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A green and facile method is presented for in situ synthesis of a novel photoluminescence-quenching nanopaper with a highly-efficient quenching ability, rapid reaction time and long-term storage. The as-prepared nanopaper is further used to construct an aptasensor platform with high performance, rapidness and robustness.

Human body fluids contain biomarkers which are used extensively for prognostication, diagnosis, monitoring, and evaluation of different treatments for a variety of diseases and disorders. The application of biosensors based on cell-free protein synthesis (CFPS) offers numerous advantages including on-demand and at-home use for fast, accurate detection of a variety of biomarkers in human fluids at an affordable price. However, current CFPS-based biosensors use commercial RNase inhibitors to inhibit different RNases present in human fluids and this reagent is approximately 90% of the expense of these biosensors. Here the flexible nature of Escherichia coli-lysate-based CFPS was used for the first time to produce murine RNase Inhibitor (m-RI) and to optimize its soluble and active production by tuning reaction temperature, reaction time, reduced potential, and addition of GroEL/ES folding chaperons. Furthermore, RNase inhibition activity of m-RI with the highest activity and stability was determined against increasing amounts of three human fluids of serum, saliva, and urine (0%-100% v/v) in lyophilized CFPS reactions. To further demonstrate the utility of the CFPS-produced m-RI, a lyophilized saliva-based glutamine biosensor was demonstrated to effectively work with saliva samples. Overall, the use of CFPS-produced m-RI reduces the total reagent costs of CFPS-based biosensors used in human body fluids approximately 90%., (© 2021 Wiley Periodicals LLC.)

Butyl butyrate (BB) has been widely used as a flavor and fragrance compound in the beverage, food, perfume, and cosmetic industries. Currently, BB is produced through two-step processes; butanol and butyrate are first produced and are used as precursors for the esterification reactions to yield BB in the next step. Recently, an alternative process to the current process has been developed by using microorganisms for the one-pot BB production. In the one-pot BB process, alcohol acyl transferases (AATs) and lipases play roles in the esterification of butanol together with their co-substrates butyryl-CoA and butyrate, respectively. In this paper, we review the characteristics of two enzymes including AAT and lipase in the esterification reaction. Also, we review the one-pot processes for BB production by employing the wild-type and engineered Clostridium species and the engineered Escherichia coli strains, with the combination of AATs and lipases.

Bacterial ghosts (BGs) are nonliving empty bacterial shells without cytoplasm retaining original morphology and identical antigenicity of natural bacteria, making them high potential and promising vaccine candidates and delivery vehicles. However, the low yield of commonly used BGs preparation methods limits its mass production and widely application. In order to improve BGs production, E. coli phage ID52 lysis protein E was introduced to generating BGs for the first time. Above all, we compared the lysis activity of lysis protein of E. coli phage φX174 and E. coli phage ID52 as well as the effects of promoters on the lysis activity of ID52-E, which shown that the lysis activity and BGs formation rate of protein ID52-E was significantly higher than protein φX174-E. Further, the lysis activity of ID52-E was significantly improved under the control of L-arabinose inducible promoter which initial induction OD600 reached as high as 2.0. The applicability of lysis protein ID52-E induced by L-arabinose was proved by preparing probiotic E. coli Nissle 1917 BGs and pathogenic Salmonella typhimurium BGs in mass production. This paper introduced a novel and highly efficient method for BGs preparation depending on recombinant expression of E. coli phage ID52-E under eco-friendly and reasonable price inducer L-arabinose. [ABSTRACT FROM AUTHOR]

Several studies have searched for new antigens to produce pneumococcal vaccines that are more effective and could provide broader coverage, given the great number of serotypes causing pneumococcal diseases. One of the promising subunit vaccine candidates is untagged recombinant pneumococcal surface protein A (PspA4Pro), obtainable in high quantities using recombinant Escherichia coli as a microbial factory. However, lipopolysaccharides (LPS) present in E. coli cell extracts must be removed, in order to obtain the target protein at the required purity, which makes the downstream process more complex and expensive. Endotoxin-free E. coli strains, which synthesize a nontoxic mutant LPS, may offer a cost-effective alternative way to produce recombinant proteins for application as therapeutics. This paper presents an investigation of PspA4Pro production employing the endotoxin-free recombinant strain ClearColi® BL21(DE3) with different media (defined, auto-induction, and other complex media), temperatures (27, 32, and 37 °C), and inducers. In comparison to conventional E. coli cells in a defined medium, ClearColi presented similar PspA4Pro yields, with lower productivities. Complex medium formulations supplemented with salts favored PspA4Pro yields, titers, and ClearColi growth rates. Induction with isopropyl-β-d-thiogalactopyranoside (0.5 mM) and lactose (2.5 g/L) together in a defined medium at 32 °C, which appeared to be a promising cultivation strategy, was reproduced in 5 L bioreactor culture, leading to a yield of 146.0 mg PspA4Pro/g dry cell weight. After purification, the cell extract generated from ClearColi led to 98% purity PspA4Pro, which maintained secondary structure and biological function. ClearColi is a potential host for industrial recombinant protein production. Key points: • ClearColi can produce as much PspA4Pro as conventional E. coli BL21(DE3) cells. • 10.5 g PspA4Pro produced in ClearColi bioreactor culture using a defined medium. • Functional PspA4Pro (98% of purity) was obtained in ClearColi bioreactor culture. [ABSTRACT FROM AUTHOR]

The microbiological quality of drinking water is a concern to consumers, water suppliers, regulators, and public health authorities alike. Monitoring the microbiological quality of drinking water largely relies on the examination of indicator bacteria such as coliforms like Escherichia coli. E. coli is widely used as an indicator of fecal pollution when monitoring the microbial quality of drinking water, because it is abundant in all mammal feces and, therefore, is found in sewage and in natural waters contaminated with fecal matter, from human origin, wild animals, or derived from agricultural activity. This paper describes the development of a novel handheld fluorimeter for the rapid detection of E. coli in drinking water based on a specific cellular biomarker. The measurement system is based on a photomultiplier tube that captures the fluorescence signal produced by the cellular biomarker when it is excited by an ultraviolet LED. The cellular biomarker is also developed and it consists of a chimeric protein with a Green fluorescent protein (GFP) in the N-terminal domain and a specific amino acid sequence in the C-terminal domain (Colicin S4) that targets specifically the structure of the microorganism to be detected. The instrument is simple to use, lightweight, and can be powered by either an ac/dc power adapter or a rechargeable battery, making it an excellent choice for the rapid detection of E. coli in drinking water in field studies and laboratory measurements. [ABSTRACT FROM PUBLISHER]

CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π − π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications. Here, the authors show that CRISPR-associated transposons can co-opt other CRISPR arrays found in the same cell. Such interactions must be considered when porting these systems into new hosts. [ABSTRACT FROM AUTHOR]

The YggS/PLPBP protein (also called COG0325 or PLPHP) is a conserved pyridoxal 5 '-phosphate (PLP) -binding protein present in all 3 domains of life. Recent studies have demonstrated that disruption or mutation of this protein has multifaceted effects in various organisms, including vitamin B6 -dependent epilepsy in humans. In Escherichia coli, disruption of this protein--encoded by yggS--perturbs Thr-Ile/Val metabolism, one-carbon metabolism, coenzyme A synthesis, and vitamin B6 homeostasis. This protein is critical for maintaining low levels of pyridoxine 5 '-phosphate (PNP) in various organisms. In the yggS -deficient E. coli strain, inhibition of PLP-dependent enzymes, such as the glycine cleavage system by PNP, is the root cause of metabolic perturbation. Our data suggest that the YggS/PLPBP protein may be involved in the balancing of B6 vitamers by mediating efficient turnover of protein-bound B6 vitamers. This paper reviews recent findings on the function of the YggS/PLPBP protein. [ABSTRACT FROM AUTHOR]

Heme, a Fe-centred hexacoordinated organometallic compound mainly present in blood proteins, acts as a major source of iron (Fe2+) for various hemolytic microorganisms. This makes blood an essential mediumsupplement for characterizing such organisms. Considering the increasing blood demand for this purpose, we are proposing an alternative approach for hemolytic characterization of bacteria using plant derivative. For the first time, we present the kinetic model of growth of Staphylococcus aureus, a beta-hemolytic, gram-positive rod-shaped bacterium, and subsequent heme uptake at an optimized concentration of crude proteins of Beta vulgaris (red beetroot). In this paper, we have determined the heme content of the beetroot juice and demonstrated the ability of S. aureus to uptake heme from beetroot protein solution. According to the determined growth kinetics, the doubling time of the bacterium in the beetroot protein supplemented medium was 25 minutes. The heme uptake analysis showed a zeroth order kinetics with a constant decline rate of 0.19 μM h-1. [ABSTRACT FROM AUTHOR]

Protein turnover is critical for proteostasis, but turnover quantification is challenging, and even in well-studied E. coli, proteome-wide measurements remain scarce. Here, we quantify the turnover rates of ~3200 E. coli proteins under 13 conditions by combining heavy isotope labeling with complement reporter ion quantification and find that cytoplasmic proteins are recycled when nitrogen is limited. We use knockout experiments to assign substrates to the known cytoplasmic ATP-dependent proteases. Surprisingly, none of these proteases are responsible for the observed cytoplasmic protein degradation in nitrogen limitation, suggesting that a major proteolysis pathway in E. coli remains to be discovered. Lastly, we show that protein degradation rates are generally independent of cell division rates. Thus, we present broadly applicable technology for protein turnover measurements and provide a rich resource for protein half-lives and protease substrates in E. coli, complementary to genomics data, that will allow researchers to study the control of proteostasis. Gupta, Johnson et al. quantify the turnover rates of ~3200 E. coli proteins, demonstrating that cytoplasmic proteins are recycled when nitrogen is limited and that protein degradation rates are generally uncoupled from cell division rates. [ABSTRACT FROM AUTHOR]

Stop codon readthrough events give rise to longer proteins, which may alter the protein's function, thereby generating short-lasting phenotypic variability from a single gene. In order to systematically assess the frequency and origin of stop codon readthrough events, we designed a library of reporters. We introduced premature stop codons into mScarlet, which enabled high-throughput quantification of protein synthesis termination errors in E. coli using fluorescent microscopy. We found that under stress conditions, stop codon readthrough may occur at rates as high as 80%, depending on the nucleotide context, suggesting that evolution frequently samples stop codon readthrough events. The analysis of selected reporters by mass spectrometry and RNA-seq showed that not only translation but also transcription errors contribute to stop codon readthrough. The RNA polymerase was more likely to misincorporate a nucleotide at premature stop codons. Proteome-wide detection of stop codon readthrough by mass spectrometry revealed that temperature regulated the expression of cryptic sequences generated by stop codon readthrough in E. coli. Overall, our findings suggest that the environment affects the accuracy of protein production, which increases protein heterogeneity when the organisms need to adapt to new conditions. This study unveils that stop codon readthrough is prevalent in E. coli, particularly under stress conditions. It highlights the influence of stop codon type and genetic context, with both transcriptional and translational origins. [ABSTRACT FROM AUTHOR]

We report the de novo design of small (<20 kDa) and highly soluble synthetic intrinsically disordered proteins (SynIDPs) that confer solubility to a fusion partner with minimal effect on the activity of the fused protein. To identify highly soluble SynIDPs, we create a pooled gene-library utilizing a one-pot gene synthesis technology to create a large library of repetitive genes that encode SynIDPs. We identify three small (<20 kDa) and highly soluble SynIDPs from this gene library that lack secondary structure and have high solvation. Recombinant fusion of these SynIDPs to three known inclusion body forming proteins rescue their soluble expression and do not impede the activity of the fusion partner, thereby eliminating the need for removal of the SynIDP tag. These findings highlight the utility of SynIDPs as solubility tags, as they promote the soluble expression of proteins in E. coli and are small, unstructured proteins that minimally interfere with the biological activity of the fused protein. Insoluble protein expression continues to be a bottleneck for biotechnology. Here, Chilkoti and colleagues report a method for generating and identifying hypersoluble intrinsically disordered protein fusion tags to improve soluble protein expression and rescue protein function. [ABSTRACT FROM AUTHOR]

The Min protein system creates a dynamic spatial pattern in Escherichia coli cells where the proteins MinD and MinE oscillate from pole to pole. MinD positions MinC, an inhibitor of FtsZ ring formation, contributing to the mid-cell localization of cell division. In this paper, Fourier analysis is used to decompose experimental and model MinD spatial distributions into time-dependent harmonic components. In both experiment and model, the second harmonic component is responsible for producing a mid-cell minimum in MinD concentration. The features of this harmonic are robust in both experiment and model. Fourier analysis reveals a close correspondence between the time-dependent behaviour of the harmonic components in the experimental data and model. Given this, each molecular species in the model was analysed individually. This analysis revealed that membrane-bound MinD dimer shows the mid-cell minimum with the highest contrast when averaged over time, carrying the strongest signal for positioning the cell division ring. This concurs with previous data showing that the MinD dimer binds to MinC inhibiting FtsZ ring formation. These results show that non-linear interactions of Min proteins are essential for producing the mid-cell positioning signal via the generation of second-order harmonic components in the time-dependent spatial protein distribution. [ABSTRACT FROM AUTHOR]

Background: Pathogenic bacteria have evolved various strategies to counteract host defences. They are also exposed to environments that are undergoing constant changes. Hence, in order to survive, bacteria must adapt themselves to the changing environmental conditions by performing regulations at the transcriptional and/or post-transcriptional levels. Roles of RNA-binding proteins (RBPs) as virulence factors have been very well studied. Here, we have used a sequence search-based method to compare and contrast the proteomes of 16 pathogenic and three non-pathogenic E. coli strains as well as to obtain a global picture of the RBP landscape (RBPome) in E. coli. Results: Our results show that there are no significant differences in the percentage of RBPs encoded by the pathogenic and the non-pathogenic E. coli strains. The differences in the types of Pfam domains as well as Pfam RNA-binding domains, encoded by these two classes of E. coli strains, are also insignificant. The complete and distinct RBPome of E. coli has been established by studying all known E. coli strains till date. We have also identified RBPs that are exclusive to pathogenic strains, and most of them can be exploited as drug targets since they appear to be non-homologous to their human host proteins. Many of these pathogen-specific proteins were uncharacterised and their identities could be resolved on the basis of sequence homology searches with known proteins. Detailed structural modelling, molecular dynamics simulations and sequence comparisons have been pursued for selected examples to understand differences in stability and RNA-binding. Conclusions: The approach used in this paper to cross-compare proteomes of pathogenic and non-pathogenic strains may also be extended to other bacterial or even eukaryotic proteomes to understand interesting differences in their RBPomes. The pathogen-specific RBPs reported in this study, may also be taken up further for clinical trials and/or experimental validations. [ABSTRACT FROM AUTHOR]

In this paper, we investigate the oscillatory expression in Escherichia coli mediated by microRNA with time delays and reaction-diffusion terms. First of all, the integrated effects of delays and diffusions are first introduced into the genetic regulatory networks involving microRNAs, and a general model of genetic regulatory networks is then formulated. Second, two functional issues on gene regulatory networks, i.e. stability and oscillation of such model, are addressed in detail, and an explicit algorithm determining the properties of periodic oscillation is also presented. We demonstrate that the oscillatory expression of Escherichia coli is not only crucially dependent on the transcriptional and translational delays, but also heavily influenced by the diffusion coefficients. The conclusion is practically verified by a lot of biological experiments and observations. We also find that if the diffusion coefficients of miRNA, mRNA, and protein are suitably small, it can predict that inhomogeneous periodic oscillations can occur unless there only exhibits spatially homogeneous periodic oscillations. The obtained results indicate that the effects of transcriptional and translational delays are essential factors for designing or controlling genetic regulatory networks, in the meantime the functions of reaction-diffusion must beconsidered. Finally, numerical examples are presented to illustrate and visualize theoretical results. [ABSTRACT FROM PUBLISHER]

The discovery of insulin led to a revolution in diabetes management. Since then, many improvements have been introduced to insulin preparations. The availability of molecular genetic techniques has enabled the creation of insulin analogs by changing the structure of the native protein in order to improve the therapeutic properties. A new expression vector pIBAINS for production of four recombinant human insulin (INS) analogs (GKR, GEKR, AKR, SR) was constructed and overexpressed in the new E. coli 20 strain as a fusion protein with modified human superoxide dismutase (SOD). The SOD gene was used as a signal peptide to enhance the expression of insulin. SOD::INS was manufactured in the form of insoluble inclusion bodies. After cleavage of the fusion protein with trypsin, the released insulin analogs were refolded and purified by reverse-phase high performance liquid chromatography (RP-HPLC). Elongation of chain A, described here for the first time, considerably improved the stability of the selected analogs. Their identity was confirmed with mass spectrometric techniques. The biological activity of the insulin derivatives was tested on rats with experimental diabetes. The obtained results proved that the new analogs described in this paper have the potential to generate prolonged hypoglycemic activity and may allow for even less frequent subcutaneous administration than once-a-day. When applied, all the analogs demonstrate a rapid onset of action. Such a combination renders the proposed biosynthetic insulin unique among already known related formulations. [ABSTRACT FROM AUTHOR]

Background: Although different protein-protein physical interaction (PPI) datasets exist for Escherichia coli, no common methodology exists to integrate these datasets and extract reliable modules reflecting the existing biological process and protein complexes. Naïve Bayesian formula is the highly accepted method to integrate different PPI datasets into a single weighted PPI network, but detecting proper weights in such network is still a major problem. Results: In this paper, we proposed a new methodology to integrate various physical PPI datasets into a single weighted PPI network in a way that the detected modules in PPI network exhibit the highest similarity to available functional modules. We used the co-expression modules as functional modules, and we shown that direct functional modules detected from Gene Ontology terms could be used as an alternative dataset. After running this integrating methodology over six different physical PPI datasets, orthologous high-confidence interactions from a related organism and two AP-MS PPI datasets gained high weights in the integrated networks, while the weights for one AP-MS PPI dataset and two other datasets derived from public databases have converged to zero. The majority of detected modules shaped around one or few hub protein(s). Still, a large number of highly interacting protein modules were detected which are functionally relevant and are likely to construct protein complexes. Conclusions: We provided a new high confidence protein complex prediction method supported by functional studies and literature mining. [ABSTRACT FROM AUTHOR]

Cysteine S–H bonds have a spectroscopically convenient stretching frequency of ∼2550 cm−1. However, their cross section is low, and the band can be strongly broadened in heterogeneous environments, making detection very challenging. With two-dimensional infrared (2D-IR) setups achieving ever higher sensitivities in recent years, systematic use of the weak cysteine sulfhydryls (Cys–SHs) absorption band is now within reach, even at low millimolar protein concentrations. Here, we demonstrate the capabilities of Cys–SH as an intrinsic 2D-IR label in pyruvate oxidase from E. coli, an enzyme with ten cysteines in its native sequence. 1D-IR measurements on the wild-type and individual cysteine knock-out variants show that two such residues have especially narrow SH signatures, caused by their intrahelical hydrogen bonding. 2D-IR analysis of these bands reveals an extraordinarily high anharmonicity (∼110 cm−1) and a long vibrational lifetime (∼4 ps). This allows monitoring spectral diffusion via center line slope analysis for up to 10 ps—separately for both the ground and excited states. The unique spectroscopic features and its ease of introduction make Cys–SH a useful IR spectroscopic label. [ABSTRACT FROM AUTHOR]

Bio-mitigation of carbon dioxide has recently attracted more research focus because it is a sustainable and ecofriendly way of carbon conversion to a variety of indispensable chemicals. However, studies regarding the recombinant strain development toward simultaneous chemical production and CO2 assimilation in E. coli are limited and the reported studies are challenged with critically low efficiency. In this context, we first expressed ribulose-1,5-bisphosphate carboxylase/oxygenases (RuBisCOs) and phosphoribulokinases (PRKs) from the Calvin cycle to optimize the CO2 assimilation capability in E. coli with a value of −4.9 g-CO2 per g-DCW. To enhance the CO2 assimilation capability and broaden the applicability of RuBisCO-equipped E. coli, we conducted stepwise integration of the recombinant genes (RuBisCO and PRK) as well as introduced the CRISPRi system for redirecting the carbon flux to the RuBisCO pathway. The final strain, SSCI with CRISPRi targeted on zwf and pfkAB, enhanced the CO2 assimilation capability to −1.58 g-CO2 per g-DCW. Finally, RuBisCO-equipped E. coli was applied for simultaneous CO2 recycling and production of high-value molecules from various proteins, including 5-aminoleuvinic acid (ALA) by ALA synthetase, carbon dioxide sequestration via carbonic anhydrase (CA), and 1,5-diaminopentane (DAP) through lysine decarboxylase. This genomic RuBisCO-equipped E. coli neutralizer is denoted as a GREEN workhorse, with the great opportunity to be a low-carbon footprint microbial cell factory. [ABSTRACT FROM AUTHOR]

The occurrence of NAD+ as a non-canonical RNA cap has been demonstrated in diverse organisms. TIR domain-containing proteins present in all kingdoms of life act in defense responses and can have NADase activity that hydrolyzes NAD+. Here, we show that TIR domain-containing proteins from several bacterial and one archaeal species can remove the NAM moiety from NAD-capped RNAs (NAD-RNAs). We demonstrate that the deNAMing activity of AbTir (from Acinetobacter baumannii) on NAD-RNA specifically produces a cyclic ADPR-RNA, which can be further decapped in vitro by known decapping enzymes. Heterologous expression of the wild-type but not a catalytic mutant AbTir in E. coli suppressed cell propagation and reduced the levels of NAD-RNAs from a subset of genes before cellular NAD+ levels are impacted. Collectively, the in vitro and in vivo analyses demonstrate that TIR domain-containing proteins can function as a deNAMing enzyme of NAD-RNAs, raising the possibility of TIR domain proteins acting in gene expression regulation. Toll/interleukin-1 receptor domain-containing proteins can catabolize NAD+. Here, Wang et al show that these proteins can also function as NAD-RNA decapping enzymes by releasing the NAM moiety from the NAD-RNA, resulting in the regulation of gene expression. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Escherichia coli (E. coli) strains are widely distributed and spreading rapidly, creating significant challenges for clinical therapeutics. NDM-5, a novel mutant of New Delhi Metallo-b-Lactamase-1 (NDM-1), exhibits high hydrolase activity toward carbapenems. Since the genetic backgrounds of clinically isolated carbapenem-resistant E. coli are heterogeneous, it is difficult to accurately evaluate the impact of blaNDM-5 on antibiotic resistance. Herein, E. coli BL21 was transformed with a plasmid harboring blaNDM-5, and the resultant strain was named BL21 (pET-28a-blaNDM-5). Consistent with the findings of previous studies, the introduction of exogenous blaNDM-5 resulted in markedly greater resistance of E. coli to multiple b-lactam antibiotics. Compared with BL21 (pET-28a), BL21 (pET-28a-blaNDM-5) exhibited reduced motility but a significant increase in biofilm formation capacity. Furthermore, transcriptome sequencing was conducted to compare the transcriptional differences between BL21 (pET-28a) and BL21 (pET-28a-blaNDM-5). A total of 461 differentially expressed genes were identified, including those related to antibiotic resistance, such as genes associated with the active efflux system (yddA, mcbR and emrY), pili (csgC, csgF and fimD), biofilm formation (csgD, csgB and ecpR) and antioxidant processes (nuoG). Finally, the pGS21a plasmid harboring blaNDM-5 was transformed into E. coli Rosetta2, after which the expression of the NDM-5 protein was induced using isopropyl-b-D-thiogalactoside (IPTG). Using glutathione-S-transferase (GST) pull-down assays, total proteins from E. coli were scanned to screen out 82 proteins that potentially interacted with NDM-5. Our findings provide new insight into the identified proteins to identify potential antibiotic targets and design novel inhibitors of carbapenem-resistant bacteria. [ABSTRACT FROM AUTHOR]

RNA modification C2-methyladenosine (m2A) exists in both rRNA and tRNA of Escherichia coli (E. coli), installed by the methyltransferase RlmN using a radical-S-adenosylmethionine (SAM) mechanism. However, the precise function of m2A in tRNA and its ubiquity in plants have remained unclear. Here we discover the presence of m2A in chloroplast rRNA and tRNA, as well as cytosolic tRNA, in multiple plant species. We identify six m2A-modified chloroplast tRNAs and two m2A-modified cytosolic tRNAs across different plants. Furthermore, we characterize three Arabidopsis m2A methyltransferases—RLMNL1, RLMNL2, and RLMNL3—which methylate chloroplast rRNA, chloroplast tRNA, and cytosolic tRNA, respectively. Our findings demonstrate that m2A37 promotes a relaxed conformation of tRNA, enhancing translation efficiency in chloroplast and cytosol by facilitating decoding of tandem m2A-tRNA-dependent codons. This study provides insights into the molecular function and biological significance of m2A, uncovering a layer of translation regulation in plants. Duan et al. demonstrate that the m2A modification is ubiquitous in plants and tRNA m2A37 promotes a relaxed conformation of tRNA, enhancing translation efficiency by facilitating decoding of tandem m2A-tRNA-dependent codons. [ABSTRACT FROM AUTHOR]

Lawsonia intracellularis, the etiologic agent of proliferative enteropathy (PE), is an obligate intracellular Gram-negative bacterium possessing a type III secretion system (T3SS), which enables the pathogen to translocate effector proteins into targeted host cells to modulate their functions. T3SS is a syringe-like apparatus consisting of a base, an extracellular needle, a tip, and a translocon. The translocon proteins assembled by two hydrophobic membrane proteins can form pores in the host-cell membrane, and therefore play an essential role in the function of T3SS. To date, little is known about the T3SS and translocon proteins of L. intracellularis. In this study, we first analyzed the conservation of the T3S apparatus between L. intracellularis and Yersinia, and characterized the putative T3S hydrophobic major translocon protein LI1158 and minor translocon protein LI1159 in the L. intracellularis genome. Then, by using Yersinia pseudotuberculosis as a surrogate system, we found that the full-length LI1158 and LI1159 proteins, but not the putative class II chaperone LI1157, were secreted in a − Ca2+ and T3SS-dependent manner and the secretion signal was located at the N terminus (aa 1–40). Furthermore, yeast-two hybrid experiments revealed that LI1158 and LI1159 could self-interact, and LI1159 could interact with LI1157. However, unlike CPn0809 and YopB, which are the major hydrophobic translocon proteins of the T3SS of C. pneumoniae and Yersinia, respectively, full-length LI1158 was non-toxic to both yeast and Escherichia coli cells, but full-length LI1159 showed certain toxicity to E. coli cells. Taken together, despite some differences from the findings in other bacteria, our results demonstrate that LI1158 and LI1159 may be the translocon proteins of L. intracellularis T3SS, and probably play important roles in the translocation of effector proteins at the early pathogen infection stage. [ABSTRACT FROM AUTHOR]

In many species highly expressed genes (HEGs) over-employ the synonymous codons that match the more abundant iso-acceptor tRNAs. Bacterial transgene codon randomization experiments report, however, that enrichment with such "translationally optimal" codons has little to no effect on the resultant protein level. By contrast, consistent with the view that ribosomal initiation is rate limiting, synonymous codon usage following the 5' ATG greatly influences protein levels, at least in part by modifying RNA stability. For the design of bacterial transgenes, for simple codon based in silico inference of protein levels and for understanding selection on synonymous mutations, it would be valuable to computationally determine initiation optimality (IO) scores for codons for any given species. One attractive approach is to characterize the 5' codon enrichment of HEGs compared with the most lowly expressed genes, just as translational optimality scores of codons have been similarly defined employing the full gene body. Here we determine the viability of this approach employing a unique opportunity: for Escherichia coli there is both the most extensive protein abundance data for native genes and a unique large-scale transgene codon randomization experiment enabling objective definition of the 5' codons that cause, rather than just correlate with, high protein abundance (that we equate with initiation optimality, broadly defined). Surprisingly, the 5' ends of native genes that specify highly abundant proteins avoid such initiation optimal codons. We find that this is probably owing to conflicting selection pressures particular to native HEGs, including selection favouring low initiation rates, this potentially enabling high efficiency of ribosomal usage and low noise. While the classical HEG enrichment approach does not work, rendering simple prediction of native protein abundance from 5' codon content futile, we report evidence that initiation optimality scores derived from the transgene experiment may hold relevance for in silico transgene design for a broad spectrum of bacteria. Author summary: Transgene experiments in Escherichia coli report that codon usage in the first few amino acids after the initiating ATG has a profound influence on the resulting protein level by promoting ribosomal initiation, rather than enabling speedy elongation. For the design of bacterial transgenes and for simple codon-based in silico inference of protein levels, it would be valuable to computationally determine initiation optimality scores for codons for any given species. An attractive approach is to characterize the 5' codon enrichment of highly expressed genes compared with the most lowly expressed genes, just as translational optimality scores of codons (affecting elongation) have been similarly defined employing the full gene body. Using unique resources provided for E. coli we show that, unexpectedly, this doesn't work: the 5' ends of highly expressed genes are enriched, compared to lowly expressed ones, in codons that objectively are associated with low initiation rates. This likely reflects conflicting selection pressures in highly expressed genes which can favour low initiation to promote low noise or high efficiency. While simple prediction of native protein abundance from 5' codon content is then somewhat futile, the objective initiation optimality scores may hold relevance for in silico transgene design for a broad spectrum of bacteria. [ABSTRACT FROM AUTHOR]

Anti-CRISPR proteins (Acrs) block the activity of CRISPR-associated (Cas) proteins, either by inhibiting DNA interference or by preventing crRNA loading and complex formation. Although the main use of Acrs in genome engineering applications is to lower the cleavage activity of Cas proteins, they can also be instrumental for various other CRISPR-based applications. Here, we explore the genome editing potential of the thermoactive type II-C Cas9 variants from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus stearothermophilus (GeoCas9) in Escherichia coli. We then demonstrate that the AcrIIC1 protein from Neisseria meningitidis robustly inhibits their DNA cleavage activity, but not their DNA binding capacity. Finally, we exploit these AcrIIC1:Cas9 complexes for gene silencing and base-editing, developing Acr base-editing tools. With these tools we pave the way for future engineering applications in mesophilic and thermophilic bacteria combining the activities of Acr and CRISPR-Cas proteins. Utilising AcrIIC1, which can provide an 'off-switch' by inhibiting the DNA cleavage activity of ThermoCas9 and GeoCas9, a Class 2 CRISPR-Acr tool is described for gene silencing and base-editing applications. [ABSTRACT FROM AUTHOR]

Heavy single-chain antibodies (VHH or nanobodies) are popular in the medical and analytical fields due to its small size, high solubility, stability, and other advantageous features. However, the usage of VHHs is limited by the low yield of its production and purification. In order to determine the optimal purification strategy for VHH to improve the yield, a method to monitor purification at the intermediate steps is needed. In this study, a simple, sensitive, low-cost sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantitate VHHs throughout the purification steps. Under optimized conditions, the assay has a sensitivity of 0.149 OD·mL/ng and a limit of detection (LOD) of 0.029 ng/mL. The average recoveries of the assay against the spiked samples were 101.9–106.0% and 100.7–108.0%. The method was applied to a variety of real samples for the detection of different VHHs in bacterial cell media. High amount of VHHs (up to 41.3 mg/mL), which are comparable to the average yield of VHH in standard production protocols, were detected in the media. This study raises attention to the problem of protein losses in cell culture supernatants and provides a method for the continuous detection of the protein abundance to optimize the expression and purification protocols especially for nanobodies. [ABSTRACT FROM AUTHOR]

The study was aimed to characterize the antioxidant and anti-microbial activities of Cynodon dactylon with special reference on its precise biochemical analysis. Physiological analysis that total carotenoids content (0.3884 ± 0.0172 mg/g), total chlorophyll content (6.1460 ± 0.2915 mg/g), total phenolic contents (13.4703 ± 0.1494 mg/g), chlorophyll a (3.7708 ± 0.1528 mg/g, catalase (CAT) contents (40.2844 ± 0.1515 units/mg), total anthocyanin contents (5.0166 ± 0.2966 g–1 FW) total soluble proteins (2.9916 ± 0.1734 mg/g) and total flavonoids content (TFC) (4.7863 ± 0.0442 μg/g) was found higher in the leaves of the Cynodon dactylon whereas, chlorophyll b (2.4881 ± 0.1326 mg/g) was found higher in the stem of Cynodon dactylon, while, peroxidase (POD) contents (81.8763 ± 4.6609 units/mg) and superoxidase dismutase (SOD) activity (80.4346 ± 5.9367 units/mg) was investigated higher in roots of Cynodon dactylon. The anti-microbial activity of Cynodon dactylon extracts was performed using a good diffusion technique against two microbial strains. Among all the plant extracts, the methanolic extracts showed a maximum inhibition zone (26.87 mm) against anti-bacterial strain Escherichia coli whereas n-hexane extract showed a maximum inhibition zone (17.88 mm) against anti-fungal strain Candida albicans. This study reported the antimicrobial activity of Cynodon dactylon against some common pathogens such as Staphylococcus aureus, Escherichia coli, and Candida albicans, which are highly associated with nosocomial infection. From the given results it is concluded that Cynodon dactylon could be exploited in pharmacology due to its antioxidant and anti-microbial properties. [ABSTRACT FROM AUTHOR]

Recombination-promoting nuclease (Rpn) proteins are broadly distributed across bacterial phyla, yet their functions remain unclear. Here, we report that these proteins are toxin-antitoxin systems, comprised of genes-within-genes, that combat phage infection. We show the small, highly variable Rpn C-terminal domains (RpnS), which are translated separately from the full-length proteins (RpnL), directly block the activities of the toxic RpnL. The crystal structure of RpnAS revealed a dimerization interface encompassing a helix that can have four amino acid repeats whose number varies widely among strains of the same species. Consistent with strong selection for the variation, we document that plasmid-encoded RpnP2L protects Escherichia coli against certain phages. We propose that many more intragenic-encoded proteins that serve regulatory roles remain to be discovered in all organisms. [ABSTRACT FROM AUTHOR]

Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell‐surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b‐C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed 'OaAEP1b‐C247A‐∆351' could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid‐activated OaAEP1b‐C247A enzyme. This constitutively active protein can be stored for up to 2 years at −80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E. coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation. [ABSTRACT FROM AUTHOR]

In the present study, silver nanoparticles (AgNPs) were synthesised by adding 1 mM Ag nitrate solution to different concentrations (1%, 2.5%, 5%) of branch extracts of Eurycoma longifolia, a well known medicinal plant in South–East Asian countries. Characterisation of AgNPs was carried out using techniques such as ultraviolet–visible spectrophotometry, X‐ray diffractrometry, Fourier transform infrared–attenuated total reflection spectroscopy (FTIR–ATR), scanning electron microscopy. XRD analysis revealed face centre cubic structure of AgNPs and FTIR–ATR showed that primary and secondary amide groups in combination with the protein molecules present in the branch extract were responsible for the reduction and stabilisation of AgNPs. Furthermore, antioxidant [2,2‐diphenyl‐1‐picrylhydrazyl and 2,2′‐Azino‐bis(3‐ethylbenzthiazoline‐6‐sulphonic acid)], antimicrobial and anticancer activities of AgNPs were investigated. The highest bactericidal activity of these biogenic AgNPs was found against Escherichia coli with zone inhibition of 11 mm. AgNPs exhibited significant anticancer activity against human glioma cells (DBTRG and U87) and human breast adenocarcinoma cells (MCF‐7 and MDA‐MB‐231) with IC50 values of 33, 42, 60 and 38 µg/ml. [ABSTRACT FROM AUTHOR]

O-GlcNAcylation is a single glycosylation of GlcNAc mediated by OGT, which regulates the function of substrate proteins and is closely related to many diseases. However, a large number of O-GlcNAc-modified target proteins are costly, inefficient, and complicated to prepare. In this study, an OGT binding peptide (OBP)-tagged strategy for improving the proportion of O-GlcNAc modification was established successfully in E. coli. OBP (P1, P2, or P3) was fused with target protein Tau as tagged Tau. Tau or tagged Tau was co-constructed with OGT into a vector expressed in E. coli. Compared with Tau, the O-GlcNAc level of P1Tau and TauP1 increased 4~6-fold. Moreover, the P1Tau and TauP1 increased the O-GlcNAc-modified homogeneity. The high O-GlcNAcylation on P1Tau resulted in a significantly slower aggregation rate than Tau in vitro. This strategy was also used successfully to increase the O-GlcNAc level of c-Myc and H2B. These results indicated that the OBP-tagged strategy was a successful approach to improve the O-GlcNAcylation of a target protein for further functional research. [ABSTRACT FROM AUTHOR]

Bacterial ribosomes contain over 50 ribosome core proteins (r-proteins). Tens of non-ribosomal proteins bind to ribosomes to promote various steps of translation or suppress protein synthesis during ribosome hibernation. This study sets out to determine how translation activity is regulated during the prolonged stationary phase. Here, we report the protein composition of ribosomes during the stationary phase. According to quantitative mass-spectrometry analysis, ribosome core proteins bL31B and bL36B are present during the late log and first days of the stationary phase and are replaced by corresponding A paralogs later in the prolonged stationary phase. Ribosome hibernation factors Rmf, Hpf, RaiA, and Sra are bound to the ribosomes during the onset and a few first days of the stationary phase when translation is strongly suppressed. In the prolonged stationary phase, a decrease in ribosome concentration is accompanied by an increase in translation and association of translation factors with simultaneous dissociation of ribosome hibernating factors. The dynamics of ribosome-associated proteins partially explain the changes in translation activity during the stationary phase. [ABSTRACT FROM AUTHOR]

Reporter genes are important tools in many biological disciplines. The discovery of novel reporter genes is relatively rare. However, known reporter genes are constantly applied to novel applications. This study reports the performance of the bilirubin-dependent fluorescent protein UnaG from the Japanese eel Anguilla japonicas in live Escherichia coli cells in response to the disruption of outer membrane (OM) integrity at low bilirubin (BR) concentrations. Using the E. coli wild-type strain MC4100, its isogenic OM-deficient mutant strain NR698, and different OM-active compounds, we show that BR uptake and UnaG fluorescence depend on a leaky OM at concentrations of 10 µM BR and below, while fluorescence is mostly OM integrity-independent at concentrations above 50 µM BR. We suggest that these properties of the UnaG–BR couple might be applied as a biosensor as an alternative to the OM integrity assays currently in use. [ABSTRACT FROM AUTHOR]

The optimal use of many biotherapeutics is restricted by Anti-drug antibodies (ADAs) and hypersensitivity responses which can affect potency and ability to administer a treatment. Here we demonstrate that Re-surfacing can be utilized as a generalizable approach to engineer proteins with extensive surface residue modifications in order to avoid binding by pre-existing ADAs. This technique was applied to E. coli Asparaginase (ASN) to produce functional mutants with up to 58 substitutions resulting in direct modification of 35% of surface residues. Re-surfaced ASNs exhibited significantly reduced binding to murine, rabbit and human polyclonal ADAs, with a negative correlation observed between binding and mutational distance from the native protein. Reductions in ADA binding correlated with diminished hypersensitivity responses in an in vivo mouse model. By using computational design approaches to traverse extended distances in mutational space while maintaining function, protein Re-surfacing may provide a means to generate novel or second line therapies for life-saving drugs with limited therapeutic alternatives. [ABSTRACT FROM AUTHOR]

The study of the interaction between temperate phages and bacteria is vital to understand their role in the development of human diseases. In this study, a novel temperate Escherichia coli phage, vB_EcoP_ZX5, with a genome size of 39,565 bp, was isolated from human fecal samples. It has a short tail and belongs to the genus Uetakevirus and the family Podoviridae. Phage vB_EcoP_ZX5 encodes three lysogeny-related proteins (ORF12, ORF21, and ORF4) and can be integrated into the 3′-end of guaA of its host E. coli YO1 for stable transmission to offspring bacteria. Phage vB_EcoP_ZX5 in lysogenized E. coli YO1+ was induced spontaneously, with a free phage titer of 107 PFU/mL. The integration of vB_EcoP_ZX5 had no significant effect on growth, biofilm, environmental stress response, antibiotic sensitivity, adherence to HeLa cells, and virulence of E. coli YO1. The ORF4 anti-repressor, ORF12 integrase, and ORF21 repressors that affect the lytic–lysogenic cycle of vB_EcoP_ZX5 were verified by protein overexpression. We could tell from changes of the number of total phages and the transcription level of phage genes that repressor protein is the key determinant of lytic-to-lysogenic conversion, and anti-repressor protein promotes the conversion from lysogenic cycle to lytic cycle. [ABSTRACT FROM AUTHOR]

The surface stiffness of the microenvironment is a mechanical signal regulating biofilm growth without the risks associated with the use of bioactive agents. However, the mechanisms determining the expansion or prevention of biofilm growth on soft and stiff substrates are largely unknown. To answer this question, we used PDMS (polydimethylsiloxane, 9–574 kPa) and HA (hyaluronic acid gels, 44 Pa–2 kPa) differing in their hydration. We showed that the softest HA inhibited Escherichia coli biofilm growth, while the stiffest PDMS activated it. The bacterial mechanical environment significantly regulated the MscS mechanosensitive channel in higher abundance on the least colonized HA-44Pa, while Type-1 pili (FimA) showed regulation in higher abundance on the most colonized PDMS-9kPa. Type-1 pili regulated the free motion (the capacity of bacteria to move far from their initial position) necessary for biofilm growth independent of the substrate surface stiffness. In contrast, the total length travelled by the bacteria (diffusion coefficient) varied positively with the surface stiffness but not with the biofilm growth. The softest, hydrated HA, the least colonized surface, revealed the least diffusive and the least free-moving bacteria. Finally, this shows that customizing the surface elasticity and hydration, together, is an efficient means of affecting the bacteria's mobility and attachment to the surface and thus designing biomedical surfaces to prevent biofilm growth. [ABSTRACT FROM AUTHOR]

Advances in synthetic biology permit the genetic encoding of synthetic chemistries at monomeric precision, enabling the synthesis of programmable proteins with tunable properties. Bacterial pili serve as an attractive biomaterial for the development of engineered protein materials due to their ability to self-assemble into mechanically robust filaments. However, most biomaterials lack electronic functionality and atomic structures of putative conductive proteins are not known. Here, we engineer high electronic conductivity in pili produced by a genomically-recoded E. coli strain. Incorporation of tryptophan into pili increased conductivity of individual filaments >80-fold. Computationally-guided ordering of the pili into nanostructures increased conductivity 5-fold compared to unordered pili networks. Site-specific conjugation of pili with gold nanoparticles, facilitated by incorporating the nonstandard amino acid propargyloxy-phenylalanine, increased filament conductivity ~170-fold. This work demonstrates the sequence-defined production of highly-conductive protein nanowires and hybrid organic-inorganic biomaterials with genetically-programmable electronic functionalities not accessible in nature or through chemical-based synthesis. Bacterial hairs called pili become highly-conductive electric wires upon addition of both natural and synthetic amino acids conjugated with gold nanoparticles. Here the authors use computationally-guided ordering further increasing their conductivity, thus yielding genetically-programmable materials. [ABSTRACT FROM AUTHOR]

Attaching and effacing (AE) lesion formation on enterocytes by enteropathogenic Escherichia coli (EPEC) requires the EPEC type III secretion system (T3SS). Two T3SS effectors injected into the host cell during infection are the atypical kinases, NleH1 and NleH2. However, the host targets of NleH1 and NleH2 kinase activity during infection have not been reported. Here phosphoproteomics identified Ser775 in the microvillus protein Eps8 as a bona fide target of NleH1 and NleH2 phosphorylation. Both kinases interacted with Eps8 through previously unrecognized, noncanonical "proline-rich" motifs, PxxDY, that bound the Src Homology 3 (SH3) domain of Eps8. Structural analysis of the Eps8 SH3 domain bound to a peptide containing one of the proline-rich motifs from NleH showed that the N-terminal part of the peptide adopts a type II polyproline helix, and its C-terminal "DY" segment makes multiple contacts with the SH3 domain. Ser775 phosphorylation by NleH1 or NleH2 hindered Eps8 bundling activity and drove dispersal of Eps8 from the AE lesion during EPEC infection. This finding suggested that NleH1 and NleH2 altered the cellular localization of Eps8 and the cytoskeletal composition of AE lesions during EPEC infection. [ABSTRACT FROM AUTHOR]

The present study reports the synthesis of silver nanoparticles (AgNPs) using both biological and chemical routes to find out the best method for control of their size and activity. The fungal agent (Fusarium oxysporum) and the plant (Azadirachta indica) were found to be the best source for AgNPs synthesis. Both biosynthesis and chemosynthesis were achieved by challenging filtrate with AgNO3 (1 mM) solution. The synthesised nanoparticles were characterised by ultraviolet–visible spectroscopy, Fourier transform infrared spectroscopy, nanoparticle tracking analysis (LM20), zeta potential measurement and transmission electron microscopy. The biologically synthesised nanoparticles were spherical, polydispersed and in the range of 10–40 nm, while chemically synthesised nanoparticles were highly monodispersed with a size of 5 nm. The antimicrobial assay against Escherichia coli and Staphylococcus aureus proved biogenic AgNPs to be more potent antibacterial agents than chemically synthesised AgNPs. The possible antibacterial mechanism of AgNPs has also been discussed. Biogenic AgNPs have shown more activity because of the protein capping and their mode of entry into the bacterial cell. These findings may encourage the use of biosynthesis over the chemosynthesis method. [ABSTRACT FROM AUTHOR]

Biochemical separations are the heart of diagnostic assays and purification methods for biologics. On-chip miniaturization and modularization of separation procedures will enable the development of customized, portable devices for personalized health-care diagnostics and point-of-use production of treatments. In this report, we describe the design and fabrication of miniature ion exchange, size exclusion and affinity chromatography modules for on-chip clean-up of recombinantly-produced proteins. Our results demonstrate that these common separations techniques can be implemented in microfluidic modules with performance comparable to conventional approaches. We introduce embedded 3-D microfluidic interconnects for integrating micro-scale separation modules that can be arranged and reconfigured to suit a variety of fluidic operations or biochemical processes. We demonstrate the utility of the modular approach with a platform for the enrichment of enhanced green fluorescent protein (eGFP) from Escherichia coli lysate through integrated affinity and size-exclusion chromatography modules. [ABSTRACT FROM AUTHOR]

Massively parallel measurements of dominant-negative inhibition by protein fragments have been used to map protein interaction sites and discover peptide inhibitors. However, the underlying principles governing fragment-based inhibition have thus far remained unclear. Here, we adapted a high-throughput inhibitory fragment assay for use in Escherichia coli, applying it to a set of 10 essential proteins. This approach yielded single amino acid resolution maps of inhibitory activity, with peaks localized to functionally important interaction sites, including oligomerization interfaces and folding contacts. Leveraging these data, we performed a systematic analysis to uncover principles of fragment-based inhibition. We determined a robust negative correlation between susceptibility to inhibition and cellular protein concentration, demonstrating that inhibitory fragments likely act primarily by titrating native protein interactions. We also characterized a series of trade-offs related to fragment length, showing that shorter peptides allow higher-resolution mapping but suffer from lower inhibitory activity. We employed an unsupervised statistical analysis to show that the inhibitory activities of protein fragments are largely driven not by generic properties such as charge, hydrophobicity, and secondary structure, but by the more specific characteristics of their bespoke macromolecular interactions. Overall, this work demonstrates fundamental characteristics of inhibitory protein fragment function and provides a foundation for understanding and controlling protein interactions in vivo. [ABSTRACT FROM AUTHOR]

Paracoccus denitrificans ArsH is encoded by two identical genes located in two distinct putative arsenic resistance (ars) operons. Escherichia coli-produced recombinant N-His6-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH2) and formation of phenylarsonous acid were observed. Growth experiments with P. denitrificans and E. coli revealed increased toxicity of phenylarsonic acid to cells expressing arsH, which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of ars operon. [ABSTRACT FROM AUTHOR]

Correction for 'Novel insights into the metal binding ability of ZinT periplasmic protein from Escherichia coli and Salmonella enterica' by Denise Bellotti et al., Dalton Trans., 2020, 49, 9393–9403, DOI: 10.1039/D0DT01626H. [ABSTRACT FROM AUTHOR]

P1 is a model temperate myovirus. It infects different Enterobacteriaceae and can develop lytically or form lysogens. Only some P1 adaptation strategies to propagate in different hosts are known. An atypical feature of P1 is the number and organization of cell lysis-associated genes. In addition to SAR-endolysin Lyz, holin LydA, and antiholin LydB, P1 encodes other predicted holins, LydC and LydD. LydD is encoded by the same operon as Lyz, LydA and LydB are encoded by an unlinked operon, and LydC is encoded by an operon preceding the lydA gene. By analyzing the phenotypes of P1 mutants in known or predicted holin genes, we show that all the products of these genes cooperate with the P1 SAR-endolysin in cell lysis and that LydD is a pinholin. The contributions of holins/pinholins to cell lysis by P1 appear to vary depending on the host of P1 and the bacterial growth conditions. The pattern of morphological transitions characteristic of SAR-endolysin–pinholin action dominates during lysis by wild-type P1, but in the case of lydC lydD mutant it changes to that characteristic of classical endolysin-pinholin action. We postulate that the complex lytic system facilitates P1 adaptation to various hosts and their growth conditions. [ABSTRACT FROM AUTHOR]