KdpD is a tandem serine histidine kinase that controls K+ pump KdpFABC transcriptionally and post-translationally.

Two-component systems, consisting of a histidine kinase and a response regulator, serve signal transduction in bacteria, often regulating transcription in response to environmental stimuli. Here, we identify a tandem serine histidine kinase function for KdpD, previously described as a histidine kinase of the KdpDE two-component system, which controls production of the potassium pump KdpFABC. We show that KdpD additionally mediates an inhibitory serine phosphorylation of KdpFABC at high potassium levels, using not its C-terminal histidine kinase domain but an N-terminal atypical serine kinase domain. Sequence analysis of KdpDs from different species highlights that some KdpDs are much shorter than others. We show that, while Escherichia coli KdpD's atypical serine kinase domain responds directly to potassium levels, a shorter version from Deinococcus geothermalis is controlled by second messenger cyclic di-AMP. Our findings add to the growing functional diversity of sensor kinases while simultaneously expanding the framework for regulatory mechanisms in bacterial potassium homeostasis.

Antibiotic class with potent in vivo activity targeting lipopolysaccharide synthesis in Gram-negative bacteria.

Here, we describe the identification of an antibiotic class acting via LpxH, a clinically unexploited target in lipopolysaccharide synthesis. The lipopolysaccharide synthesis pathway is essential in most Gram-negative bacteria and there is no analogous pathway in humans. Based on a series of phenotypic screens, we identified a hit targeting this pathway that had activity on efflux-defective strains of Escherichia coli. We recognized common structural elements between this hit and a previously published inhibitor, also with activity against efflux-deficient bacteria. With the help of X-ray structures, this information was used to design inhibitors with activity on efflux-proficient, wild-type strains. Optimization of properties such as solubility, metabolic stability and serum protein binding resulted in compounds having potent in vivo efficacy against bloodstream infections caused by the critical Gram-negative pathogens E. coli and Klebsiella pneumoniae. Other favorable properties of the series include a lack of pre-existing resistance in clinical isolates, and no loss of activity against strains expressing extended-spectrum-ß-lactamase, metallo-ß-lactamase, or carbapenemase-resistance genes. Further development of this class of antibiotics could make an important contribution to the ongoing struggle against antibiotic resistance.

Identification of autophagy receptors for the Crohn's disease-associated adherent-invasive Escherichia coli.

Introduction: Crohn's disease (CD) is a chronic inflammatory bowel disease, of which the etiology involves genetic, environmental and microbial factors. Adherent-invasive Escherichia coli (AIEC) and polymorphisms in autophagy-related genes have been implicated in CD etiology. Autophagy is a key process for the maintenance of cellular homeostasis, which allows the degradation of damaged cytoplasmic components and pathogens via lysosome. We have shown that a functional autophagy is necessary for AIEC clearance. Here, we aimed at identifying the autophagy receptor(s) responsible to target AIEC to autophagy for degradation. Methods: The levels of autophagy receptors p62, NDP52, NBR1, TAX1BP1 and Optineurin were knocked down in human intestinal epithelial cells T84 using siRNAs. The NDP52 knock-out (KO) and p62 KO HeLa cells, as well as NDP52 KO HeLa cells expressing the wild-type NDP52 or the mutated NDP52Val248Ala protein were used. Results and discussion: We showed that, among the tested autophagy receptors (p62, NDP52, NBR1, TAX1BP1 and Optineurin), diminished expression of p62 or NDP52 increased the number of the clinical AIEC LF82 strain inside epithelial cells. This was associated with increased pro-inflammatory cytokine production. Moreover, p62 or NDP52 directly colocalized with AIEC LF82 and LC3, an autophagy marker. As the NDP52Val248Ala polymorphism has been associated with increased CD susceptibility, we investigated its impact on AIEC control. However, in HeLa cell and under our experimental condition, no effect of this polymorphism neither on AIEC LF82 intracellular number nor on pro-inflammatory cytokine production was observed. Together, our results suggest that p62 and NDP52 act as autophagy receptors for AIEC recognition, controlling AIEC intracellular replication and inflammation.

Characterization of a secondary hydroxy-acyltransferase for lipid A in Vibrio parahaemolyticus.

Lipid A plays a crucial role in Vibrio parahaemolyticus. Previously we have reported the diversity of secondary acylation of lipid A in V. parahaemolyticus and four V. parahaemolyticus genes VP_RS08405, VP_RS01045, VP_RS12170, and VP_RS00880 exhibiting homology to the secondary acyltransferases in Escherichia coli. In this study, the gene VP_RS12170 was identified as a specific lipid A secondary hydroxy-acyltransferase responsible for transferring a 3-hydroxymyristate to the 2'-position of lipid A. Four E. coli mutant strains WHL00, WHM00, WH300, and WH001 were constructed, and they would synthesize lipid A with different structures due to the absence of genes encoding lipid A secondary acyltransferases or Kdo transferase. Then V. parahaemolyticus VP_RS12170 was overexpressed in W3110, WHL00, WHM00, WH300, and WH001, and lipid A was isolated from these strains and analyzed by using thin-layer chromatography and high-performance liquid chromatography-tandem mass spectrometry. The detailed structural changes of lipid A in these mutant strains with and without VP_RS12170 overexpression were compared and conclude that VP_RS12170 can specifically transfer a 3-hydroxymyristate to the 2'-position of lipid A. This study also demonstrated that the function of VP_RS12170 is Kdo-dependent and its favorite substrate is Kdo-lipid IVA. These findings give us better understanding the biosynthetic pathway and the structural diversity of V. parahaemolyticus lipid A.

Constructing an Antibiotic-Free Protein Expression System for Ovalbumin Biosynthesis in Probiotic Escherichia coli Nissle 1917.

Ovalbumin (OVA) is the principal protein constituent of eggs. As an alternative to eggs, cell-cultured OVA can reduce the environmental impact of global warming and land use. Escherichia coli Nissle 1917 (EcN), a probiotic with specific endogenous cryptic plasmids that stably exist in cells without the addition of antibiotics, was chosen as the host for the efficient heterologous expression of the OVA. OVA yield reached 20 mg·L-1 in shake flasks using the OVA expression cassette containing a tac promoter (Ptac) upstream of the OVA-coding sequences on the endogenous plasmid pMUT2. Subsequently, we improved the level of the expression of the OVA by employing a dual promoter (PP5 combined with Ptac via a sigma factor binding site 24) and ribosome binding site (RBS) substitution. These enhancements increased the level of production of OVA in shake flasks to 30 and 42 mg·L-1, respectively. OVA by EcNP-P28 harboring plasmid L28 equipped with both dual promoter and the strong RBS8 reached 3.70 g·L-1 in a 3 L bioreactor. Recombinant OVA and natural OVA showed similar biochemical characteristics, including secondary structure, isoelectric point, amino acid composition, and thermal stability. This is currently the highest OVA production reported among prokaryotes. We successfully constructed an antibiotic-free heterologous protein expression system for EcN.

The Treatment of Tubal Inflammatory Infertility using Yinjia Tablets through EGFR/MEK/ERK Signaling Pathway based on Network Pharmacology.

Background: Salpingitis obstructive infertility (SOI) refers to infertility caused by abnormal conditions such as tubal adhesion and blockage caused by acute and chronic salpingitis. SOI has a serious impact on women's physical and mental health and family harmony, and it is a clinical problem that needs to be solved urgently.

Objective: The purpose of the present study was to explore the potential pharmacological mechanisms of the Yinjia tablets (Yin Jia Pian, YJP) on tubal inflammation.

Methods: Networks of YJP-associated targets and tubal inflammation-related genes were constructed through the STRING database. Potential targets and pathway enrichment analysis related to the therapeutic efficacy of YJP were identified using Cytoscape and Database for Annotation, Visualization, and Integrated Discovery (metascape). E. coli was used to establish a rat model of tubal inflammation and to validate the predictions of network pharmacology and the therapeutic efficacy of YJP. H&E staining was used to observe the pathological changes in fallopian tubes. TEM observation of the ultrastructure of the fallopian tubes. ELISA was used to detect the changes of IL-6 and TNF-α in fallopian tubes. Immunohistochemistry was used to detect the expression of ESR1. The changes of Bcl-2, ERK1/2, p-ERK1/2, MEK, p-MEK, EGFR, and p-EGFR were detected by western blot.

Results: Through database analysis, it was found that YJP shared 105 identical targets with the disease. Network pharmacology analysis showed that IL-6, TNF, and EGFR belong to the top 5 core proteins associated with salpingitis, and EGFR/MEK/ERK may be the main pathway involved. The E. coli-induced disease rat model of fallopian tube tissue showed damage, mitochondrial disruption, and increased levels of the inflammatory factors IL-6 and TNF-α. Tubal inflammatory infertility rats have increased expression of Bcl-2, p-ERK1/2, p-MEK, and p-EGFR, and decreased expression of ESR1. In vivo, experiments showed that YJP improved damage of tissue, inhibited shedding of tubal cilia, and suppressed the inflammatory response of the body. Furthermore, YJP inhibited EGFR/MEK/ERK signaling, inhibited the apoptotic protein Bcl-2, and upregulated ESR1.

Conclusion: This study revealed that YJP Reducing tubal inflammation and promoting tissue repair may be associated with inhibition of the EGFR/MEK/ERK signaling pathway.

Characterizing arginine, ornithine, and putrescine pathways in enteric pathobionts.

Arginine-ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell-free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine-ornithine metabolism in enteric pathobionts.

The importance of the fourth Greek key motif of human γD-crystallin in maintaining lens transparency-the tale told by the tail.

Purpose: Congenital cataract affects 1-15 per 10,000 newborns worldwide, and 20,000-40,000 children are born every year with developmental bilateral cataracts. Mutations in the crystallin genes are known to cause congenital cataracts. Crystallins, proteins present in the eye lens, are made up of four Greek key motifs separated into two domains. Greek key motifs play an important role in compact folding to provide the necessary refractive index and transparency. The present study was designed to understand the importance of the fourth Greek key motif in maintaining lens transparency by choosing a naturally reported Y134X mutant human γD- crystallin in a Danish infant and its relationship to lens opacification and cataract. Methods: Human γD-crystallin complementary DNA (cDNA) was cloned into the pET-21a vector, and the Y134X mutant clone was generated by site-directed mutagenesis. Wild-type and mutant proteins were overexpressed in the BL21 DE3 pLysS cells of E. coli. Wild-type protein was purified from the soluble fraction using the ion exchange and gel filtration chromatography methods. Mutant protein was predominantly found in insoluble fraction and purified from inclusion bodies. The structure, stability, aggregational, and amyloid fibril formation properties of the mutant were compared to those of the wild type using the fluorescence and circular dichroism spectroscopy methods. Results: Loss of the fourth Greek key motif in human γD-crystallin affects the backbone conformation, alters the tryptophan micro-environment, and exposes a nonpolar hydrophobic core to the surface. Mutant is less stable and opens its Greek key motifs earlier with a concentration midpoint (CM) of unfolding curve of 1.5 M compared to the wild type human γD-crystallin (CM: 2.5 M). Mutant is capable of forming self-aggregates immediately in response to heating at 48.6 °C. Conclusions: Loss of 39 amino acids in the fourth Greek key motif of human γD-crystallin affects the secondary and tertiary structures and exposes the hydrophobic residues to the solvent. These changes make the molecule less stable, resulting in the formation of light-scattering particles, which explains the importance of the fourth Greek key in the underlying mechanism of opacification and cataract.

Myxococcus xanthus translesion DNA synthesis protein ImuA is an ATPase enhanced by DNA.

Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well-studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error-prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C-terminus, as loss of the ImuA C-terminus results in DNA-independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl-imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self-interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the ß-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.

Construction and characterization of a hypervesiculation strain of Escherichia coli Nissle 1917.

Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in a host. OMVs secreted by probiotic probiotic strain Escherichia coli Nissle 1917 (EcN) have been reported to induce an immune response. In this study, we aimed to increase the OMV production of EcN. The double gene knockout of mlaE and nlpI was conducted in EcN because the ΔmlaEΔnlpI of experimental strain E. coli K12 showed the highest OMV production in our previous report. The ΔmlaEΔnlpI of EcN showed approximately 8 times higher OMV production compared with the parental (wild-type) strain. Quick-freeze, deep-etch replica electron microscopy revealed that plasmolysis occurred in the elongated ΔmlaEΔnlpI cells and the peptidoglycan (PG) had numerous holes. While these phenomena are similar to the findings for the ΔmlaEΔnlpI of K12, there were more PG holes in the ΔmlaEΔnlpI of EcN than the K12 strain, which were observed not only at the tip of the long axis but also in the whole PG structure. Further analysis clarified that the viability of ΔmlaEΔnlpI of EcN decreased compared with that of the wild-type. Although the amount of PG in ΔmlaEΔnlpI cells was about half of that in wild-type, the components of amino acids in PG did not change in ΔmlaEΔnlpI. Although the viability decreased compared to the wild-type, the ΔmlaEΔnlpI grew in normal culture conditions. The hypervesiculation strain constructed here is expected to be used as an enhanced probiotic strain.

Influence of PhoPQ and PmrAB two component system alternations on colistin resistance from non-mcr colistin resistant clinical E. Coli strains.

BACKGROUND: The current understanding of acquired chromosomal colistin resistance mechanisms in Enterobacterales primarily involves the disruption of the upstream PmrAB and PhoPQ two-component system (TCS) control caused by mutations in the regulatory genes. Interestingly, previous studies have yielded conflicting results regarding the interaction of regulatory genes related to colistin resistance in Escherichia coli, specifically those surrounding PhoPQ and PmrAB TCS. RESULTS: In our study, we focused on two clinical non-mcr colistin-resistant strains of E. coli, TSAREC02 and TSAREC03, to gain a better understanding of their resistance mechanisms. Upon analysis, we discovered that TSAREC02 had a deletion (Δ27-45) in MgrB, as well as substitutions (G206R, Y222H) in PmrB. On the other hand, TSAREC03 exhibited a long deletion (Δ84-224) in PhoP, along with substitutions (M1I, L14P, P178S, T235N) in PmrB. We employed recombinant DNA techniques to explore the interaction between the PhoPQ and PmrAB two-component systems (TCSs) and examine the impact of the mutated phoPQ and pmrB genes on the minimum inhibitory concentrations (MICs) of colistin. We observed significant changes in the expression of the pmrD gene, which encodes a connector protein regulated by the PhoPQ TCS, in the TSAREC02 wild-type (WT)-mgrB replacement mutant and the TSAREC03 WT-phoP replacement mutant, compared to their respective parental strains. However, the expressions of pmrB/pmrA, which reflect PmrAB TCS activity, and the colistin MICs remained unchanged. In contrast, the colistin MICs and pmrB/pmrA expression levels were significantly reduced in the pmrB deletion mutants from both TSAREC02 and TSAREC03, compared to their parental strains. Moreover, we were able to restore colistin resistance and the expressions of pmrB/pmrA by transforming a plasmid containing the parental mutated pmrB back into the TSAREC02 and TSAREC03 mutants, respectively. CONCLUSION: While additional data from clinical E. coli isolates are necessary to validate whether our findings could be broadly applied to the E. coli population, our study illuminates distinct regulatory pathway interactions involving colistin resistance in E. coli compared to other species of Enterobacterales. The added information provided by our study contribute to a deeper understanding of the complex pathway interactions within Enterobacterales.

Pyrrole-based inhibitors of RND-type efflux pumps reverse antibiotic resistance and display anti-virulence potential.

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

The role of genetic polymorphisms in the sulfation of pregnenolone by human cytosolic sulfotransferase SULT2B1a.

Pregnenolone is a key intermediate in the biosynthesis of many steroid hormones and neuroprotective steroids. Sulfotransferase family cytosolic 2B member 1 (SULT2B1a) has been reported to be highly selective to sulfate pregnenolone. This study aimed to clarify the effect of missense single nucleotide polymorphisms (SNPs) of the human SULT2B1 gene on the sulfating activity of coded SULT2B1a allozymes toward Pregnenolone. To investigate the effects of single nucleotide polymorphisms of the SULT2B1 gene on the sulfation of pregnenolone by SULT2B1a allozymes, 13 recombinant SULT2B1a allozymes were generated, expressed, and purified using established procedures. Human SULT2B1a SNPs were identified by a comprehensive database search. 13 SULT2B1a nonsynonymous missense coding SNPs (cSNPs) were selected, and site-directed mutagenesis was used to generate the corresponding cDNAs, packaged in pGEX-2TK expression vector, encoding these 13 SULT2B1a allozymes, which were bacterially expressed in BL21 E. coli cells and purified by glutathione-Sepharose affinity chromatography. Purified SULT2B1a allozymes were analyzed for sulfating activities towards pregnenolone. In comparison with the wild-type SULT2B1a, of the 13 allozymes, 11 showed reduced activity toward pregnenolone at 0.1 µM. Specifically, P134L and R259Q allozymes, reported to be involved in autosomal-recessive congenital ichthyosis, displayed low activity (1-10%) toward pregnenolone. The findings of this study may demonstrate the impact of genetic polymorphism on the sulfation of pregnenolone in individuals with different SULT2B1 genotypes.

Metabolic engineering of Candida tropicalis for efficient 1,2,4-butanetriol production.

1,2,4-Butanetriol serves as a precursor in the manufacture of diverse pharmaceuticals and the energetic plasticizer 1,2,4-butanetriol trinitrate. The study involved further modifications to an engineered Candida tropicalis strain, aimed at improving the production efficiency of 1,2,4-butanetriol. Faced with the issue of xylonate accumulation due to the low activity of heterologous xylonate dehydratase, we modulated iron metabolism at the transcriptional level to boost intracellular iron ion availability, thus enhancing the enzyme activity by 2.2-fold. Addressing the NADPH shortfall encountered during 1,2,4-butanetriol biosynthesis, we overexpressed pivotal genes in the NADPH regeneration pathway, achieving a 1,2,4-butanetriol yield of 3.2 g/L. The introduction of calcium carbonate to maintain pH balance led to an increased yield of 4 g/L, marking a 111% improvement over the baseline strain. Finally, the use of corncob hydrolysate as a substrate culminated in 1,2,4-butanetriol production of 3.42 g/L, thereby identifying a novel host for the conversion of corncob hydrolysate to 1,2,4-butanetriol.

Reduced OxyR positively regulates the prodigiosin biosynthesis in Serratia marcescens FS14.

OxyR, a LysR family transcriptional regulator, plays vital roles in bacterial oxidative stress response. In this study, we found that the deletion of oxyR not only inhibited the antioxidant capacity of S. marcescens FS14, but also decreased the production of prodigiosin. Further study revealed that OxyR activated the prodigiosin biosynthesis at the transcriptional level. Complementary results showed that not only the wild-type OxyR but also the reduced form OxyRC199S could activate the prodigiosin biosynthesis. We further demonstrated that reduced form of wild type OxyR could bind to the promoter of pig gene cluster, and identified the binding sites which is different from oxidized OxyR binding sites in E. coli. Our results demonstrated that OxyR in FS14 uses oxidized form to regulate the expression of the antioxidant related genes and utilizes reduced form to activate prodigiosin production. Further in silico analysis suggested that the activation of prodigiosin biosynthesis by reduced OxyR should be general in S. marcesencs. To our knowledge, this is the first report to show that OxyR uses the reduced form to activate the gene's expression, therefore, our results provide a novel regulation mechanism of OxyR.

From Natural Microbe Screening to Sustained Chitinase Activity in Exogenous Hosts.

Genetic parts and hosts can be sourced from nature to realize new functions for synthetic biology or to improve performance in a particular application environment. Here, we proceed from the discovery and characterization of new parts to stable expression in new hosts with a particular focus on achieving sustained chitinase activity. Chitinase is a key enzyme for various industrial applications that require the breakdown of chitin, the second most abundant biopolymer on the earth. Diverse microbes exhibit chitinase activity, but for applications, the environmental conditions for optimal enzyme activity and microbe fitness must align with the application context. Achieving sustained chitinase activity under broad conditions in heterologous hosts has also proven difficult due to toxic side effects. Toward addressing these challenges, we first screen ocean water samples to identify microbes with chitinase activity. Next, we perform whole genome sequencing and analysis and select a chitinase gene for heterologous expression. Then, we optimize transformation methods for target hosts and introduce chitinase. Finally, to achieve robust function, we optimize ribosome binding sites and discover a beneficial promoter that upregulates chitinase expression in the presence of colloidal chitin in a sense-and-respond fashion. We demonstrate chitinase activity for >21 days in standard (Escherichia coli) and nonstandard (Roseobacter denitrificans) hosts. Besides enhancing chitinase applications, our pipeline is extendable to other functions, identifies natural microbes that can be used directly in non-GMO contexts, generates new parts for synthetic biology, and achieves weeks of stable activity in heterologous hosts.

Rational Engineering of Escherichia coli Nissle 1917 as Live Biotherapeutic to Degrade Uremic Toxin Precursors.

Uremic toxins (UTs) are microbiota-derived metabolites that accelerate the progression of kidney damage in patients with chronic kidney disease (CKD). One of the major UTs involved in CKD progression is p-cresol-sulfate (PCS), derived from dietary l-tyrosine (l-Tyr). Here, we engineered a probiotic strain of Escherichia coli Nissle 1917, to convert l-Tyr to the nontoxic compound p-coumaric acid via tyrosine ammonia lyase (TAL). First, a small metagenomic library was assessed to identify the TAL with the greatest whole-cell activity. Second, accessory genes implicated in the import of l-Tyr and export of PCA were overexpressed to enhance l-Tyr degradation by 106% and 56%, respectively. Last, random mutagenesis coupled to a novel selection and screening strategy was developed that identified a TAL variant with a 25% increase in whole-cell activity. Taken together, the final strain exhibits a 183% improvement over initial whole-cell activity and provides a promising candidate to degrade l-Tyr mediated PCS accumulation.

Plasmid expression of Deinococcus radiodurans RecA confers UV-A protection to Escherichia coli with an inverse protein dose dependence, which does not exceed conspecific RecA protection.

Low level expression in Escherichia coli of the RecA protein from the radiation resistant bacterium Deinococcus radiodurans protects a RecA deficient strain of E. coli from UV-A irradiation by up to ∼160% over basal UV-A resistance. The protection effect is inverse protein dose dependent: increasing the expression level of the D. radiodurans RecA (DrRecA) protein decreases the protection factor. This inverse protein dose dependence effect helps resolve previously conflicting reports of whether DrRecA expression is protective or toxic for E. coli. In contrast to the D. radiodurans protein effect, conspecific plasmid expression of E. coli RecA protein in RecA deficient E. coli is consistently protective over several protein expression levels, as well as consistently more protective to higher levels of UV-A exposure than that provided by the D. radiodurans protein. The results indicate that plasmid expression of D. radiodurans RecA can modestly enhance the UV resistance of living E. coli, but that the heterospecific protein shifts from protective to toxic as expression is increased.

Structure and biological activity in vitro of Flagellin and its mutants from Escherichia coli Nissle 1917.

Bacterial flagellin is a potent immunomodulatory agent. Previously, we successfully obtained flagellin from Escherichia coli Nissle 1917 (FliCEcN) and constructed two mutants with varying degrees of deletion in its highly variable regions (HVRs). We found that there was a difference in immune stimulation levels between the two mutants, with the mutant lacking the D2-D3 domain pair of FliCEcN having a better adjuvant effect. Therefore, this study further analyzed the structural characteristics of the aforementioned FliCEcN and its two mutants and measured their levels of Caco-2 cell stimulation to explore the impact of different domains in the HVRs of FliCEcN on its structure and immune efficacy. This study utilized AlphaFold2, SERS (Surface-enhanced Raman spectroscopy), and CD (circular dichroism) techniques to analyze the structural characteristics of FliCEcN and its mutants, FliCΔ174-506 and FliCΔ274-406, and tested their immune effects by stimulating Caco-2 cells in vitro. The results indicate that the D2 and D3 domains of FliCEcN have more complex interactions compared to the D1-D2 domain pair., and these domains also play a role in molecular docking with TLR5 (Toll-like receptor 5). Furthermore, FliCΔ274-406 has more missing side chain and characteristic amino acid peaks than FliCΔ174-506. The FliCEcN group was found to stimulate higher levels of IL-10 (interleukin 10) secretion, while the FliCΔ174-506 and FliCΔ274-406 groups had higher levels of IL-6 (interleukin 6) and TNF-α (tumor necrosis factor-α) secretion. In summary, the deletion of different domains in the HVRs of FliCEcN affects its structural characteristics, its interaction with TLR5, and the secretion of immune factors by Caco-2 cells.

Type 1 secretion necessitates a tight interplay between all domains of the ABC transporter.

Type I secretion systems (T1SS) facilitate the secretion of substrates in one step across both membranes of Gram-negative bacteria. A prime example is the hemolysin T1SS which secretes the toxin HlyA. Secretion is energized by the ABC transporter HlyB, which forms a complex together with the membrane fusion protein HlyD and the outer membrane protein TolC. HlyB features three domains: an N-terminal C39 peptidase-like domain (CLD), a transmembrane domain (TMD) and a C-terminal nucleotide binding domain (NBD). Here, we created chimeric transporters by swapping one or more domains of HlyB with the respective domain(s) of RtxB, a HlyB homolog from Kingella kingae. We tested all chimeric transporters for their ability to secrete pro-HlyA when co-expressed with HlyD. The CLD proved to be most critical, as a substitution abolished secretion. Swapping only the TMD or NBD reduced the secretion efficiency, while a simultaneous exchange abolished secretion. These results indicate that the CLD is the most critical secretion determinant, while TMD and NBD might possess additional recognition or interaction sites. This mode of recognition represents a hierarchical and extreme unusual case of substrate recognition for ABC transporters and optimal secretion requires a tight interplay between all domains.