleles. CONCLUSION: This rare case highlighted the importance of identifying rare haemoglobin variant during prenatal screening. The clinical and genetic data provides useful information on the pathogenicity of this variant and further insight into the role of distal histidine residue of α-globin.

Widespread pfhrp2/3 deletions and HRP2-based false-negative results in southern Ethiopia.

BACKGROUND: Rapid diagnostic tests (RDTs) play a significant role in expanding case management in peripheral healthcare systems. Histidine-rich protein-2 (HRP2) antigen detection RDTs are predominantly used to diagnose Plasmodium falciparum infection. However, the evolution and spread of P. falciparum parasite strains with deleted hrp2/3 genes, causing false-negative results, have been reported. This study assessed the diagnostic performance of HRP2-detecting RDTs for P. falciparum cases and the prevalence of pfhrp2/3 deletions among symptomatic patients seeking malaria diagnosis at selected health facilities in southern Ethiopia. METHODS: A multi-health facilities-based cross-sectional study was conducted on self-presenting febrile patients seeking treatment in southern Ethiopia from July to September 2022. A purposive sampling strategy was used to enroll patients with microscopically confirmed P. falciparum infections. A capillary blood sample was obtained to prepare a blood film for microscopy and a RDT using the SD Bioline™ Malaria Pf/Pv Test. Dried blood spot samples were collected for further molecular analysis. DNA was extracted using gene aid kits and amplification was performed using nested PCR assay. Exon 2 of hrp2 and hrp3, which are the main protein-coding regions, was used to confirm its deletion. The diagnostic performance of RDT was evaluated using PCR as the gold standard test for P. falciparum infections. RESULTS: Of 279 P. falciparum PCR-confirmed samples, 249 (89.2%) had successful msp-2 amplification, which was then genotyped for hrp2/3 gene deletions. The study revealed that pfhrp2/3 deletions were common in all health centres, and it was estimated that 144 patients (57.8%) across all health facilities had pfhrp2/3 deletions, leading to false-negative PfHRP2 RDT results. Deletions spanning exon 2 of hrp2, exon 2 of hrp3, and double deletions (hrp2/3) accounted for 68 (27.3%), 76 (30.5%), and 33 (13.2%) of cases, respectively. The study findings revealed the prevalence of P. falciparum parasites lacking a single pfhrp2-/3-gene and that both genes varied across the study sites. This study also showed that the sensitivity of the SD Bioline PfHRP2-RDT test was 76.5% when PCR was used as the reference test. CONCLUSION: This study confirmed the existence of widespread pfhrp2/3- gene deletions, and their magnitude exceeded the WHO-recommended threshold (> 5%). False-negative RDT results resulting from deletions in Pfhrp2/3- affect a country's attempts at malaria control and elimination. Therefore, the adoption of non-HRP2-based RDTs as an alternative measure is required to avoid the consequences associated with the continued use of HRP-2-based RDTs, in the study area in particular and in Ethiopia in general.

pH Dependence of HSF1 trimerization is shaped by intramolecular interactions.

Heat shock factor 1 (HSF1) primarily regulates various cellular stress responses. Previous studies have shown that low pH within the physiological range directly activates HSF1 function in vitro. However, the detailed molecular mechanisms remain unclear. This study proposes a molecular mechanism based on the trimerization behavior of HSF1 at different pH values. Extensive mutagenesis of human and goldfish HSF1 revealed that the optimal pH for trimerization depended on the identity of residue 103. In particular, when residue 103 was occupied by tyrosine, a significant increase in the optimal pH was observed, regardless of the rest of the sequence. This behavior can be explained by the protonation state of the neighboring histidine residues, His101 and His110. Residue 103 plays a key role in trimerization by forming disulfide or non-covalent bonds with Cys36. If tyrosine resides at residue 103 in an acidic environment, its electrostatic interactions with positively charged histidine residues prevent effective trimerization. His101 and His110 are neutralized at a higher pH, which releases Tyr103 to interact with Cys36 and drives the effective trimerization of HSF1. This study showed that the protonation state of a histidine residue can regulate the intramolecular interactions, which consequently leads to a drastic change in the oligomerization behavior of the entire protein.

Supercharged coiled-coil protein with N-terminal decahistidine tag boosts siRNA complexation and delivery efficiency of a lipoproteoplex.

Short interfering RNA (siRNA) therapeutics have soared in popularity due to their highly selective and potent targeting of faulty genes, providing a non-palliative approach to address diseases. Despite their potential, effective transfection of siRNA into cells requires the assistance of an accompanying vector. Vectors constructed from non-viral materials, while offering safer and non-cytotoxic profiles, often grapple with lackluster loading and delivery efficiencies, necessitating substantial milligram quantities of expensive siRNA to confer the desired downstream effects. We detail the recombinant synthesis of a diverse series of coiled-coil supercharged protein (CSP) biomaterials systematically designed to investigate the impact of two arginine point mutations (Q39R and N61R) and decahistidine tags on liposomal siRNA delivery. The most efficacious variant, N8, exhibits a twofold increase in its affinity to siRNA and achieves a twofold enhancement in transfection activity with minimal cytotoxicity in vitro. Subsequent analysis unveils the destabilizing effect of the Q39R and N61R supercharging mutations and the incorporation of C-terminal decahistidine tags on α-helical secondary structure. Cross-correlational regression analyses reveal that the amount of helical character in these mutants is key in N8's enhanced siRNA complexation and downstream delivery efficiency.

Protocol for detecting histidine polyphosphate modification of human proteins via MBP-tagged expression in E. coli.

Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM. For complete details on the use and execution of this protocol, please refer to Neville et al.1.

A non-canonical nucleophile unlocks a new mechanistic pathway in a designed enzyme.

Directed evolution of computationally designed enzymes has provided new insights into the emergence of sophisticated catalytic sites in proteins. In this regard, we have recently shown that a histidine nucleophile and a flexible arginine can work in synergy to accelerate the Morita-Baylis-Hillman (MBH) reaction with unrivalled efficiency. Here, we show that replacing the catalytic histidine with a non-canonical Nδ-methylhistidine (MeHis23) nucleophile leads to a substantially altered evolutionary outcome in which the catalytic Arg124 has been abandoned. Instead, Glu26 has emerged, which mediates a rate-limiting proton transfer step to deliver an enzyme (BHMeHis1.8) that is more than an order of magnitude more active than our earlier MBHase. Interestingly, although MeHis23 to His substitution in BHMeHis1.8 reduces activity by 4-fold, the resulting His containing variant is still a potent MBH biocatalyst. However, analysis of the BHMeHis1.8 evolutionary trajectory reveals that the MeHis nucleophile was crucial in the early stages of engineering to unlock the new mechanistic pathway. This study demonstrates how even subtle perturbations to key catalytic elements of designed enzymes can lead to vastly different evolutionary outcomes, resulting in new mechanistic solutions to complex chemical transformations.

Revisiting catalytic His and Glu residues in coproporphyrin ferrochelatase - unexpected activities of active site variants.

The identification of the coproporphyrin-dependent heme biosynthetic pathway, which is used almost exclusively by monoderm bacteria in 2015 by Dailey et al. triggered studies aimed at investigating the enzymes involved in this pathway that were originally assigned to the protoporphyrin-dependent heme biosynthetic pathway. Here, we revisit the active site of coproporphyrin ferrochelatase by a biophysical and biochemical investigation using the physiological substrate coproporphyrin III, which in contrast to the previously used substrate protoporphyrin IX has four propionate substituents and no vinyl groups. In particular, we have compared the reactivity of wild-type coproporphyrin ferrochelatase from the firmicute Listeria monocytogenes with those of variants, namely, His182Ala (H182A) and Glu263Gln (E263Q), involving two key active site residues. Interestingly, both variants are active only toward the physiological substrate coproporphyrin III but inactive toward protoporphyrin IX. In addition, E263 exchange impairs the final oxidation step from ferrous coproheme to ferric coproheme. The characteristics of the active site in the context of the residues involved and the substrate binding properties are discussed here using structural and functional means, providing a further contribution to the deciphering of this enigmatic reaction mechanism.

Mutation of conserved histidine residues of dengue virus envelope protein impairs viral like particle maturation and secretion.

Dengue virus (DENV) envelope protein plays crucial role in virus entry and maturation of virus during infection. Maturation of DENV occurs in the trans Golgi network at slightly acidic pH which is close to pKa of histidine. When exposed to the acidic environment of the late secretory pathway, dengue virus particles go through a significant conformational change, whereby interactions of structural proteins envelope (E) and prM proteins are reorganised and enable furin protease to cleave prM resulting in mature virus. In order to study the role of histidine of E protein in DENV maturation, we mutated 7 conserved histidine residues of envelope protein and assessed the percent of budding using viral like particle (VLP) system. Histidine mutants; H144A, H244A, H261A and H282A severely disrupted VLP formation without any significant change in expression in cell and its oligomerization ability. Treatment with acidotropic amine reversed the defect for all 4 mutants suggesting that these histidines could be involved in maturation and release. Over expression of capsid protein slightly enhanced VLP release of H244A and H261A. Similarly, furin over expression increased VLP release of these mutants. Co-immunoprecipitation studies revealed that prM and E interaction is lost for H244A, H261A and H282A mutants at acidic pH but not at neutral pH indicating that they could be involved in histidine switch during maturation at acidic pH. Detailed analysis of the mutants could provide novel insights on the interplay of envelop protein during maturation and aid in target for drug development.

Time-resolved RNA-seq analysis to unravel the in vivo competence induction by Streptococcus pneumoniae during pneumonia-derived sepsis.

Competence development in Streptococcus pneumoniae (pneumococcus) is tightly intertwined with virulence. In addition to genes encoding genetic transformation machinery, the competence regulon also regulates the expression of allolytic factors, bacteriocins, and cytotoxins. Pneumococcal competence system has been extensively interrogated in vitro where the short transient competent state upregulates the expression of three distinct phases of "early," "late," and "delayed" genes. Recently, we have demonstrated that the pneumococcal competent state develops naturally in mouse models of pneumonia-derived sepsis. To unravel the underlying adaptive mechanisms driving the development of the competent state, we conducted a time-resolved transcriptomic analysis guided by the spatiotemporal live in vivo imaging system of competence induction during pneumonia-derived sepsis. Mouse lungs infected by the serotype 2 strain D39 expressing a competent state-specific reporter gene (D39-ssbB-luc) were subjected to RNA sequencing guided by monitoring the competence development at 0, 12, 24, and, at the moribund state, >40 hours post-infection (hpi). Transcriptomic analysis revealed that the competence-specific gene expression patterns in vivo were distinct from those under in vitro conditions. There was significant upregulation of early, late, and some delayed phase competence-specific genes as early as 12 hpi, suggesting that the pneumococcal competence regulon is important for adaptation to the lung environment. Additionally, members of the histidine triad (pht) gene family were sharply upregulated at 12 hpi followed by a steep decline throughout the rest of the infection cycle, suggesting that Pht proteins participate in the early adaptation to the lung environment. Further analysis revealed that Pht proteins execute a metal ion-dependent regulatory role in competence induction.IMPORTANCEThe induction of pneumococcal competence for genetic transformation has been extensively studied in vitro but poorly understood during lung infection. We utilized a combination of live imaging and RNA sequencing to monitor the development of a competent state during acute pneumonia. Upregulation of competence-specific genes was observed as early as 12 hour post-infection, suggesting that the pneumococcal competence regulon plays an important role in adapting pneumococcus to the stressful lung environment. Among others, we report novel finding that the pneumococcal histidine triad (pht) family of genes participates in the adaptation to the lung environment and regulates pneumococcal competence induction.

Deciphering the role of Zn2+-binding histidines from TIA-1 on the assembly and dynamics of stress granules.

T-cell intracellular antigen-1 (TIA-1) is a key RNA-binding protein that participates in translation regulation and RNA splicing. TIA-1 undergoes liquid-liquid phase separation as a fundamental mechanism that enables the condensation of RNA and proteins into membraneless organelles called stress granules (SGs). However, this dynamic behavior can lead to aberrant fibril formation, implicated in neurodegenerative disorders, and must be tightly regulated. In this study, we investigated the role in the cell of histidine residues His94 and His96, responsible for Zn2+ binding. Using fluorescence microscopy, we found that the specific binding site formed by these residues is critical for SG assembly. Furthermore, it also plays a role maintaining the dynamic behavior of SG-assembled TIA-1. Collectively, our findings confirm the physiological relevance of TIA-1 His94 and His96 in the Zn2+-mediated regulatory mechanism for protection against fibril formation in SGs.

Histidine transport is essential for the growth of Staphylococcus aureus at low pH.

Staphylococcus aureus is an opportunistic pathogen capable of causing many different human diseases. During colonization and infection, S. aureus will encounter a range of hostile environments, including acidic conditions such as those found on the skin and within macrophages. However, little is known about the mechanisms that S. aureus uses to detect and respond to low pH. Here, we employed a transposon sequencing approach to determine on a genome-wide level the genes required or detrimental for growth at low pH. We identified 31 genes that were essential for the growth of S. aureus at pH 4.5 and confirmed the importance of many of them through follow up experiments using mutant strains inactivated for individual genes. Most of the genes identified code for proteins with functions in cell wall assembly and maintenance. These data suggest that the cell wall has a more important role than previously appreciated in promoting bacterial survival when under acid stress. We also identified several novel processes previously not linked to the acid stress response in S. aureus. These include aerobic respiration and histidine transport, the latter by showing that one of the most important genes, SAUSA300_0846, codes for a previously uncharacterized histidine transporter. We further show that under acid stress, the expression of the histidine transporter gene is increased in WT S. aureus. In a S. aureus SAUSA300_0846 mutant strain expression of the histidine biosynthesis genes is induced under acid stress conditions allowing the bacteria to maintain cytosolic histidine levels. This strain is, however, unable to maintain its cytosolic pH to the same extent as a WT strain, revealing an important function specifically for histidine transport in the acid stress response of S. aureus.