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In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition., Competing Interests: Declaration of competing interest The authors declare no competing financial interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Plants are often seen as a potent tool in the recombinant protein production industry. However, unlike bacterial expression, it is not a popular method due to the low yield and difficulty of protein extraction and purification. Therefore, developing a new high efficient and easy to purify platform is crucial. One of the best approaches to make extraction easier is to utilize the Extensin Signal peptide (EXT) to translocate the recombinant protein to the outside of the cell, along with incorporating an Elastin-like polypeptide tag (ELP) to enhance purification and accumulation rates. In this research, we transiently expressed Shigella dysenteriae's IpaDSTxB fused to both NtEXT and ELP in both Nicotiana tabacum and Medicago sativa. Our results demonstrated that N. tabacum, with an average yield of 6.39 ng/μg TSP, outperforms M. sativa, which had an average yield of 3.58 ng/μg TSP. On the other hand, analyzing NtEXT signal peptide indicated that merging EXT to the constructs facilitates translocation of IpaDSTxB to the apoplast by 78.4% and 65.9% in N. tabacum and M. sativa, respectively. Conversely, the mean level for constructs without EXT was below 25% for both plants. Furthermore, investigation into the orientation of ELP showed that merging it to the C-terminal of IpaDSTxB leads to a higher accumulation rate in both N. tabacum and M. sativa by 1.39 and 1.28 times, respectively. It also facilitates purification rate by over 70% in comparison to 20% of the 6His tag. The results show a highly efficient and easy to purify platform for the expression of heterologous proteins in plant., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nucleotide sugars (UDP-Sugars) are essential for the production of polysaccharides and glycoconjugates utilized in medicines, cosmetics, and food industries. The enzyme Galactose-1-phosphate uridylyltransferase (GalU; EC 2.7.7.12) is responsible for the synthesis of UDP-galactose from α-d-galactose-1-phosphate (Gal-1P) and UTP. A novel bacterial GalU (TiGalU) encoded from a thermophilic bacterium, Thermodesulfatator indicus, was successfully purified using the Ni-NTA column after being expressed in Escherichia coli. The optimal pH for recombinant TiGalU was determined to be 5.5. The optimum temperature of the enzyme was 45 °C. The activity of TiGalU was not dependent on Mg 2+ and was strongly inhibited by SDS. When coupled with galactose kinase (GALK1) and β-1,4-galactosyltransferase 1 (B4GALT1), the enzyme enabled the one-pot synthesis of Gal-β-1,4-GlcNAc-X by utilizing galactose and UTP as substrates. This study reported the in vitro biosynthesis of Gal-β-1,4-GlcNAc-X for the first time, providing an environmentally friendly way to biosynthesis glycosides and other polysaccharides., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Chiral amino acids and their deamination products, α-keto acids, have important applications in food, medicine, and fine chemicals. In this study, two l-amino acid deaminase genes from Proteus mirabilis, PM473 of type Ⅰ and PM471 of type Ⅱ were cloned and expressed in Escherichia coli respectively, expected to achieve the chiral separation of amino acids. Extensive substrate preference testing showed that both deaminases had catalytic effects on the d-amino acid component of the D, l-amino acids, and PM473 has a wider catalytic range for amino acids. When D, L-Cys was used as the substrate, all L-Cys components and 75.1 % of D-Cys were converted to mercapto pyruvate, and the remaining D-Cys was a single chiral enantiomer. Molecular docking analysis showed that the interaction between the substrate and the key residues affected the stereoselectivity of enzymes. The compatibility of hydrophobicity between the binding pocket and substrate may be the basic factor that affects the substrate selectivity. This work provides an alternative method for the production of α-keto acids and the resolution of chiral amino acids., Competing Interests: Declaration of competing interest All authors agree to publish and declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Staphylococcus aureus (S. aureus) presents a significant challenge in both nosocomial and community settings due to its pathogenicity. The emergence of drug-resistant strains exacerbates S. aureus infections, leading to increased mortality rates. PyrG, a member of the cytidine triphosphate (CTP) synthase family, serves as a crucial therapeutic target against S. aureus due to the pivotal role of CTP in cellular metabolism. However, the structural and mechanistic details of S. aureus PyrG remains unknown. Here, we successfully expressed and purified monomeric PyrG. Mutational experiments were conducted based on the results of molecular docking. Based on the results of the molecular docking, we carried out mutation experiments and found that Q386A dramatically decreased the CTP synthase activity compared to the wild-type protein, while Y54A almost completely abolished the activity. Exposure of S. aureus to the kinase inhibitor crizotinib increased expression of gene pyrG. Our results identify the two key sites on PyrG for the CTP synthase activity, and present PyrG gene expression increased during the treatment of crizotinib, which may eventually provide valuable guidance for the development of new drugs against S. aureus infections., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Thermostable apurinic/apyrimidinic (AP) endonuclease (TtAP), cloned from Caldanaerobacter subterraneus subsp. tengcongensis, is an exonuclease III (Exo III) family protein with high-heat resistance, has activities of AP site endonuclease, 3'-5' exonuclease, and 3'-nuclease, and facilitates efficient amplification of lengthy DNA fragments in PCR. However, the research of the combinant TtAP in Escherichia coli with its expression, large-scale extraction and purification of its protein was limited. In this study, we optimized the codons of TtAP gene for expression in E. coli and constructed a fusion gene encoding TtAP with a 6His tag (TtAP-6His). TtAP-6His was put into vector pET-30a (+) to form the expression vector pET-30a (+) -TtAP-6His, and was then introduced into E. coli strain Rosetta (DE3). We established a systematic process for the extraction of TtAP protein using 5 liters of bacterial suspension, including the optimization of IPTG induction time (6 h), followed by protein extraction using enzymolysis buffers, the heat treatment of temperature (70 °C) with 60 min to remove impurity, precipitation with ammonium sulfate (55 %), protein purification with Ni-affinity chromatography, and the enzyme activities finally were determined. The purification yield of TtAP-6His ranged from 73.67 to 115.25 mg/L (47 KU/mg)., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Previously, we identified the human annexin A1 as a purification tag for column-free purification with gentler calcium-responsive precipitation. In this work, we used the annexin A1 tagged green fluorescent protein constructs for detecting extracellular production in Escherichia coli, Bacillus subtilis, and Pichia pastoris, and identified that the leaderless fusion protein was transported extracellularly in E. coli with supply of additives including Triton X-100. The coexpressed enzymes, culture compositions, and induction conditions in E. coli extracellular expression systems were optimized. With coexpression of phospholipase C from Bacillus cereus and addition of 0.2 % Triton X-100 after induction for 60 h at 28 °C, the annexin A1 tagged green fluorescent protein and 5-aminolevulinate dehydratase from E. coli were overexpressed and purified from lysogeny broth by precipitation with 20 mM Ca 2+ and redissolution with 25 mM EDTA with the acceptable protein purities and recoveries. The silica binding peptide was fused to the annexin A1 tagged fluorescent protein fusion for successive affinity precipitation and purification. With incubation of the specific protease, the released tag-free protein displayed higher purity via on-resin cleavage than that through cleavage of the free fusion protein. The tandem tag is applicable for two-step purification of small or large amounts of other fusion proteins in the culture and recovery of tag-free proteins at low cost., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The recognition and characterization of gene-encoded nitric oxide synthase (NOS) from Exiguobacterium profundum are reported in this study. A new gene was sequenced and cloned from E. profundum and heterologously expressed in E. coli for functional identification, followed by protein purification using the His-tag. The stability and activity characteristics of the recombinant NOS were evaluated using different concentrations of IPTG at various time points. A band of approximately 42 kDa was observed by SDS-PAGE. The K m value of NOS, calculated based on the Michaelis-Menten equation was 0.59 μmol/L. Additionally, homologous sequence alignment analysis indicated that the new NOS shared 80.48 % similarity with the same protein from Bacillus subtilis and Umezawaea. The construction of the NOS expression vector and the purification of the recombinant protein provide a foundation for further functional research and inhibitor development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Infections that are acquired due to a prolonged hospital stay and manifest 2 days following the admission of a patient to a health-care institution can be classified as hospital-acquired infections. Klebsiella pneumoniae (K. pneumoniae) has become a critical pathogen, posing serious concern globally due to the rising incidences of hypervirulent and carbapenem-resistant strains. Glutaredoxin is a redox protein that protects cells from oxidative stress as it associates with glutathione to reduce mixed disulfides. Protein adenylyltransferase (PrAT) is a pseudokinase with a proposed mechanism of transferring an AMP group from ATP to glutaredoxin. Inducing oxidative stress to the bacterium by inhibiting the activity of PrAT is a promising approach to combating its contribution to hospital-acquired infections. Thus, this study aims to overexpress, purify, and analyse the effects of ATP and Mg 2+ binding to Klebsiella pneumoniae PrAT (KpPrAT). The pET expression system and nickel affinity chromatography were effective in expressing and purifying KpPrAT. Far-UV CD spectroscopy demonstrates that the protein is predominantly α-helical, even in the presence of Mg 2+ . Extrinsic fluorescence spectroscopy with ANS indicates the presence of a hydrophobic pocket in the presence of ATP and Mg 2+ , while mant-ATP studies allude to the potential nucleotide binding ability of KpPrAT. The presence of Mg 2+ increases the thermostability of the protein. Isothermal titration calorimetry provides insight into the binding affinity and thermodynamic parameters associated with the binding of ATP to KpPrAT, with or without Mg 2+ . Conclusively, the presence of Mg 2+ induces a conformation in KpPrAT that favours nucleotide binding., (© 2024. The Author(s).)

The production of fermentable sugars from lignocellulosic biomass is achieved by the synergistic action of a group of enzymes called cellulases. Cellulose is a long chain of chemically linked glucoses by β-1,4 bonds. The enzyme β-1,4-endoglucanase is the first cellulase involved in the degradation, breaking the bond of the amorphous regions. A β-1,4-endoglucanase enzyme with high activity was obtained from a Bacillus subtilis strain isolated from wastewater of a pulp and paper mill. Sequencing and bioinformatic analysis showed that the gene amplified by PCR consisting of 1407 nucleotides and coding for a β-1,4-endoglucanase enzyme of approximately 55 kDa. The open reading frame (ORF) encoding the mature endoglucanase (eglS) was successfully inserted in a modified cloning plasmid (pITD03) and into the pYD1 plasmid used for its expression in yeast. Carboxymethylcellulose (CMC) plate assay, SDS-PAGE, and zymogram confirmed the production and secretion by the transformed E. coli BL21-SI strain of a 39 kDa β-1,4-endoglucanase consistent with the catalytic domain without the cellulose-binding module (CBM). The results showed that the truncated β-1,4-endoglucanase had higher activity and stability., Competing Interests: Declaration of competing interest The author(s) declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

New thermostable β-1,3-1,4-glucanase (lichenase) designated as Blg29 was expressed and purified from a locally isolated alkaliphilic bacteria Bacillus lehensis G1. The genome sequence of B. lehensis predicted an open reading frame of Blg29 with a deduced of 249 amino acids and a molecular weight of 28.99 kDa. The gene encoding for Blg29 was successfully amplified via PCR and subsequently expressed as a recombinant protein using the E. coli expression system. Recombinant Blg29 was produced as a soluble form and further purified via immobilized metal ion affinity chromatography (IMAC). Based on biochemical characterization, recombinant Blg29 showed optimal activity at pH9 and temperature 60 °C respectively. This enzyme was stable for more than 2 h, incubated at 50 °C, and could withstand ∼50 % of its activity at 70 °C for an hour and a half. No significant effect on Blg29 was observed when incubated with metal ions except for a small increase with ion Ca 2+ . Blg29 showed high substrate activity towards lichenan where V m , K m , K cat, and k cat /K m values were 2040.82 μmolmin ‾1 mg ‾1 , 4.69 mg/mL, and 986.39 s‾ 1 and 210.32 mLs ‾1 mg‾ 1 respectively. The high thermostability and activity make this enzyme useable for a broad prospect in industry applications., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Transglutaminase (TGase) from Streptomyces mobaraensis commonly used to improve protein-based foods due to its unique enzymatic reactions, which imply considerable attention in its production. Recently, TGase exhibit broad market potential in non-food industries. However, achieving efficient synthesis of TGase remains a significant challenge. Herein, we achieved a substantial amount of a fully functional and kinetically stable TGase produced by Komagataella phaffii (Pichia pastoris) using multiple strategies including Geneticin (G418) screening, combinatorial mutations, promoter optimization, and co-expression. The active TGase expression reached a maximum of 10.1 U mL -1 in shake flask upon 96 h of induction, which was 3.8-fold of the wild type. Also, the engineered strain exhibited a 6.4-fold increase in half-life and a 2-fold increase in specific activity, reaching 172.67 min at 60 °C (t 1/2 (60 °C)) and 65.3 U mg -1 , respectively. Moreover, the high-cell density cultivation in 5-L fermenter was also applied to test the productivity at large scale. Following optimization at a fermenter, the secretory yield of TGase reached 47.96 U mL -1 in the culture supernatant. Given the complexity inherent in protein expression and secretion, our research is of great significance and offers a comprehensive guide for improving the production of a wide range of heterologous proteins., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Effective utilization of glucose, xylose, and acetate, common carbon sources in lignocellulose hydrolysate, can boost biomanufacturing economics. However, carbon leaks into biomass biosynthesis pathways instead of the intended target product remain to be optimized. This study aimed to enhance α-carotene production by optimizing glucose, xylose, and acetate utilization in a high-efficiency Corynebacterium glutamicum cell factory. Heterologous xylose pathway expression in C. glutamicum resulted in strain m4, exhibiting a two-fold increase in α-carotene production from xylose compared to glucose. Xylose utilization was found to boost the biosynthesis of pyruvate and acetyl-CoA, essential precursors for carotenoid biosynthesis. Additionally, metabolic engineering including pck, pyc, ppc, and aceE deletion, completely disrupted the metabolic connection between glycolysis and the TCA cycle, further enhancing α-carotene production. This strategic intervention directed glucose and xylose primarily towards target chemical production, while acetate supplied essential metabolites for cell growth recovery. The engineered strain C. glutamicum m8 achieved 30 mg/g α-carotene, 67% higher than strain m4. In fed-batch fermentation, strain m8 produced 1802 mg/L of α-carotene, marking the highest titer reported to date in microbial fermentation. Moreover, it exhibited excellent performance in authentic lignocellulosic hydrolysate, producing 216 mg/L α-carotene, 1.45 times higher than the initial strain (m4). These labor-division strategies significantly contribute to the development of clean processes for producing various valuable chemicals from lignocellulosic resources., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

D-Allulose 3-epimerase (DAE) is a vital biocatalyst for the industrial synthesis of D-allulose, an ultra-low calorie rare sugar. However, limited thermostability of DAEs hinders their use at high-temperature production. In this research, hyperthermophilic TI-DAE (Tm = 98.4 ± 0.7 ℃) from Thermotoga sp. was identified via in silico screening. A comparative study of the structure and function of site-directed saturation mutagenesis mutants pinpointed the residue I100 as pivotal in maintaining the high-temperature activity and thermostability of TI-DAE. Employing TI-DAE as a biocatalyst, D-allulose was produced from D-fructose with a conversion rate of 32.5%. Moreover, TI-DAE demonstrated excellent catalytic synergy with glucose isomerase CAGI, enabling the one-step conversion of D-glucose to D-allulose with a conversion rate of 21.6%. This study offers a promising resource for the enzyme engineering of DAEs and a high-performance biocatalyst for industrial D-allulose production., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Polymyxin is a lipopeptide antibiotic that is effective against multidrug-resistant Gram-negative bacteria. However, its clinical development is limited due to low titer and the presence of homologs. To address this, the polymyxin gene cluster was integrated into Bacillus subtilis, and sfp from Paenibacillus polymyxa was expressed heterologously, enabling recombinant B. subtilis to synthesize polymyxin B. Regulating NRPS domain inhibited formation of polymyxin B2 and B3. The production of polymyxin B increased to 329.7 mg/L by replacing the native promoters of pmxA, pmxB, and pmxE with PfusA, C2up, and PfusA, respectively. Further enhancement in this production, up to 616.1 mg/L, was achieved by improving the synthesis ability of 6-methyloctanoic acid compared to the original strain expressing polymyxin heterologously. Additionally, incorporating an anikasin-derived domain into the hybrid nonribosomal peptide synthase of polymyxin increased the B1 ratio in polymyxin B from 57.5% to 62.2%. Through optimization of peptone supply in the fermentation medium and fermentation in a 5.0-L bioreactor, the final polymyxin B titer reached 962.1 mg/L, with a yield of 19.24 mg/g maltodextrin and a productivity of 10.02 mg/(L·h). This study demonstrates a successful approach for enhancing polymyxin B production and increasing the B1 ratio through combinatorial metabolic engineering., (Copyright © 2024 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

Background: Staphylococcus aureus is a common pathogen with strains that are resistant to existing antibiotics. MurJ from S. aureus (SaMurJ), an integral membrane protein functioning as Lipid II flippase, is a potential target for developing new antibacterial agents against this pathogen. Successful expression and purification of this protein shall be useful in the development of drugs against this target., Objective: In this study, we demonstrated the optimized expression and purification procedures of SaMurJ, identified suitable detergent for extracting and solubilizing the protein, and examined the peptidisc system to generate a detergent-free environment., Methods: SaMurJ fused with N-terminal ten-His tag was expressed without induction. Six detergents were selected for screening the most efficient candidate for extraction and solubilization of the protein. The thermostability of the detergent-solubilized protein was assessed by evaluated temperature incubation. Different ratios of peptidisc bi-helical peptide (NSPr) to SaMurJ were mixed and the on-bead peptidisc assembly method was applied., Results: SaMurJ expressed in BL21(DE3) was confirmed by peptide fingerprinting, with a yield of 1 mg SaMurJ per liter culture. DDM was identified as the optimum detergent for solubilization and the nickel affinity column enabled SaMurJ purification with a purity of ~88%. However, NSPr could not stabilize SaMurJ., Conclusion: The expression and purification of SaMurJ were successful, with high purity and good yield. SaMurJ can be solubilized and stabilized by a DDM-containing buffer., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The role of RNA G-quadruplexes (rG4s) in bacteria remains poorly understood. High G-quadruplex densities have been linked to organismal stress. Here we investigate rG4s in mycobacteria, which survive highly stressful conditions within the host. We show that rG4-enrichment is a unique feature exclusive to slow-growing pathogenic mycobacteria, and Mycobacterium tuberculosis (Mtb) transcripts contain an abundance of folded rG4s. Notably, the PE/PPE family of genes, unique to slow-growing pathogenic mycobacteria, contain over 50% of rG4s within Mtb transcripts. We found that RNA oligonucleotides of putative rG4s in PE/PPE genes form G-quadruplex structures in vitro, which are stabilized by the G-quadruplex ligand BRACO19. Furthermore, BRACO19 inhibits the transcription of PE/PPE genes and selectively suppresses the growth of Mtb but not Mycobacterium smegmatis or other rapidly growing bacteria. Importantly, the stabilization of rG4s inhibits the translation of Mtb PE/PPE genes (PPE56, PPE67, PPE68, PE_PGRS39, and PE_PGRS41) ectopically expressed in M. smegmatis or Escherichia coli. In addition, the rG4-mediated reduction in PE/PPE protein levels attenuates proinflammatory response upon infection of THP-1 cells. Our findings shed new light on the regulation of PE/PPE genes and highlight a pivotal role for rG4s in Mtb transcripts as regulators of post-transcriptional translational control. The rG4s in mycobacterial transcripts may represent potential drug targets for newer therapies., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Lactococcus lactis displaying recombinant proteins on its surface can be used as a potential drug delivery vector in prophylactic medication and therapeutic treatments for many diseases. These applications enable live-cell mucosal and oral administration, providing painless, needle-free solutions and triggering robust immune response at the site of pathogen entry. Immunization requires quantitative control of antigens and, ideally, a complete understanding of the bacterial processing mechanism applied to the target proteins. In this study, we propose a double-labeling method based on a conjugated dye specific for a recombinantly introduced polyhistidine tag (to visualize surface-exposed proteins) and a membrane-permeable dye specific for a tetra-cysteine tag (to visualize cytoplasmic proteins), combined with a method to block the labeling of surface-exposed tetra-cysteine tags, to clearly obtain location-specific signals of the two dyes. This allows simultaneous detection and quantification of targeted proteins on the cell surface and in the cytoplasm. Using this method, we were able to detect full-length peptide chains for the model proteins HtrA and BmpA in L. lactis , which are associated with the cell membrane by two different attachment modes, and thus confirm that membrane-associated proteins in L. lactis are secreted using the Sec-dependent post-translational pathway. We were able to quantitatively follow cytoplasmic protein production and accumulation and subsequent export and surface attachment, which provides a convenient tool for monitoring these processes for cell surface display applications.

The Gac/Rsm system is a global regulator of Pseudomonas aeruginosa gene expression. The primary effectors are RsmA and RsmF. Both are RNA-binding proteins that interact with target mRNAs to modulate protein synthesis. RsmA/RsmF recognize GGA sequences presented in the loop portion of stem-loop structures. For repressed targets, the GGA sites usually overlap the ribosome binding site (RBS) and RsmA/RsmF binding inhibits translation initiation. RsmA/RsmF activity is controlled by several small non-coding RNAs (sRNA) that sequester RsmA/RsmF from target mRNAs. The most important sequestering sRNAs are RsmY and RsmZ. Transcription of rsmY / rsmZ is directly controlled by the GacSA two-component regulatory system. GacSA activity is antagonized by RetS, a hybrid sensor kinase. In the absence of retS , rsmY / rsmZ transcription is derepressed and RsmA/RsmF are sequestered by RsmY/RsmZ. Gac/Rsm system homeostasis is tightly controlled by at least two mechanisms. First, direct binding of RsmA to the rsmA and rsmF mRNAs inhibits further synthesis of both proteins. Second, RsmA stimulates rsmY/rsmZ transcription through an undefined mechanism. In this study we demonstrate that RsmA stimulates rsmY/rsmZ transcription by directly inhibiting RetS synthesis. RetS protein levels are elevated 2.5-fold in an rsmA mutant. Epistasis experiments demonstrate that the rsmA requirement for rsmY/rsmZ transcription is entirely suppressed in an rsmA, retS double mutant. RsmA directly interacts with the retS mRNA and requires two distinct GGA sites, one of which overlaps the RBS. We propose a model wherein RsmA inhibits RetS synthesis to promote rsmY / rsmZ transcription and that this acts as a checkpoint to limit RsmA/RsmF availability. IMPORTANCE The Pseudomonas aeruginosa Gac/Rsm system controls ∼500 genes and governs a critical lifestyle switch by inversely regulating factors that favor acute or chronic colonization. Control of gene expression by the Gac/Rsm system is mediated through RsmA and RsmF, small RNA-binding proteins that interact with target mRNAs to inhibit or promote protein synthesis and/or mRNA stability. RsmA/RsmF activity is governed by two small non-coding RNAs (RsmY and RsmZ) that sequester RsmA/RsmF from target mRNAs. The GacSA two-component regulatory system plays a pivotal role in the Gac/Rsm system by controlling rsmYZ transcription. This study provides insight into the control of homeostasis by demonstrating that RsmA directly targets and inhibits expression of RetS, an orphan sensor kinase critical for rsmYZ transcription.

Many integral membrane proteins are produced by translocon-associated ribosomes. The assembly of ribosomes translating membrane proteins on the translocons is mediated by a conserved system, composed of the signal recognition particle and its receptor (FtsY in Escherichia coli). FtsY is a peripheral membrane protein, and its role late during membrane protein targeting involves interactions with the translocon. However, earlier stages in the pathway have remained obscure, namely, how FtsY targets the membrane in vivo and where it initially docks. Our previous studies have demonstrated co-translational membrane-targeting of FtsY translation intermediates and identified a nascent FtsY targeting-peptide. Here, in a set of in vivo experiments, we utilized tightly stalled FtsY translation intermediates, pull-down assays and site-directed cross-linking, which revealed FtsY-nascent chain-associated proteins in the cytosol and on the membrane. Our results demonstrate interactions between the FtsY-translating ribosomes and cytosolic chaperones, which are followed by directly docking on the translocon. In support of this conclusion, we show that translocon over-expression increases dramatically the amount of membrane associated FtsY-translating ribosomes. The co-translational contacts of the FtsY nascent chains with the translocon differ from its post-translational contacts, suggesting a major structural maturation process. The identified interactions led us to propose a model for how FtsY may target the membrane co-translationally. On top of our past observations, the current results may add another tier to the hypothesis that FtsY acts stoichiometrically in targeting ribosomes to the membrane in a constitutive manner., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Bacillus subtilis has many attributes that make it a popular host for recombinant protein production. Although its protein production ability has been enhanced through protease gene disruption, residual proteases like quality control HtrA and HtrB can limit protein yield by degrading inadequately folded proteins present during overexpression. In this study, two strategies were employed to increase production of industrial enzyme pullulanase: enhancing extracellular pullulanase folding and optimizing its signal peptide. The hypothesis was that disruption of dltB gene of expression host B. subtilis WS9 would enhance recombinant extracellular folding by increasing cation binding to the cell's outer envelope. Consistent with this hypothesis, disrupting dltB enhanced pullulanase production by 49% in shake-flask cultures. The disruption also enhanced extracellular α-CGTase and β-CGTase production by 25% and 35%, respectively. Then, more effective signal peptide for pullulanase production was identified through high-throughput screening of 173 unique B. subtilis signal peptides. Replacing the native signal peptide of pullulanase with that encoded by ywtF increased extracellular pullulanase activity by an additional 12%. Three-liter fermenter scale-up production yielded the highest extracellular pullulanase activity reported to date: 8037.91 U·mL -1 . This study highlights the usefulness of dltB deletion and signal peptide optimization in enhancing extracellular protein production., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Many opportunistic bacteria that infect the upper respiratory tract decorate their cell surface with phosphorylcholine to support colonisation and outgrowth. These surface modifications require the active import of choline from the host environment, a process thought to be mediated by a family of dedicated integral membrane proteins that act as choline permeases. Here, we present the expression and purification of the archetype of these choline transporters, LicB from Haemophilus influenzae. We show that LicB can be recombinantly produced in Escherichia coli and purified to homogeneity in a stable, folded state using the detergent n-dodecyl-β-d-maltopyranoside. Equilibrium binding studies with the fluorescent ligand dansylcholine suggest that LicB is selective towards choline, with reduced affinity for acetylcholine and no apparent activity towards other small molecules including glycine, carnitine and betaine. We also identify a conserved sequence motif within the LicB family and show that mutations within this motif compromise protein structure and function. Our results are consistent with previous observations that LicB is a specific high-affinity choline transporter, and provide an experimental platform for further studies of this permease family., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The urease enzyme has been an important target for the discovery of effective pharmacological and agricultural products. Thirteen regio-selectively alkylated benzimidazole-2-thione derivatives have been designed to carry the essential features of urease inhibitors. The urease enzyme was isolated from Helicobacter pylori as a recombinant urease utilizing the His-tag method. The isolated enzyme was purified and characterized using chromatographic and FPLC techniques showing a maximal activity of 200 mg/mL. Additionally, the commercial Jack bean urease was purchased and included in this study for comparative and mechanistic investigations. The designed compounds were synthesized and screened for their inhibitory activity against the two ureases. Compound 2 inhibited H. pylori and Jack bean ureases with IC 50 values of 0.11; and 0.26 mM; respectively. While compound 5 showed IC 50 values of 0.01; and 0.29 mM; respectively. Compounds 2 and 5 were docked against Helicobacter pylori urease (PDB ID: 1E9Y; resolution: 3.00 Å) and exhibited correct binding modes with free energy (ΔG) values of -9.74 and -13.82 kcal mol -1 ; respectively. Further; the in silico ADMET and toxicity properties of 2 and 5 indicated their general safeties and likeness to be used as drugs. Finally, the compounds' safety was authenticated by an in vitro cytotoxicity assay against fibroblast cells.

Bacterial protein secretion is crucial to the maintenance of viability and pathogenicity. Although many bacterial secretion systems have been identified, the underlying mechanisms regulating their expression are less well explored. Yersinia entomophaga MH96, an entomopathogenic bacterium, releases an abundance of proteins including the Yen-Tc into the growth medium when cultured in Luria Bertani broth at ≤ 25°C. Through the development of a high-throughput exoproteome screening assay (HESA), genes involved in MH96 exoprotein production were identified. Of 4,080 screened transposon mutants, 34 mutants exhibited a decreased exoprotein release, and one mutation located in the intergenic region of the Yen-Tc operon displayed an elevated exoprotein release relative to the wild-type strain MH96. DNA sequencing revealed several transposon insertions clustered in gene regions associated with lipopolysaccharide (LPSI and LPSII), and N-acyl-homoserine lactone synthesis (quorum sensing). Twelve transposon insertions were located within transcriptional regulators or intergenic regions. The HESA will have broad applicability for identifying genes associated with exoproteome production in a range of microorganisms., Competing Interests: The authors have declared that no competing interests exist.

We used a rapid antigen test for the detection of carbapenemases directly from positive blood culture bottles of pediatric hemato-oncologic patients, known carriers of carbapenemase-producing enterobacteriaceae. Resistance mechanism was detected within 15 minutes of observing Gram-negative bacilli from a positive bottle, leading to treatment modification. This simple-to-use, inexpensive assay shortens the interval between empiric to tailored antimicrobial therapy., Competing Interests: The study was funded by the research group with no external support. The authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Proteins with novel functions or advanced activities developed by various protein engineering techniques must have sufficient solubility to retain their bioactivity. However, inactive protein aggregates are frequently produced during heterologous protein expression in Escherichia coli. To prevent the formation of inclusion bodies, fusion tag technology has been commonly employed, owing to its good performance in soluble expression of target proteins, ease of application, and purification feasibility. Thus, researchers have continuously developed novel fusion tags to expand the expression capacity of high-value proteins in E. coli., Results: A novel fusion tag comprising carbohydrate-binding module 66 (CBM66) was developed for the soluble expression of heterologous proteins in E. coli. The target protein solubilization capacity of the CBM66 tag was verified using seven proteins that are poorly expressed or form inclusion bodies in E. coli: four human-derived signaling polypeptides and three microbial enzymes. Compared to native proteins, CBM66-fused proteins exhibited improved solubility and high production titer. The protein-solubilizing effect of the CBM66 tag was compared with that of two commercial tags, maltose-binding protein and glutathione-S-transferase, using poly(ethylene terephthalate) hydrolase (PETase) as a model protein; CBM66 fusion resulted in a 3.7-fold higher expression amount of soluble PETase (approximately 370 mg/L) compared to fusion with the other commercial tags. The intact PETase was purified from the fusion protein upon serial treatment with enterokinase and affinity chromatography using levan-agarose resin. The bioactivity of the three proteins assessed was maintained even when the CBM66 tag was fused., Conclusions: The use of the CBM66 tag to improve soluble protein expression facilitates the easy and economic production of high-value proteins in E. coli., (© 2021. The Author(s).)

Building iron-sulfur (Fe-S) clusters and assembling Fe-S proteins are essential actions for life on Earth. The three processes that sustain life, photosynthesis, nitrogen fixation, and respiration, require Fe-S proteins. Genes coding for Fe-S proteins can be found in nearly every sequenced genome. Fe-S proteins have a wide variety of functions, and therefore, defective assembly of Fe-S proteins results in cell death or global metabolic defects. Compared to alternative essential cellular processes, there is less known about Fe-S cluster synthesis and Fe-S protein maturation. Moreover, new factors involved in Fe-S protein assembly continue to be discovered. These facts highlight the growing need to develop a deeper biological understanding of Fe-S cluster synthesis, holo-protein maturation, and Fe-S cluster repair. Here, we outline bacterial strategies used to assemble Fe-S proteins and the genetic regulation of these processes. We focus on recent and relevant findings and discuss future directions, including the proposal of using Fe-S protein assembly as an antipathogen target.

This work addresses the amino acid sequence, structural analysis, biochemical characterization and glycosidase activity of two recombinant α-rhamnosidases, Ram1 and Ram2, from Lactobacillus plantarum WCFS1. The substrate specificity of both enzymes towards the disaccharide rutinose and natural dietary flavonoids naringin and rutin was also determined and compared to that of a commercial multienzyme complex (Pectinex Ultra Passover, PPO). Ram1 is a less acidic- and heat-active enzyme than Ram2 and exhibited a high activity towards pNP-α-L-rhamnopyranoside, but it was unable to hydrolyze neither rutinose, naringin or rutin. In contrast, Ram2 enzyme showed a substrate specificity towards α-(1➔6) glycosidic flavonoids, such as rutin, and the disaccharide rutinose. The mechanism of action of Ram2 towards rutin was elucidated and revealed the potential cost-effective and selective production of the monoglycosylated flavonoid isoquercetin (quercetin-3-O-glucoside). PPO efficiently converted both naringin and rutin into their corresponding aglycones. These findings revealed the potential usefulness of PPO for the improvement of sensory properties of beverages through debittering of citrus juices, as well as the potential use of Ram2 to selectively produce isoquercetin, a highly valued and bioactive flavonoid whose production is not currently affordable., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

The regulatory network of gene expression in Pseudomonas aeruginosa , an opportunistic human pathogen, is very complex. In the PAO1 reference strain, about 10% of genes encode transcriptional regulators, many of which have undefined regulons and unknown functions. The aim of this study is the characterization of PA2577 protein, a representative of the Lrp/AsnC family of transcriptional regulators. This family encompasses proteins involved in the amino acid metabolism, regulation of transport processes or cell morphogenesis. The transcriptome profiling of P. aeruginosa cells with mild PA2577 overproduction revealed a decreased expression of the PA2576 gene oriented divergently to PA2577 and encoding an EamA-like transporter. A gene expression analysis showed a higher mRNA level of PA2576 in P. aeruginosa Δ PA2577 , indicating that PA2577 acts as a repressor. Concomitantly, ChIP-seq and EMSA assays confirmed strong interactions of PA2577 with the PA2577 / PA2576 intergenic region. Additionally, phenotype microarray analyses indicated an impaired metabolism of Δ PA2576 and Δ PA2577 mutants in the presence of polymyxin B, which suggests disturbances of membrane functions in these mutants. We show that PA2576 interacts with two proteins, PA5006 and PA3694, with a predicted role in lipopolysaccharide (LPS) and membrane biogenesis. Overall, our results indicate that PA2577 acts as a repressor of the PA2576 gene coding for the EamA-like transporter and may play a role in the modulation of the cellular response to stress conditions, including antimicrobial peptides, e.g., polymyxin B.

Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations., Competing Interests: Acknowledgements This work was supported by Human Frontier Science Program Grant RGP0041/2017 (J.M.S.-R. and E.A.G.), National Science Foundation Award #2032315 (E.A.G.), National Institutes of Health Award #R01AR069137 (E.A.G.), Department of Defense MURI Award #W911NF-16-1-0372 (E.A.G.), Spanish Ministry of Science and Innovation/FEDER Funds Grants RTI-2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.) and the Science, Engineering and Research Board (SERB, India) Grant MTR/2019/000392 (A.N.N.). We are grateful to the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and the staff at ID23-1 beamline for assistance during data collection. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

In our previous study, the chitosanase AqCoA and the chitooligosaccharides it produced were found to exhibit significant protective effects against fungal diseases. In this study, we enhanced the expression of AqCoA using the novel pMC-GAP that enables stable transformation of Escherichia coli, and built an integrated model based on the gene copy number, molecular chaperones, and protein production of AqCoA. In terms of gene dosage, the highest hydrolase activity was 0.32 U/ml in the strain with four copies, which was 1.78-fold higher than that of the strain with only one copy (0.18 U/ml). In addition, we found the chaperones such as PDI, ERO1, HAC1, YDJ1, SSE1, SSA4, and SSO2 improved protein expression. Furthermore, the PDI/ERO1, SSA4/SSE1, and YDJ1/SSO2 pairs synergistically increased the expression levels by 61%, 31%, and 42%, respectively. Finally, we investigated the combined effects of gene copy numbers and molecular chaperones on protein expression. The highest activity reached 2.32 U/ml in the strain with six integrated molecular chaperone expression cassettes and sixteen copies of the target gene, which was 13-fold higher than that of the control strain with only one copy (GAP-1AqCoA). Combined optimization of gene dosage and molecular chaperone combinations significantly increased the expression level of AqCoA, providing a powerful strategy to improve the expression of other heterologous proteins in P. pastoris., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Homoserine dehydrogenase (HSD), encoded by the hom gene, is a key enzyme in the aspartate pathway, which reversibly catalyzes the conversion of l-aspartate β-semialdehyde to l-homoserine (l-Hse), using either NAD(H) or NADP(H) as a coenzyme. In this work, we presented the first characterization of the HSD from the symbiotic Polynucleobacter necessaries subsp. necessarius (PnHSD) produced in Escherichia coli. Sequence analysis showed that PnHSD is an ACT domain-containing monofunctional HSD with 436 amnio acid residues. SDS-PAGE and Western blot demonstrated that PnHSD could be overexpressed in E. coli BL21(DE3) cell as a soluble form by using SUMO fusion technique. It could be purified to apparent homogeneity for biochemical characterization. Size-exclusion chromatography revealed that the purified PnHSD has a native molecular mass of ∼160 kDa, indicating a homotetrameric structure. The oxidation activity of PnHSD was studied in this work. Kinetic analysis revealed that PnHSD displayed an up to 1460-fold preference for NAD + over NADP + , in contrast to its homologs. The purified PnHSD displayed maximal activity at 35 °C and pH 11. Similar to its NAD + -dependent homolog, neither NaCl and KCl activation nor L-Thr inhibition on the enzymatic activity of PnHSD was observed. These results will contribute to a better understanding of the coenzyme specificity of the HSD family and the aspartate pathway of P. necessarius., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Using the USA300, methicillin-resistant Staphylococcus aureus strain LAC, we previously examined the impact of regulatory mutations implicated in biofilm formation on protease production and virulence in a murine sepsis model. Here we examined the impact of these mutations in the USA200, methicillin-sensitive strain UAMS-1. Mutation of agr, mgrA, rot, sarA and sigB attenuated the virulence of UAMS-1. A common characteristic of codY, rot, sigB , and sarA mutants was increased protease production, with mutation of rot having the least impact followed by mutation of codY, sigB and sarA , respectively. Protein A was undetectable in conditioned medium from all four mutants, while extracellular nuclease was only present in the proteolytically cleaved NucA form. The abundance of high molecular weight proteins was reduced in all four mutants. Biofilm formation was reduced in codY, sarA and sigB mutants, but not in the rot mutant. Eliminating protease production partially reversed these phenotypes and enhanced biofilm formation. This was also true in LAC codY, rot, sarA and sigB mutants. Eliminating protease production enhanced the virulence of LAC and UAMS-1 sarA, sigB and rot mutants in a murine sepsis model but did not significantly impact the virulence of the codY mutant in either strain. Nevertheless, these results demonstrate that repressing protease production plays an important role in defining critical phenotypes in diverse clinical isolates of S. aureus and that Rot, SigB and SarA play critical roles in this regard.

Absract: BACKGROUND: To describe the course and intervention of an hospital-wide IMI-Producing Enterobacter ludwigii outbreak., Methods: This was an outbreak interventional study, done at a tertiary care center in Tel-Aviv, Israel. Data was collected on the course of the outbreak and the demographic and clinical characteristics of all patients involved in the outbreak. The intervention measures included patients' cohorting, contact isolation precautions, environmental cleaning and screening of contacts. The molecular features and phylogeny of outbreak-related isolates were studied by whole-genome based analysis., Results: The outbreak included 34 patients that were colonized by IMI-Producing E. ludwigii and were identified in 24 wards throughout the hospital. Colonization was identified in the first 72 h of admission in 13/34 patients (38.2%). Most patients (91.2%) were admitted from home and had relatively low level of comorbidities. The majority of them (88%) had no recent use of invasive catheters and none had previous carriage of other multi-drug resistant bacteria. All available isolates harbored the bla IMI-17 allele and belonged to Sequence-Type 385. With the exception of two isolates, all isolates were closely related with less than a 20-SNP difference between them., Conclusions: This outbreak had most likely originated in the community and subsequently disseminated inside our institution. More studies are required in order to elucidate the epidemiology of IMI-Producing E. ludwigii and the possible role of environmental sources in its dissemination., (© 2021. The Author(s).)

Tryptophan metabolism plays a role in the occurrence and development of hepatocellular carcinoma cells. By degrading certain amino acids, tumor growth can be limited while maintaining the body's normal nutritional requirements. Tryptophan side-chain oxidase (TSO) enzyme can degrade tryptophan, and its inhibitory effect on hepatocellular carcinoma cells is worthy of further study. To investigate the degradation effect on tryptophan, TSO was isolated and purified from qq Pseudomonas. The reaction products were identified with high performance liquid chromatography (HPLC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS). De novo sequencing provided the complete amino acid sequence of TSO. The results of CCK-8, colony formation, transwell, and qPCR confirmed that TSO had inhibitory effects on the proliferation and migration of HCCLM3 (human hepatocarcinoma cell line) and HepG2 cells. The results of flow cytometry confirmed its apoptotic activity. In animal experiments, we found that the tumor-suppressive effect was better in the oncotherapy group than the intraperitoneal injection group. The results of immunohistochemistry also suggested that TSO could inhibit proliferation and promote apoptosis. In conclusion, a specific enzyme that can degrade tryptophan and inhibit the growth of hepatoma cells was authenticated, and its basic information was obtained by extraction/purification and amino acid sequencing.

The present study focuses on the immobilization of the bacterial ribonuclease barnase (Bn) into submicron porous calcium carbonate (CaCO 3 ) particles. For encapsulation, we apply adsorption, freezing-induced loading and co-precipitation methods and study the effects of adsorption time, enzyme concentration and anionic polyelectrolytes on the encapsulation efficiency of Bn. We show that the use of negatively charged dextran sulfate (DS) and ribonucleic acid from yeast (RNA) increases the loading capacity (LC) of the enzyme on CaCO 3 particles by about 3-fold as compared to the particles with Bn itself. The ribonuclease (RNase) activity of encapsulated enzyme depends on the LC of the particles and transformation of metastable vaterite to stable calcite, as studied by the assessment of enzyme activities in particles.

Overproduction of the membrane proteins in Escherichia coli cells is a common approach to obtain sufficient material for their functional and structural studies. However, the efficiency of this process can be limited by toxic effects which decrease the viability of the host and lead to low yield of the product. During the expression of the esterase autotransporter AT877 from Psychrobacter cryohalolentis K5 T , we observed significant growth inhibition of the C41(DE3) cells in comparison with the same cells producing other recombinant proteins. Induction of AT877 synthesis also resulted in the elevated expression of a magnesium transporter MgtA and decreased ATP content of the cells. To characterize the response to overexpression of the autotransporter in bacterial cells, we performed a comparative analysis of their proteomic profile by mass spectrometry. According to the obtained data, E. coli cells which synthesize AT877 experience complex stress condition presumably associated with secretion apparatus overloading and improper localization of the recombinant protein. Several response pathways were shown to be activated by AT877 overproduction including Cpx, PhoP/PhoQ, Psp, and σ E The obtained results open new opportunities for optimization of the recombinant membrane protein expression in E. coli for structural studies and biotechnological applications., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bacteria must balance the different needs for substrate assimilation, growth functions, and resilience in order to thrive in their environment. Of all cellular macromolecules, the bacterial proteome is by far the most important resource and its size is limited. Here, we investigated how the highly versatile 'knallgas' bacterium Cupriavidus necator reallocates protein resources when grown on different limiting substrates and with different growth rates. We determined protein quantity by mass spectrometry and estimated enzyme utilization by resource balance analysis modeling. We found that C. necator invests a large fraction of its proteome in functions that are hardly utilized. Of the enzymes that are utilized, many are present in excess abundance. One prominent example is the strong expression of CBB cycle genes such as Rubisco during growth on fructose. Modeling and mutant competition experiments suggest that CO 2 -reassimilation through Rubisco does not provide a fitness benefit for heterotrophic growth, but is rather an investment in readiness for autotrophy., Competing Interests: MJ, NC, MJ, AH, BF, KK, AM, QC, JA, EH No competing interests declared, (© 2021, Jahn et al.)

Background: 3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17β-HSD. Previous reports showed that three tetR genes are located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first tetR gene encoded a potential repressor of 3,17β-HSD by sensing environmental signals. However, whether the other proposed tetR genes act as repressors of 3,17β-HSD are still unknown., Methods and Results: In the present study, we cloned the second tetR gene and analyzed the regulatory mechanism of the protein on 3,17β-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second tetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17β-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17β-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second tetR gene knocked-out mutant expresses good biological genetic stability, and the expression of 3,17β-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction., Conclusions: The second tetR gene acts as a negative regulator in 3,17β-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

Streptococcus pneumoniae, the most common cause of community-acquired pneumonia, causes severe invasive infections, including meningitis and bacteremia. The widespread use of macrolides has been reported to increase the prevalence of macrolide-resistant S. pneumoniae (MRSP), thereby leading to treatment failure in patients with pneumococcal pneumonia. However, previous studies have demonstrated that several macrolides and lincosamides have beneficial effects on MRSP infection since they inhibit the production and release of pneumolysin, a pneumococcal pore-forming toxin released during autolysis. In this regard, we previously demonstrated that the mechanisms underlying the inhibition of pneumolysin release by erythromycin involved both the transcriptional downregulation of the gene encoding pneumolysin and the impairment of autolysis in MRSP. Here, using a cell supernatant of the culture, we have shown that clarithromycin inhibits pneumolysin release in MRSP. However, contrary to previous observations in erythromycin-treated MRSP, clarithromycin upregulated the transcription of the pneumococcal autolysis-related lytA gene and enhanced autolysis, leading to the leakage of pneumococcal DNA. On the other hand, compared to erythromycin, clarithromycin significantly downregulated the gene encoding pneumolysin. In a mouse model of MRSP pneumonia, the administration of both clarithromycin and erythromycin significantly decreased the pneumolysin protein level in bronchoalveolar lavage fluid and improved lung injury and arterial oxygen saturation without affecting bacterial load. Collectively, these in vitro and in vivo data reinforce the benefits of macrolides on the clinical outcomes of patients with pneumococcal pneumonia. IMPORTANCE Pneumolysin is a potent intracellular toxin possessing multiple functions that augment pneumococcal virulence. For over 10 years, sub-MICs of macrolides, including clarithromycin, have been recognized to decrease pneumolysin production and release from pneumococcal cells. However, this study indicates that macrolides significantly slowed pneumococcal growth, which may be related to decreased pneumolysin release recorded by previous studies. In this study, we demonstrated that clarithromycin decreases pneumolysin production through downregulation of ply gene transcription, regardless of its inhibitory activity against bacterial growth. Additionally, administration of clarithromycin resulted in the amelioration of lung injury in a mouse model of pneumonia induced by macrolide-resistant pneumococci. Therefore, therapeutic targeting of pneumolysin offers a good strategy to treat pneumococcal pneumonia.

Almost 140 years after the identification of Mycobacterium tuberculosis as the etiological agent of tuberculosis, important aspects of its biology remain poorly described. Little is known about the role of posttranscriptional control of gene expression and RNA biology, including the role of most of the small RNAs (sRNAs) identified to date. We have carried out a detailed investigation of the M. tuberculosis sRNA F6 and shown it to be dependent on SigF for expression and significantly induced in starvation conditions in vitro and in a mouse model of infection. Further exploration of F6 using an in vitro starvation model of infection indicates that F6 affects the expression of the essential chaperonins GroEL2 and GroES. Our results point toward a role for F6 during periods of low metabolic activity typically associated with long-term survival of M. tuberculosis in human granulomas. IMPORTANCE Control of gene expression via small regulatory RNAs (sRNAs) is poorly understood in one of the most successful pathogens, Mycobacterium tuberculosis. Here, we present an in-depth characterization of the sRNA F6, including its expression in different infection models and the differential gene expression observed upon deletion of the sRNA. Our results demonstrate that deletion of F6 leads to dysregulation of the two essential chaperonins GroEL2 and GroES and, moreover, indicate a role for F6 in the long-term survival and persistence of M. tuberculosis in the human host.

Nisin is synthesized by a putative membrane-associated lantibiotic synthetase complex composed of the dehydratase NisB, the cyclase NisC, and the ABC transporter NisT in Lactococcus lactis. Earlier work has demonstrated that NisB and NisT are linked via NisC to form such a complex. Here, we conducted for the first time the isolation of the intact NisBTC complex and NisT-associated subcomplexes from the cytoplasmic membrane by affinity purification. A specific interaction of NisT, not only with NisC but also with NisB, was detected. The cellular presence of NisB and/or NisC in complex with precursor nisin (NisA) was determined, which shows a highly dynamic and transient assembly of the NisABC complex via an alternating binding mechanism during nisin dehydration and cyclization. Mutational analyses, with cysteine-to-alanine mutations in NisA, suggest a tendency for NisA to lose affinity to NisC concomitant with an increasing number of completed lanthionine rings. Split NisBs were able to catalyze glutamylation and elimination reactions in an alternating way as efficiently as full-length NisB, with no significant influence on the following cyclization and transport. Notably, the harvest of the leader peptide in complex with the independent elimination domain of NisB points to a second leader peptide binding motif that is located in the C-terminal region of NisB, giving rise to a model where the leader peptide binds to different sites in NisB for glutamylation and elimination. Overall, these combined studies provide new insights into the cooperative biosynthesis mechanism of nisin and thereby lay a foundation for further structural and functional characterization of the NisBTC complex. IMPORTANCE Lantibiotics are ribosomally synthesized and posttranslationally modified peptide antibiotics. Although the membrane-associated lantibiotic biosynthesis machinery has long been proposed to exist, the isolation of such a complex has not been reported yet, which limits the elucidation of the processive mechanism of lantibiotic biosynthesis. In this work, we present direct evidence for the existence of the nisin biosynthetic complex at the cytoplasmic membrane of L. lactis, producing fully modified precursor nisin. By analyses of the interactions within the intact NisBTC complex and the modification machinery NisABC, we were able to elucidate the cooperative action for the modification and transport of nisin. Inspired by the natural and documented degradation process of NisB, artificial split-NisBs were made and thoroughly characterized, demonstrating a crucial clue to the evolution of the LanB family. Importantly, our study also suggests that the leader peptide of NisA binds to two different recognition motifs, i.e., one for glutamylation and one for elimination.

In this study, we report the characterization of three glycosyltransferases involved in the biosynthesis of ligupurpuroside B, a complex acylated phenolic glycoside in Ligustrum robustum . UGT85AF8 catalyzed the formation of salidroside from tyrosol. UGT79G7, an osmanthuside A 1,3-rhamnosyltransferase, and UGT79A19, an osmanthuside B 1,4-rhamnosyltransferase, sequentially converted osmanthuside A into ligupurpuroside B. Orthologs of UGT79G7 were also discovered from other plants producing verbascoside. These rhamnosyltransferases expand the toolbox for the biosynthesis of natural products with various sugar chains.

SR1 is a dual-function sRNA from Bacillus subtilis. It inhibits translation initiation of ahrC mRNA encoding the transcription activator of the arginine catabolic operons. Base-pairing is promoted by the RNA chaperone CsrA, which induces a slight structural change in the ahrC mRNA to facilitate SR1 binding. Additionally, SR1 encodes the small protein SR1P that interacts with glyceraldehyde-3P dehydrogenase A to promote binding to RNase J1 and enhancing J1 activity. Here, we describe a new target of SR1, kinA mRNA encoding the major histidine kinase of the sporulation phosphorelay. SR1 and kinA mRNA share 7 complementary regions. Base-pairing between SR1 and kinA mRNA decreases kinA translation without affecting kinA mRNA stability and represses transcription of the KinA/Spo0A downstream targets spoIIE, spoIIGA and cotA. The initial interaction between SR1 and kinA mRNA occurs 10 nt downstream of the kinA start codon and is decisive for inhibition. The sr1 encoded peptide SR1P is dispensable for kinA regulation. Deletion of sr1 accelerates sporulation resulting in low quality spores with reduced stress resistance and altered coat protein composition which can be compensated by sr1 overexpression. Neither CsrA nor Hfq influence sporulation or spore properties., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein-glutaminase (EC 3.5.1.44, PG) converts protein glutamine residues in proteins and peptides into glutamic acid residue, and markedly improves the solubility, emulsification, and foaming properties of food proteins. However, the source bacteria, Chryseobacterium proteolyticum, have low enzyme production ability, inefficient genetic operation, and high production cost. Therefore, it is critical to establish an efficient expression system for active PG. Here, combinatorial engineering was developed for high-yield production of PG in Bacillus subtilis. First, we evaluated different B. subtilis strains for PG self-activation. Then, combinatorial optimization involving promoters, signal peptides, and culture medium was applied to produce active recombinant PG in a B. subtilis expression system. Through combinatorial engineering, PG enzyme activity reached 3.23 U/mL in shaken-flask cultures. Active PG with the yield of 7.07 U/mL was obtained at 40 h by the P SecA -YdeJ combination in fed-batch fermentation, which is the highest yield of PG in existing reports., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The ZIP family transport zinc ions from the extracellular medium across the plasma membrane or from the intracellular compartments across endomembranes, which play fundamental roles in metal homeostasis and are broadly involved in physiological and pathological processes. Desulfovibrio is the predominant sulphate-reducing bacteria in human colonic microbiota, but also a potential choice for metal bioremediation. while, there are no published studies describing the zinc transporters from Desulfovibrio up to now. In this study, we obtained for the first time a heterologously expressed ZIP homolog from Desulfovibrio vulgaris, termed dvZip. The purified dvZip was reconstituted into proteoliposomes, and confirmed its zinc transport ability in vitro. By combining topology prediction, homology modeling and phylogenetic approaches, we also noticed that dvZip belongs to the GufA and probably have 8 transmembrane α-helical segments (TM 1-8) in which both termini are located on the extracellular, with TM2, 4, 5 and 7 create an inner bundle. We believe that purification and characterization of zinc (probably also cadmium) transporters from Desulfovibrio vulgaris such as dvZip could shed light on understanding of metal homeostasis of Desulfovibrio and provided protein products for future detailed function and structural studies., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Expression of recombinant proteins requires at times the aid of molecular chaperones for efficient post-translational folding into functional structure. However, predicting the compatibility of a protein substrate with the right type of chaperone to produce functional proteins is a daunting issue. To study the difference in effects of chaperones on His-tagged recombinant proteins with different characteristics, we performed in vitro proteins expression using Escherichia coli overexpressed with several chaperone 'teams': Trigger Factor (TF), GroEL/GroES and DnaK/DnaJ/GrpE, alone or in combinations, with the aim to determine whether protein secondary structure can serve as predictor for chaperone success. Protein A, which has a helix dominant structure, showed the most efficient folding with GroES/EL or TF chaperones alone, whereas Protein B, which has less helix in the structure, showed a remarkable effect on the DnaK/J/GrpE system alone. This tendency was also seen with other recombinant proteins with particular properties. With the chaperons' assistance, both proteins were synthesized more efficiently in the culture at 22.5 °C for 20 h than at 37 °C for 3 h. These findings suggest a novel avenue to study compatibility of chaperones with substrate proteins and optimal culture conditions for producing functional proteins with a potential for predictive analysis of the success of chaperones based on the properties of the substrate protein., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The combined catalysis of glucose isomerase (GI) and D-psicose 3-epimerase (DPEase) provided a convenient route for the direct synthesis of D-allulose from d-glucose, whose cost is lower than d-fructose. In the present research, the weak activity of DPEase was the key rate-limiting step and resulted in the accumulation of d-fructose in engineered Bacillus subtilis. Then, the 5'-untranslated region (5'-UTR) structure of the mRNA translational initiation region was optimized for the precise control of DPEase expression. The manipulation of the 5'-UTR region promoted the accessibility to ribosome binding and the stability of mRNA, resulting in a maximum of 1.73- and 1.98-fold increase in DPEase activity and intracellular mRNA amount, respectively. Under the optimal catalytic conditions of 75 °C, pH 6.5, 110 g/L d-glucose, and 1 mmol/L Co 2+ , the reaction equilibrium time was reduced from 7.6 h to 6.1 h. We hope that our results could provide a facilitated strategy for large-scale production of D-allulose at low-cost., (Copyright © 2021 Elsevier B.V. All rights reserved.)

To adapt to various ecological niches, the members of genus Bacillus display a wide spectrum of glycoside hydrolases (GH) responsible for the hydrolysis of cellulose and lignocellulose. Being abundant and renewable, cellulose-containing plant biomass may be applied as a substrate in second-generation biotechnologies for the production of platform chemicals. The present study aims to enhance the natural cellulase activity of two promising 2,3-butanediol (2,3-BD) producers, Bacillus licheniformis 24 and B. velezensis 5RB, by cloning and heterologous expression of cel8A and cel48S genes of Acetivibrio thermocellus . In B. licheniformis , the endocellulase Cel8A (GH8) was cloned to supplement the action of CelA (GH9), while in B. velezensis , the cellobiohydrolase Cel48S (GH48) successfully complemented the activity of endo-cellulase EglS (GH5). The expression of the natural and heterologous cellulase genes in both hosts was demonstrated by reverse-transcription PCR. The secretion of clostridial cellulases was additionally enhanced by enzyme fusion to the subtilisin-like signal peptide, reaching a significant increase in the cellulase activity of the cell-free supernatants. The results presented are the first to reveal the possibility of genetic complementation for enhancement of cellulase activity in bacilli, thus opening the prospect for genetic improvement of strains with an important biotechnological application.

Background: Bacillus subtilis is one of the most important microorganisms for recombinant protein production. It possesses the GRAS (generally recognized as safe) status and a potent protein secretion capacity. Secretory protein production greatly facilitates downstream processing and thus significantly reduces costs. However, not all heterologous proteins are secreted and intracellular production poses difficulties for quantification. To tackle this problem, we have established a so-called intracellular split GFP (iSplit GFP) assay in B. subtilis as a tool for the in vivo protein detection during expression in batch cultures and at a single-cell level. For the iSplit GFP assay, the eleventh β-sheet of sfGFP is fused to a target protein and can complement a detector protein consisting of the respective truncated sfGFP (GFP1-10) to form fluorescent holo-GFP., Results: As proof of concept, the GFP11-tag was fused C-terminally to the E. coli β-glucuronidase GUS, resulting in fusion protein GUS11. Variable GUS and GUS11 production levels in B. subtilis were achieved by varying the ribosome binding site via spacers of increasing lengths (4-12 nucleotides) for the GUS-encoding gene. Differences in intracellular enzyme accumulation were determined by measuring the GUS11 enzymatic activity and subsequently by adding the detector protein to respective cell extracts. Moreover, the detector protein was co-produced with the GUS11 using a two-plasmid system, which enabled the in vivo detection and online monitoring of glucuronidase production. Using this system in combination with flow cytometry and microfluidics, we were able to monitor protein production at a single-cell level thus yielding information about intracellular protein distribution and culture heterogeneity., Conclusion: Our results demonstrate that the iSplit GFP assay is suitable for the detection, quantification and online monitoring of recombinant protein production in B. subtilis during cultivation as well as for analyzing production heterogeneity and intracellular localization at a single-cell level., (© 2021. The Author(s).)