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Alterations in microbiotas and endogenous enzymes have been implicated in meat deterioration. However, the factors that mediate the interactions between meat quality and microbiome profile were inadequately investigated. In this study, we collected pork samples throughout the refrigeration period and employed metaproteomics to characterize both the pork and microbial proteins. Our findings demonstrated that pork proteins associated with the catabolic process are upregulated during storage compared to the initial stage. Pseudomonas, Clostridium, Goodfellowiella, and Gonapodya contribute to the spoilage process. Notably, we observed an elevated abundance of microbial proteins related to glycolytic enzymes in refrigerated pork, identifying numerous proteins linked to biogenic amine production, thus highlighting their essential role in microbial decay. Further, we reveal that many of these microbial proteins from Pseudomonas are ribosomal proteins, promoting enzyme synthesis by enhancing transcription and translation. This study provides intrinsic insights into the underlying mechanisms by which microorganisms contribute to meat spoilage., 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. Published by Elsevier Ltd.)

The Campylobacter jejuni bacterium, which causes foodborne enteritis in humans, expresses the uncharacterized protein CJ0600. Based on sequence analysis, CJ0600 has been proposed to function as a 1-aminocyclopropane-1-carboxylate (ACC) deaminase (AccDA) or cysteine desulfhydrase (CysDS). However, it has never been investigated whether CJ0600 exerts AccDA or CysDS activity or how CJ0600 mediates its enzymatic activity. To reveal the structural features necessary for the function of CJ0600, we determined the crystal structure of CJ0600 and characterized its enzymatic activity. CJ0600 contains two domains and features an interdomain pocket, which accommodates a pyridoxal 5'-phosphate (PLP) molecule as a Schiff base with its lysine residue (K35), as observed in its structural homologs, including AccDA, CysDS, and serine deaminase (SerDA). However, unlike its structural homologs, CJ0600 exists as a monomer and exhibits unique structural features throughout its structure. Moreover, CJ0600 contains unique active site residues that are not observed in AccDA, CysDS, or SerDA. Consistently, phylogenetic analysis indicates that CJ0600 and its orthologs are evolutionarily distinct from AccDA, CysDS, and SerDA. Indeed, CJ0600 showed no CysDS or SerDA activity and extremely weak AccDA activity. These observations suggest that CJ0600 functions as a unique PLP-dependent enzyme that has not been reported., 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.)

The nitrogen-fixing rhizobia-legume symbiosis relies on a complex interchange of molecular signals between the two partners during the whole interaction. On the bacterial side, different surface polysaccharides, such as lipopolysaccharide (LPS) and exopolysaccharide (EPS), might play important roles for the success of the interaction. In a previous work we studied two Sinorhizobium fredii HH103 mutants affected in the rkpK and lpsL genes, which are responsible for the production of glucuronic acid and galacturonic acid, respectively. Both mutants produced an altered LPS, and the rkpK mutant, in addition, lacked EPS. These mutants were differently affected in symbiosis with Glycine max and Vigna unguiculata, with the lpsL mutant showing a stronger impairment than the rkpK mutant. In the present work we have further investigated the LPS structure and the symbiotic abilities of the HH103 lpsL and rkpK mutants. We demonstrate that both strains produce the same LPS, with a truncated core oligosaccharide devoid of uronic acids. We show that the symbiotic performance of the lpsL mutant with Macroptilium atropurpureum and Glycyrrhiza uralensis is worse than that of the rkpK mutant. Introduction of an exoA mutation (which avoids EPS production) in HH103 lpsL improved its symbiotic performance with G. max, M. atropurpureum, and G. uralensis to the level exhibited by HH103 rkpK, suggesting that the presence of EPS might hide the truncated LPS produced by the former mutant., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Alcohol dehydrogenase I from Zymomonas mobilis (zmADH1) is a zinc-dependent oxidoreductase that catalyses the oxidation of primary or secondary alcohols to the corresponding aldehydes or ketones using NAD + /NADH as a cofactor. Efforts to express zmADH1 in Escherichia coli in a soluble form have been laden with solubility difficulties. A soluble form of recombinant zmADH1 was achieved by the addition of 1 mM zinc into media. Zinc addition facilitates the proper folding of recombinant zmADH1 and significantly reduces the formation of inclusion bodies. The yield of recombinant zmADH1 represents approximately 30 mg/1 L Luria-Bertani media. Intensified production in fermenters showed a striking difference between the specific and total activities of zmADH1 produced at different zinc concentrations. The zmADH1 showed an affinity to medium-chain alcohols, especially 1-pentanol, which could be used in new greener routes for preparation of aldehydes and alcohols., 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.)

The heterotrimeric flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase derived from Burkholderia cepacia (BcGDH) has many exceptional features for its use in glucose sensing-including that this enzyme is capable of direct electron transfer with an electrode in its heterotrimeric configuration. However, this enzyme's high catalytic activity towards not only glucose but also galactose presents an engineering challenge. To increase the substrate specificity of this enzyme, it must be engineered to reduce its activity towards galactose while maintaining its activity towards glucose. To aid in these mutagenesis studies, the crystal structure composed of BcGDH's small subunit and catalytic subunit (BcGDHγα), in complex with D-glucono-1,5-lactone was elucidated and used to construct the three-dimensional model for targeted, site-directed mutagenesis. BcGDHγα was then mutated at three different residues, glycine 322, asparagine 474 and asparagine 475. The single mutations that showed the greatest glucose selectivity were combined to create the resulting mutant, α-G322Q-N474S-N475S. The α-G322Q-N474S-N475S mutant and BcGDHγα wild type were then characterized with dye-mediated dehydrogenase activity assays to determine their kinetic parameters. The α-G322Q-N474S-N475S mutant showed more than a 2-fold increase in V max towards glucose and this mutant showed a lower activity towards galactose in the physiological range (5 mM) of 4.19 U mg -1 , as compared to the wild type, 86.6 U mg -1 . This resulting increase in specificity lead to an 81.7 gal/glc % activity for the wild type while the α-G322Q-N474S-N475S mutant had just 10.9 gal/glc % activity at 5 mM. While the BcGDHγα wild type has high specificity towards galactose, our engineering α-G322Q-N474S-N475S mutant showed concentration dependent response to glucose and was not affected by galactose., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Koji Sode reports financial support was provided by Arkray Inc, Kyoto, Japan. Koji Sode has patent pending to Arkray Inc. Kyoto, Japan. Junko Okuda-Shimazaki has patent pending to Arkray Inc. Katsuhiro Kojima has patent pending to Arkray Inc. If there are other authors, they 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.)

Mycoplasma synoviae is an important avian pathogen that causes respiratory infections and arthritis symptoms in chickens and turkeys, resulting in significant economic damage to the poultry farming industry worldwide. Cell adhesion is a vital stage of Mycoplasma infection, and the proteins associated with this process play an important role in its pathogenesis. Elongation factor thermo stable (EF-Ts) is an important factor in prokaryotic biosynthesis that serves as a guanosine exchange factor for elongation factor thermo unstable (EF-Tu). To date, little is known about the role of EF-Ts in Mycoplasma infection. In this study, we identified EF-Ts as an immunogenic protein in M. synoviae through liquid chromatography with tandem mass spectrometry (LC-MS/MS) screening. We constructed an E. coli recombinant expression vector and prepared a highly efficient rabbit antiserum. Immunoblot analysis and suspension immunofluorescence revealed that the EF-Ts is located in both the cell membrane and cytoplasm. The prepared rabbit EF-Ts antiserum exhibited complement-dependent Mycoplasma-killing activity and inhibited the adhesion of rEF-Ts and M. synoviae to DF-1 cells. An in-vitro binding assay showed that EF-Ts could bind to fibronectin (Fn) and chicken plasminogen (cPlg) in a dose-dependent manner. In addition, EF-Ts could internalize into cells through lipid rafts and clathrin-dependent endocytosis and induce DF-1 cell proliferation. In conclusion, our studies demonstrated that MS EF-Ts is a potentially immunogenic, novel adhesion protein that acts as a critical virulence factor in M. synoviae adhesion to host cells during infection. These studies further deepen our understanding of the pathogenic mechanism of M. synoviae., Competing Interests: Declarations Ethics approval and consent to participate New Zealand rabbits were acquired from the Animal Center of Lanzhou Veterinary Research Institute (Lanzhou, China). For the animal experiment, permission from the Animal Care and Ethics Committee of Gansu Agricultural University was obtained (Lanzhou, China), and all methods were carried out in accordance with the relevant guidelines and regulations outlined in the ethics declaration. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Mercury (Hg) is a heavy metal that exhibits high biological toxicity. Monomethylmercury and dimethylmercury are neurotoxins and a significant environmental concern as they bioaccumulate and biomagnify within the aquatic food web. Microbial Hg methylation involves two proteins, HgcA and HgcB. Here, we show that HgcA and HgcB can be heterologously coexpressed, and the HgcAB complex can be purified. We demonstrated that HgcA is a membrane-associated cobalamin-dependent methyltransferase and HgcB is a ferredoxin-like protein containing two [4Fe-4S] clusters. Further, spectroscopic and kinetic results demonstrate that S-adenosyl-L-methionine (SAM) donates the methyl group to Hg in a two-step reaction involving a methylcob(III)alamin intermediate including Co-thiolate ligation from a conserved Cys residue. Our findings uncover a biological role for SAM in microbial Hg methylation., Competing Interests: Competing interests statement:The authors declare no competing interest.

Transgenic crops producing crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) have been used extensively to control some major crop pests. However, many populations of the noctuid moth Helicoverpa zea , one of the most important crop pests in the United States, have evolved practical resistance to several Cry proteins including Cry1Ac. Although mutations in single genes that confer resistance to Cry proteins have been identified in lab-selected and gene-edited strains of H. zea and other lepidopteran pests, the genetic basis of field-evolved resistance to Cry proteins in H. zea has remained elusive. We used a genomic approach to analyze the genetic basis of field-evolved resistance to Cry1Ac in 937 H. zea derived from 17 sites in seven states of the southern United States. We found evidence for extensive gene flow among all populations studied. Field-evolved resistance was not associated with mutations in 20 single candidate genes previously implicated in resistance or susceptibility to Cry proteins in H. zea or other lepidopterans. Instead, resistance in field samples was associated with increased copy number of a cluster of nine trypsin genes. However, trypsin gene amplification occurred in a susceptible sample and not in all resistant samples, implying that this amplification does not always confer resistance and mutations in other genes also contribute to field-evolved resistance to Cry1Ac in H. zea . The mismatch between lab-generated and field-evolved resistance in H. zea is unlike other cases of Bt resistance and reflects challenges for managing this pest., Competing Interests: Competing interests statement:J.A.F. and B.E.T. are coauthors of patents on engineering Bacillus thuringiensis (Bt) toxins to counter resistance (US10704059) and potentiating Bt toxins (US20090175974A1), respectively. Badische Anilin und Soda Fabrik (BASF), Corteva Agriscience, Cotton Incorporated, Syngenta, and the Agricultural Biotechnology Stewardship Technical Committee (representing a consortium of agricultural biotechnology companies) did not provide funding to support this work but have funded other work by some of the authors.

Chlamydia protein associating with death domains ( Ct CADD) is involved in the biosynthesis of p -aminobenzoic acid (pABA) for integration into folate, a critical cofactor that is required for pathogenic survival. CADD activates dioxygen and utilizes its own tyrosine and lysine as synthons to furnish the carboxylate, carbon backbone, and amine group of pABA in a complex multistep mechanism. Unlike other members of the heme oxygenase-like dimetal oxidase (HDO) superfamily that typically house an Fe 2 cofactor, previous activity studies have shown that Ct CADD likely uses a heterobimetallic Fe/Mn center. The structure of the Fe 2+ /Mn 2+ cofactor and how the conserved HDO scaffold mediates metal selectivity have remained enigmatic. Adopting an in crystallo metalation approach, Ct CADD was solved in the apo, Fe 2+ 2 , Mn 2+ 2 , and catalytically active Fe 2+ /Mn 2+ forms to identify the probable site for Mn binding. The analysis of Ct CADD active-site variants further reinforces the importance of the secondary coordination sphere on cofactor preference for competent pABA formation. Rapid kinetic optical and electron paramagnetic resonance (EPR) studies show that the heterobimetallic cofactor selectively reacts with dioxygen and likely initiates pABA assembly through the formation of a transient tyrosine radical intermediate and a resultant heterobimetallic Mn 3+ /Fe 3+ cluster.

Cyclodipeptide synthases (CDPSs) catalyze the synthesis of diverse cyclodipeptides (CDPs) by utilizing two aminoacyl-tRNA (aa-tRNA) substrates in a sequential ping-pong reaction mechanism. Numerous CDPSs have been characterized to provide precursors for diketopiperazines (DKPs) with diverse structural characteristics and biological activities. BcmA, belonging to the XYP subfamily, is a cyclo(l-Ile-l-Leu)-synthesizing CDPS involved in the biosynthesis of the antibiotic bicyclomycin. The structural basis and determinants influencing BcmA enzyme activity and substrate selectivity are not well understood. Here, we report the crystal structure of Ss BcmA from Streptomyces sapporonensis . Through structural comparison and systematic site-directed mutagenesis, we highlight the significance of key residues located in the aminoacyl-binding pocket for enzyme activity and substrate specificity. In particular, the nonconserved residues D161 and K165 in pocket P2 are essential for the activity of Ss BcmA without significant alteration of the substrate specificity, while the conserved residues F158 as well as F210 and S211 in P2 are responsible for determining substrate selectivity. These findings facilitate the understanding of how CDPSs selectively accept hydrophobic substrates and provide additional clues for the engineering of these enzymes for synthetic biology applications.

Xylanibacter ruminicola is an abundant rumen bacterium that produces propionate in a cobalamin (vitamin B 12 )-dependent manner via the succinate pathway. However, the extent to which this occurs across ruminal Xylanibacter and closely related bacteria, and the effect of cobalamin supplementation on the expression of propionate pathway genes and enzymes has yet to be investigated. To assess this, we screened 14 strains and found that almost all strains produced propionate when supplemented with cobalamin. X. ruminicola KHP1 was selected for further study, including complete genome sequencing, and comparative transcriptomics and proteomics of KHP1 cultures grown with and without supplemented cobalamin. The complete KHP1 genome was searched for cobalamin-binding riboswitches and four were predicted, though none were closely located to any of the succinate pathway genes, which were dispersed at numerous genomic loci. Cobalamin supplementation led to the differential expression of 17.5% of genes, including genes encoding the cobalamin-dependent methylmalonyl-CoA mutase and some methylmalonyl-CoA decarboxylase subunits, but most propionate biosynthesis pathway genes were not differentially expressed. The effect of cobalamin supplementation on the KHP1 proteome was much less pronounced, with the only differentially abundant propionate pathway enzyme being methylmalonyl-CoA mutase, which had greater abundance when supplemented with cobalamin. Our results demonstrate that cobalamin supplementation does not result in induction of the entire propionate biosynthesis pathway, but consistently increased expression of methylmalonyl-CoA mutase at transcriptome and proteome levels. The magnitude of the differential expression of propionate pathway genes observed was minor compared to that of genes proximate to predicted cobalamin riboswitches., Importance: In ruminants, the rumen microbial community plays a critical role in nutrition through the fermentation of feed to provide vital energy substrates for the host animal. Propionate is a major rumen fermentation end-product and increasing its production is desirable given its importance in host glucose production and impact on greenhouse gas production. Vitamin B 12 (cobalamin) can induce propionate production in the prominent rumen bacterium Xylanibacter ruminicola , but it is not fully understood how cobalamin regulates propionate pathway activity. Contrary to expectation, we found that cobalamin supplementation had little effect on propionate pathway expression at transcriptome and proteome levels, with minor upregulation of genes encoding the cobalamin-dependent enzyme of the pathway. These findings provide new insights into factors that regulate propionate production and suggest that cobalamin-dependent propionate production by X. ruminicola is controlled post-translationally., Competing Interests: The authors declare no conflict of interest.

Toxin/antitoxin (TA) systems are present in nearly every prokaryotic genome and play the important physiological roles of phage inhibition by reducing metabolism (this includes persistence for the extreme case of complete cessation of metabolism), genetic element stabilization, and biofilm formation. TA systems have also been incorporated into other cell systems, such as CRISPR-Cas and phage quorum sensing. For the simplest and best-studied case, proteinaceous toxins and antitoxins (i.e., type II), toxin activity is masked by direct binding of the antitoxin. A long-standing, unresolved question in the TA field is how toxins are activated when bound to antitoxins at nanomolar affinity. The current paradigm envisions preferential degradation of the antitoxin by a protease, but this is highly unlikely in that a protease cannot discriminate between bound toxin and bound antitoxin because both are highly structured. Strikingly, recent results from several studies show one likely mechanism for toxin activation is conformational changes in the TA complex that result in the release or activation of the toxin as a result of a protein trigger, such as that from phages, and as a result of thermally-driven refolding dynamics., Competing Interests: The authors declare no conflict of interest.

As bacterial symbionts transition from a motile free-living state to a sessile biofilm state, they must coordinate behavior changes suitable to each lifestyle. Cyclic diguanylate (c-di-GMP) is an intracellular signaling molecule that can regulate this transition, and it is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes. Generally, c-di-GMP inhibits motility and promotes biofilm formation. While c-di-GMP and the enzymes that contribute to its metabolism have been well studied in pathogens, considerably less focus has been placed on c-di-GMP regulation in beneficial symbionts. Vibrio fischeri is the sole beneficial symbiont of the Hawaiian bobtail squid ( Euprymna scolopes ) light organ, and the bacterium requires both motility and biofilm formation to efficiently colonize. c-di-GMP regulates swimming motility and cellulose exopolysaccharide production in V. fischeri . The genome encodes 50 DGCs and PDEs, and while a few of these proteins have been characterized, the majority have not undergone comprehensive characterization. In this study, we use protein overexpression to systematically characterize the functional potential of all 50 V . fischeri proteins. All 28 predicted DGCs and 10 of the 14 predicted PDEs displayed at least one phenotype consistent with their predicted function, and a majority of each displayed multiple phenotypes. Finally, active site mutant analysis of proteins with the potential for both DGC and PDE activities revealed potential activities for these proteins. This work presents a systems-level functional analysis of a family of signaling proteins in a tractable animal symbiont and will inform future efforts to characterize the roles of individual proteins during lifestyle transitions.IMPORTANCECyclic diguanylate (c-di-GMP) is a critical second messenger that mediates bacterial behaviors, and Vibrio fischeri colonization of its Hawaiian bobtail squid host presents a tractable model in which to interrogate the role of c-di-GMP during animal colonization. This work provides systems-level characterization of the 50 proteins predicted to modulate c-di-GMP levels. By combining multiple assays, we generated a rich understanding of which proteins have the capacity to influence c-di-GMP levels and behaviors. Our functional approach yielded insights into how proteins with domains to both synthesize and degrade c-di-GMP may impact bacterial behaviors. Finally, we integrated published data to provide a broader picture of each of the 50 proteins analyzed. This study will inform future work to define specific pathways by which c-di-GMP regulates symbiotic behaviors and transitions., Competing Interests: The authors declare no conflict of interest.

Bacillus coagulans, a Gram-positive thermophilic bacterium, is recognized for its probiotic properties and recent development as a microbial cell factory. Despite its importance for biotechnological applications, the current understanding of B. coagulans ' robustness is limited, especially for undomesticated strains. To fill this knowledge gap, we characterized the metabolic capability and performed functional genomics and systems analysis of a novel, robust strain, B. coagulans B-768. Genome sequencing revealed that B-768 has the largest B. coagulans genome known to date (3.94 Mbp), about 0.63 Mbp larger than the average genome of sequenced B. coagulans strains, with expanded carbohydrate metabolism and mobilome. Functional genomics identified a well-equipped genetic portfolio for utilizing a wide range of C5 (xylose, arabinose), C6 (glucose, mannose, galactose), and C12 (cellobiose) sugars present in biomass hydrolysates, which was validated experimentally. For growth on individual xylose and glucose, the dominant sugars in biomass hydrolysates, B-768 exhibited distinct phenotypes and proteome profiles. Faster growth and glucose uptake rates resulted in lactate overflow metabolism, which makes B. coagulans a lactate overproducer; however, slower growth and xylose uptake diminished overflow metabolism due to the high energy demand for sugar assimilation. Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) made up 60%-65% of the measured proteomes but were allocated differently when growing on xylose and glucose. The trade-off in proteome reallocation, with high investment in COG-C over COG-G, explains the xylose growth phenotype with significant upregulation of xylose metabolism, pyruvate metabolism, and tricarboxylic acid (TCA) cycle. Strain B-768 tolerates and effectively utilizes inhibitory biomass hydrolysates containing mixed sugars and exhibits hierarchical sugar utilization with glucose as the preferential substrate.IMPORTANCEThe robustness of B. coagulans makes it a valuable microorganism for biotechnology applications; yet, this phenotype is not well understood at the cellular level. Through phenotypic characterization and systems analysis, this study elucidates the functional genomics and robustness of a novel, undomesticated strain, B. coagulans B-768, capable of utilizing inhibitory switchgrass biomass hydrolysates. The genome of B-768, enriched with carbohydrate metabolism genes, demonstrates high regulatory capacity. The coordination of proteome reallocation in Carbohydrate Transport and Metabolism (COG-G), Translation (COG-J), and Energy Conversion and Production (COG-C) is critical for effective cell growth, sugar utilization, and lactate production via overflow metabolism. Overall, B-768 is a novel, robust, and promising B. coagulans strain that can be harnessed as a microbial biomanufacturing platform to produce chemicals and fuels from biomass hydrolysates., Competing Interests: The authors declare no conflict of interest.

Vitamin B 12 is involved in many important biochemical reactions for humans, and its deficiency can lead to serious diseases. The industrial production of vitamin B 12 is achieved through microbial fermentation. In this work, we determine the crystal structures of the l-threonine-O-3-phosphate (Thr-P) decarboxylase CobC from Sinorhizobium meliloti (SmCobC), an industrial vitamin B 12 -producing bacterium, in apo form and in complex with a reaction intermediate. Our structures supported the Thr-P decarboxylase activity of SmCobC and revealed that the positively charged substrate-binding pocket between the large and small domains determines its substrate selectivity for Thr-P. Moreover, our results provided evidence for the proposition that the AP-P linker is formed by direct incorporation of AP-P in the biosynthetic pathway of vitamin B 12 in S.meliloti., 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.)

Background: SepIVA has been reported to be an essential septation factor in Mycolicibacterium smegmatis and Mycobacterium tuberculosis. It is a coiled-coil protein with similarity to DivIVA, a protein necessary for polar growth in members of the phylum Actinomycetota. Orthologues of SepIVA are broadly distributed among actinomycetes, including in Streptomyces spp., Results: To clarify the role of SepIVA and its potential involvement in cell division in streptomycetes, we generated sepIVA deletion mutants in Streptomyces venezuelae and found that sepIVA is dispensable for growth, cell division and sporulation. Further, mNeonGreen-SepIVA fusion protein did not localize at division septa, and we found no evidence of involvement of SepIVA in cell division. Instead, mNeonGreen-SepIVA was accumulated at the tips of growing vegetative hyphae in ways reminiscent of the apical localization of polarisome components like DivIVA. Bacterial two-hybrid system analyses revealed an interaction between SepIVA and DivIVA. The results indicate that SepIVA is associated with polar growth. However, no phenotypic effects of sepIVA deletion could be detected, and no evidence was observed of redundancy with the other DivIVA-like coiled-coil proteins Scy and FilP that are also associated with apical growth in streptomycetes., Conclusions: We conclude that S. venezuelae SepIVA, in contrast to the situation in mycobacteria, is dispensable for growth and viability. The results suggest that it is associated with polar growth rather than septum formation., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Penicillin-binding proteins (PBPs) are the targets of β-lactam antibiotics; however, changes in the affinity of PBPs for beta-lactam antibiotics often affect the susceptibility of bacteria to antibiotics. The purpose of this study was to elucidate the mechanism by which cefditoren, an oral third-generation cephalosporin, binds PBPs. The minimal inhibitory concentration (MIC), bactericidal curves, and inhibition zone comparisons were assessed to evaluate the antibacterial activity of cefditoren. PBP1A and PBP2X proteins from Streptococcus pneumoniae were purified, and their ability to bind to cefditoren was investigated via microscale thermophoresis. The Kd of cefditoren toward PBP1A was 0.005 ± 0.004 µM, which was lower than those of other cephalosporins (cefcapene, cefixime and cefdinir). In contrast, the Kd of cefditoren toward PBP2X of S. pneumoniae was 9.70 ± 8.24 µM, which was lower than that of cefixime but higher than those of cefcapene and cefdinir. Additionally, the biotinylated ampicillin (BIO-AMP) method was employed to evaluate the affinity of cefditoren toward PBPs of Haemophilus influenzae, and the results demonstrated that cefditoren and PBP3A/B had the lowest IC 50 values (0.060 ± 0.002 µM). These findings indicate that cefditoren has a strong affinity for PBP1A of H. influenzae. Cefditoren has a high affinity toward the PBP1As of S. pneumoniae and PBP1A and PBP3A/B of H. influenzae, which may contribute to the effective antibacterial effects of cefditoren against clinical strains and its low propensity for inducing resistance. The data presented in this article help elucidate the mechanism by which cefditoren, an oral third-generation cephalosporin, binds to PBPs and provide theoretical support for the wider use of cefditoren as an antibiotic therapy., Competing Interests: Declarations Conflict of 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. The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Ultraviolet radiation (UV) is a major abiotic stress resulting in relative short duration of Bacillus thuringiensis (Bt) biopesticides in the field, which is expected to be solved by formation of Bt biofilm with higher UV resistance. Therefore, one of the important prerequisite works is to clarify the functions of biofilm-associated genes on biofilm formation and UV resistance of Bt. In this study, comparative genomics and bioinformatic analysis indicated that BTXL6_19475 gene involved in biofilm formation of Bt XL6 was likely to encode a galactose-1-phosphate uridylyltransferase (GalT, E.C. 2.7.7.12). Heterologous expression of the BTXL6_19475 gene in Escherichia coli and detection of its GalT enzyme activity in vitro proved that the gene did encode GalT. Comparing the wild type Bt strain XL6 with galT gene knockout mutant Bt XL6ΔgalT and its complementary strain Bt XL6ΔgalT::19,475, GalT promoted the biofilm formation and enhanced the UV-B resistance of Bt XL6 likely by increasing its D-ribose production and reducing its alanine aryldamidase activity. GalT did not affect the growth and the cell motility of Bt XL6. A regulation map had been proposed to elucidate how GalT promoted biofilm formation and enhanced UV-B resistance of Bt XL6 by the cross-talk between Leloir pathway, Embden-Meyerhof glycolysis pathway and pentose phosphate pathway. Our finding provides a theoretical basis for the efficient use of biofilm genes to improve the UV resistance of Bt biofilms and thus extend field duration of Bt formulations based on biofilm engineering., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

(+)-Bicyclogermacrene is a sesquiterpene compound found in various plant essential oils and serves as a crucial precursor for multiple biologically active compounds. Many derivatives of (+)-bicyclogermacrene have been shown to exhibit valuable bioactivities. Cytochrome P450 BM3 from Bacillus megaterium can catalyze a variety of substrates and different types of oxidation reactions, making it become a powerful tool for oxidizing terpenes. In this study, we employed P450 BM3-139-3 variant for in vitro enzymatic oxidation of (+)-bicyclogermacrene, identifying a novel oxidized derivative of (+)-bicyclogermacrene, named (+)-3,6-epoxymaaliane, and an unknown sesquiterpenoid in a ratio of 70 : 30 (by GC peak area). (+)-3,6-Epoxymaaliane showed demonstrated antibacterial activities toward Escherichia coli and Staphylococcus aureus. To obtain a better variant of the monooxygenase with a high selectivity to form (+)-3,6-epoxymaaliane, we combined alanine scanning with the "Focused Rational Iterative Site-Specific Mutagenesis" (FRISM) strategy to modify the closest residues within 5 Å radius surrounding the substrate to create a small-but-smart library of mutants. Consequently, it gave an optimal variant with 1.6-fold improvement, in a turnover number (TON) of up to 964 toward (+)-3,6-epoxymaaliane production with a higher product selectivity., (© 2024 Wiley-VCH GmbH.)

Carotenoids are essential components of the photosynthetic apparatus and precursors of plant hormones, such as strigolactones (SLs). SLs are involved in various aspects of plant development and stress-response processes, including the establishment of root and shoot architecture. SL biosynthesis begins with the reversible isomerization of all- trans -carotene into 9- cis -β-carotene, catalysed by DWARF27 β-carotene isomerase (D27). Sequence comparisons have revealed the presence of D27-related proteins in photosynthetic eukaryotes and cyanobacteria lacking SLs. To gain insight into the evolution of SL biosynthesis, we characterized the activity of a cyanobacterial D27 protein ( Ca D27) from Cyanobacterim aponinum , using carotenoid-accumulating Escherichia coli cells and in vitro enzymatic assays. Our results demonstrate that Ca D27 is an all- trans / cis and cis / cis -β-carotene isomerase, with a cis / cis conversion preference. Ca D27 catalysed 13- cis /15- cis- , all- trans /9- cis -β-carotene, and neurosporene isomerization. Compared with plant enzymes, it exhibited a lower 9- cis -/all- trans -β-carotene conversion ratio. A comprehensive genome survey revealed the presence of D27 as a single-copy gene in the genomes of 20 out of 200 cyanobacteria species analysed. Phylogenetic and enzymatic analysis of Ca D27 indicated that cyanobacterial D27 genes form a single orthologous group, which is considered an ancestral type of those found in photosynthetic eukaryotes. This article is part of the theme issue 'The evolution of plant meta‌bolism'.

Electrocatalytic carbon dioxide (CO 2 ) reduction by CO 2 reductases is a promising approach for biomanufacturing. Among all known biological or chemical catalysts, hydrogen-dependent carbon dioxide reductase from Thermoanaerobacter kivui (TkHDCR) possesses the highest activity toward CO 2 reduction. Herein, we engineer TkHDCR to generate an electro-responsive carbon dioxide reductase considering the safety and convenience. To achieve this purpose, a recombinant Escherichia coli TkHDCR overexpression system is established. The formate dehydrogenase is obtained via subunit truncation and rational design, which enables direct electron transfer (DET)-type bioelectrocatalysis with a near-zero overpotential. By applying a constant voltage of -500 mV (vs. SHE) to a mediated electrolytic cell, 22.8 ± 1.6 mM formate is synthesized in 16 h with an average production rate of 7.1 ± 0.5 μmol h -1 cm -2 , a Faradaic efficiency of 98.9% and a half-cell energy efficiency of 94.4%. This study provides an enzyme candidate for high efficient CO 2 reduction and opens up a way to develop paradigm for CO 2 -based bio-manufacturing., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The cell membrane remodeling mediated by cyclopropane fatty acid synthase (CfaS) plays a crucial role in microbial physiological processes resisting various environmental stressors, including acid. Herein, we found a relatively high proportion (24.8%-28.3%) of cyclopropane fatty acid (CFA) Cy-19:0 in the cell membrane of a newly isolated extreme acidophile, Acidithiobacillus caldus CCTCC AB 2019256, under extreme acid stress. Overexpression of the CfaS encoding gene cfaS2 in Escherichia coli conferred enhanced acid resistance. GC-MS analysis revealed a 3.52-fold increase in the relative proportion of Cy-19:0 in the cell membrane of the overexpression strain compared to the control. Correspondingly, membrane fluidity, permeability and cell surface hydrophobicity were reduced to varying degrees. Additionally, HPLC analysis indicated that the overexpression strain had 1.54-, 1.42-, 1.85-, 1.20- and 1.05-fold higher levels of intracellular glutamic acid, arginine, aspartic acid, methionine and alanine, respectively, compared to the control. Overall, our findings shed light on the role of CfaS derived from extreme acidophile in bacterial defense against environmental acid stress, potentially facilitating its application in the design and development of industrial microbial chassis cells for organic acid production., Competing Interests: Declarations Conflict of interest The authors declare no competing interests. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors., (© 2024. The Author(s), under exclusive licence to Springer Nature Japan KK.)

A critical cell cycle checkpoint for most bacteria is the onset of constriction when the septal peptidoglycan synthesis starts. According to the current understanding, the arrival of FtsN to midcell triggers this checkpoint in Escherichia coli. Recent structural and in vitro data suggests that recruitment of FtsN to the Z-ring leads to a conformational switch in actin-like FtsA, which links FtsZ protofilaments to the cell membrane and acts as a hub for the late divisome proteins. Here, we investigate this putative pathway using in vivo measurements and stochastic cell cycle modeling at moderately fast growth rates. Quantitatively upregulating protein concentrations and determining the resulting division timings shows that FtsN and FtsA numbers are not rate-limiting for the division in E. coli. However, at higher overexpression levels, they affect divisions: FtsN by accelerating and FtsA by inhibiting them. At the same time, we find that the FtsZ numbers in the cell are one of the rate-limiting factors for cell divisions in E. coli. Altogether, these findings suggest that instead of FtsN, accumulation of FtsZ in the Z-ring is one of the main drivers of the onset of constriction in E. coli at faster growth rates., Competing Interests: Competing interests The authors declare no competing interest., (© 2024. The Author(s).)

Antibiotic resistance in bacterial pathogen infections is a growing global issue that occurs due to their adaptation to changing environmental conditions. Therefore, producing an efficient vaccine as an alternative approach can improve the immune system, eradicate related pathogens, and overcome this growing problem. Streptococcus agalactiae belongs to group B Streptococcus (GBS). Colonization of GBS during pregnancy is a significant risk factor for infants and young children. S. agalactiae infected population exhibits resistance to beta-lactams, including penicillin and the second-line antibiotics erythromycin and clindamycin. On the other hand, there are currently no commercial vaccines against this pathogen. Vaccination of pregnant women is a highly effective method to protect newborns and infants from S. agalactiae infection, and it has been identified as an urgent demand by the World Health Organization. This study employed various immunoinformatic tools to develop an effective vaccine that could trigger both humoral and cell-mediated immunity and prevent disease. For this purpose, three conserved antigenic proteins of the main pathogenic strains of S. agalactiae were utilized to predict CTL, HTL, and B-cell epitopes for producing an mRNA vaccine against different strains of S. agalactiae. The selected epitopes were fused using proper linkers. The Resuscitation promoting factor E (RpfE) sequence was incorporated in the designed vaccine construct as an adjuvant to boost its immune response. Different physicochemical characteristics of the final designed vaccine, modeling of the three-dimensional structure, molecular docking, molecular dynamics simulation, and immunological response simulation were screened following vaccine administration in an in vivo model. Computational immune simulation data identified that IgG1, IgM, INF γ, IL-2, T helper, and B-cell populations increased significantly after vaccination. These findings suggested that the vaccine candidate may provide good protection against S. agalactiae infection. However, experimental and animal model studies are required for additional validation and implementation in human vaccination programs., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Bacteria invest significant resources into the continuous creation and tailoring of their essential protective peptidoglycan (PG) cell wall. Several soluble PG biosynthesis products in the periplasm are transported to the cytosol for recycling, leading to enhanced bacterial fitness. GlcNAc-1,6-anhydroMurNAc and peptide variants are transported by the essential major facilitator superfamily importer AmpG in Gram-negative pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Accumulation of GlcNAc-1,6-anhydroMurNAc-pentapeptides also results from β-lactam antibiotic induced cell wall damage. In some species, these products upregulate the β-lactamase AmpC, which hydrolyzes β-lactams to allow for bacterial survival and drug-resistant infections. Here, we have used cryo-electron microscopy and chemical synthesis of substrates in an integrated structural, biochemical, and cellular analysis of AmpG. We show how AmpG accommodates the large GlcNAc-1,6-anhydroMurNAc peptides, including a unique hydrophobic vestibule to the substrate binding cavity, and characterize residues involved in binding that inform the mechanism of proton-mediated transport., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: Plesiomonas shigelloides is a gram-negative opportunistic pathogen associated with gastrointestinal and extraintestinal diseases in humans. There have been reports of specific functional genes in the study of P. shigelloides, but there are also many unknown genes that may play a role in P. shigelloides pathogenesis as global regulatory proteins or virulence factors., Results: In this study, we found a transcriptional regulator of the PadR family in P. shigelloides and named it PstR (GenBank accession number: EON87311.1), which is present in various pathogenic bacteria but whose function has rarely been reported. RNA sequencing (RNA-Seq) was used to analyze the effects of PstR on P. shigelloides, and the results indicated that PstR regulates approximately 9.83% of the transcriptome, which includes impacts on motility, virulence, and physiological metabolism. RNA-seq results showed that PstR positively regulated the expression of the flagella gene cluster, which was also confirmed by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Luminescence screening assay. Meanwhile, the ΔpstR mutant strains lacked flagella and were non-motile, as confirmed by motility assays and transmission electron microscopy (TEM). Additionally, PstR also positively regulates T3SS expression, which aids in P. shigelloides' capacity to infect Caco-2 cells. Meanwhile, we also revealed that PstR negatively regulates fatty acid degradation and metabolism, as well as the regulatory relationship between PsrA, a regulator of fatty acid degradation and metabolism, and its downstream genes in P. shigelloides., Conclusions: Overall, we revealed the effects of PstR on motility, virulence, and physiological metabolism in P. shigelloides, which will serve as a foundation for future research into the intricate regulatory network of PstR in bacteria., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Purpose: This study analyzed antibiotic resistance mechanisms and molecular epidemiology of multidrug-resistant Pseudomonas aeruginosa (MDR-PA), aiming at providing clues for prevention and control of MDR-PA infections., Methods: The carbapenemase resistance genes (VIM, IMP, NDM, KPC, GES, OXA-40) of MDR-PA strains were detected by polymerase chain reaction (PCR) and sequencing. The efflux pump system (MexA, MexC, MexE, MexX), AmpC and OprD2 were detected by real-time fluorescent quantitative PCR (qPCR) in MDR-PA group and sensitive-Pseudomonas aeruginosa (S-PA) group. The homology analysis of MDR-PA strains was performed by multilocus sequence typing (MLST)., Results: A total of 81 MDR-PA strains were collected from the First Affiliated Hospital of Guangxi Medical University from October 2022 to October 2023. Among the carbapenemase detected, the detection rate of NDM-1 was the highest, with a rate of 34.57% (28/81). MexA had a higher expression in MDR-PA group than that in S-PA group (P<0.0001). 81 MDR-PA strains belonged to 40 different ST types, mainly including ST1971, ST244, ST357 and ST308, and the predominant ST type was ST1971 (34.57%, 28/81)., Conclusion: The mechanisms of antibiotic resistance of MDR-PA strains mainly were the production of MBLS and higher expression of MexA in our study, and ST1971 was the predominant ST type of MDR-PA strains in our hospital, our findings may assist in prevention and control of MDR-PA infections., Competing Interests: Declarations Ethics approval and consent to participate Informed consent was obtained from the patients and the study was approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Approval Number: 2024-E235-01). Consent for publication Not applicable. Competing interests The authors declare no competing interests. Clinical trial number Not applicable., (© 2024. The Author(s).)

Nowadays, the emergence of some microbial species resistant to antibiotics, both gram-positive and gram-negative bacteria, is due to changes in molecular activities, biological processes and their cellular structure in order to survive. The aim of the gene network analysis for the drug-resistant Enterococcus faecium as gram-positive and Salmonella Typhimurium as gram-negative bacteria was to gain insights into the important interactions between hub genes involved in key molecular pathways associated with cellular adaptations and the comparison of survival mechanisms of these two bacteria exposed to ciprofloxacin. To identify the gene clusters and hub genes, the gene networks in drug-resistant E. faecium and S. Typhimurium were analyzed using Cytoscape. Subsequently, the putative regulatory elements were found by examining the promoter regions of the hub genes and their gene ontology (GO) was determined. In addition, the interaction between milRNAs and up-regulated genes was predicted. RcsC and D920&#95;01853 have been identified as the most important of the hub genes in S. Typhimurium and E. faecium, respectively. The enrichment analysis of hub genes revealed the importance of efflux pumps, and different enzymatic and binding activities in both bacteria. However, E. faecium specifically increases phospholipid biosynthesis and isopentenyl diphosphate biosynthesis, whereas S. Typhimurium focuses on phosphorelay signal transduction, transcriptional regulation, and protein autophosphorylation. The similarities in the GO findings of the promoters suggest common pathways for survival and basic physiological functions of both bacteria, including peptidoglycan production, glucose transport and cellular homeostasis. The genes with the most interactions with milRNAs include dpiB, rcsC and kdpD in S. Typhimurium and EFAU004&#95;01228, EFAU004&#95;02016 and EFAU004&#95;00870 in E. faecium, respectively. The results showed that gram-positive and gram-negative bacteria have different mechanisms to survive under antibiotic stress. By deciphering their intricate adaptations, we can develop more effective therapeutic approaches and combat the challenges posed by multidrug-resistant bacteria., Competing Interests: They authors declare that they have no conflict of interest and have no relevant financial or non-financial interests to disclose., (Copyright: © 2024 Davati, Ghorbani. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Translation-targeting toxic small alarmone synthetases (toxSAS) are effectors of bacterial toxin-antitoxin systems that pyrophosphorylate the 3'-CCA end of transfer RNA (tRNA) to prevent aminoacylation. toxSAS are implicated in antiphage immunity: Phage detection triggers the toxSAS activity to shut down viral production. We show that the toxSAS FaRel2 inspects the tRNA acceptor stem to specifically select tRNA Gly and tRNA Thr . The first, second, fourth, and fifth base pairs of the stem act as the specificity determinants. We show that the toxSASs PhRel2 and CapRel SJ46 differ in tRNA specificity from FaRel2 and rationalize this through structural modeling: While the universal 3'-CCA end slots into a highly conserved CCA recognition groove, the acceptor stem recognition region is variable across toxSAS diversity. As phages use tRNA isoacceptors to overcome tRNA-targeting defenses, we hypothesize that highly evolvable modular tRNA recognition allows for the escape of viral countermeasures through tRNA substrate specificity switching.

Vibrio cholerae harbours a primary chromosome derived from the monochromosomal ancestor of the Vibrionales (ChrI) and a secondary chromosome derived from a megaplasmid (ChrII). The coordinated segregation of the replication terminus of both chromosomes (TerI and TerII) determines when and where cell division occurs. ChrI encodes a homologue of Escherichia coli MatP, a protein that binds to a DNA motif (matS) that is overrepresented in replication termini. Here, we use a combination of deep sequencing and fluorescence microscopy techniques to show that V. cholerae MatP structures TerI and TerII into macrodomains, targets them to mid-cell during replication, and delays their segregation, thus supporting that ChrII behaves as a bona fide chromosome. We further show that the extent of the segregation delay mediated by MatP depends on the number and local density of matS sites, and is independent of its assembly into tetramers and any interaction with the divisome, in contrast to what has been previously observed in E. coli., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Many complex terpenoids, predominantly isolated from plants and fungi, show drug-like physicochemical properties. Recent advances in genome mining revealed actinobacteria as an almost untouched treasure trove of terpene biosynthetic gene clusters (BGCs). In this study, we characterized a terpene BGC with an unusual architecture. The selected BGC includes, among others, genes encoding a terpene cyclase fused to a truncated reductase domain and a cytochrome P450 monooxygenase (P450) that is split over three gene fragments. Functional characterization of the BGC in a heterologous host led to the identification of several new members of the trans -eunicellane family of diterpenoids, the euthailols, that feature unique oxidation patterns. A combination of bioinformatic analyses, structural modeling studies, and heterologous expression revealed a dual function of the pathway-encoded hypothetical protein that acts as an isomerase and an oxygenase. Moreover, in the absence of other tailoring enzymes, a P450 hydroxylates the eunicellane scaffold at a position that is not modified in other eunicellanes. Surprisingly, both the modifications installed by the hypothetical protein and one of the P450s exhibit partial redundancy. Bioactivity assays revealed that some of the euthailols show growth inhibitory properties against Gram-negative ESKAPE pathogens. The characterization of the euthailol BGC in this study provides unprecedented insights into the partial functional redundancy of tailoring enzymes in complex diterpenoid biosynthesis and highlights hypothetical proteins as an important and largely overlooked family of tailoring enzymes involved in the maturation of complex terpenoids.

A cryptic cluster of genes, known as the ebo cluster, has been found in a variety of genomic contexts among bacteria and algae. In Pseudomonas fluorescens NZI7, the ebo cluster (a.k.a. EDB cluster) is involved in the bacterial repellent mechanism against nematode grazing. In cyanobacteria, the cluster plays a role in the transport of the scytonemin monomer from the cytosol to the periplasm. Despite their broad distribution and interesting phenotypes, neither the pathway nor the functions of the enzymes are known. Here we show that EboD proteins from the ebo clusters in Nostoc punctiforme and Sporocytophaga myxococcoides catalyze the cyclization of mannose 6-phosphate to a novel cyclitol, 2-deoxy-4- epi - scyllo -inosose. The enzyme product is postulated to be a precursor of a signaling molecule or a transporter in the organisms. This study sheds the first light onto ebo/EDB pathways and established a functionally distinct enzyme that extends the diversity of sugar phosphate cyclases.

Herein, we report macroscopic materials formed by the assembly of engineered bacterial spores. Spores were engineered by using a T7-driven expression system to display a high density of pH-responsive self-associating proteins on their surface. The engineered surface protein on the spore surface enabled pH-dependent binding at the protein level and enabled the assembly of granular materials. Mechanical properties remained largely constant with changing pH, but erosion stability was pH-dependent in a manner consistent with the pH-dependent interaction between the engineered surface proteins. Our finding utilizes synthetic biology for the design of macroscopic materials and illuminates the impact of coiled-coil interaction across various length scales.

Shigella is a foodborne enteropathogenic bacteria that causes severe bacillary dysentery in humans. Shigella primarily colonizes the human colon and causes disease via invasion of colon epithelial cells. However, the signal regulatory mechanisms associated with its colonization and pathogenesis in the colon remain poorly defined. Here, we report a leucine-mediated regulatory mechanism that promotes Shigella virulence gene expression and invasion of colon epithelial cells. Shigella in response to leucine, which is highly abundant in the colon, via the leucine-responsive regulator Lrp and the binding of Lrp with leucine induces the expression of a newly identified small RNA SsrV. SsrV then activates the expression of virF and downstream invasion-related virulence genes by increasing the protein level of the LysR-type transcription regulator LrhA, therefore enabling Shigella invasion of colon epithelial cells. Shigella lacking ssrV displays impaired invasion ability. Collectively, these findings suggest that Shigella employs a leucine-responsive environmental activation mechanism to establish colonization and pathogenicity., 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 Ltd. All rights reserved.)

StkP, the Ser/Thr protein kinase of the major human pathogen Streptococcus pneumoniae, monitors cell wall signals and regulates growth and division in response. In vivo, StkP interacts with GpsB, a cell division protein required for septal ring formation and closure, that affects StkP-dependent phosphorylation. Here, we report that although StkP has basal intrinsic kinase activity, GpsB promotes efficient autophosphorylation of StkP and phosphorylation of StkP substrates. Phosphoproteomic analyzes showed that GpsB is phosphorylated at several Ser and Thr residues. We confirmed that StkP directly phosphorylates GpsB in vitro and in vivo, with T79 and T83 being the major phosphorylation sites. In vitro, phosphoablative GpsB substitutions had a lower potential to stimulate StkP activity, whereas phosphomimetic substitutions were functional in terms of StkP activation. In vivo, substitutions of GpsB phosphoacceptor residues, either phosphoablative or mimetic, had a negative effect on GpsB function, resulting in reduced StkP-dependent phosphorylation and impaired cell division. The bacterial two-hybrid assay and co-immunoprecipitation of GpsB from cells with differentially active StkP indicated that increased phosphorylation of GpsB resulted in a more efficient interaction of GpsB with StkP. Our data suggest that GpsB acts as an adaptor that directly promotes StkP activity by mediating interactions within the StkP signaling hub, ensuring StkP recruitment into the complex and substrate specificity. We present a model that interaction of StkP with GpsB and its phosphorylation and dephosphorylation dynamically modulate kinase activity during exponential growth and under cell wall stress of S. pneumoniae, ensuring the proper functioning of the StkP signaling pathway., 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 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The 91 kDa oligomeric ring-shaped ligand binding protein TRAP (trp RNA binding attenuation protein) regulates the expression of a series of genes involved in tryptophan (Trp) biosynthesis in bacilli. When cellular Trp levels rise, the free amino acid binds to sites buried in the interfaces between each of the 11 (or 12, depending on the species) protomers in the ring. Crystal structures of Trp-bound TRAP show the Trp ligands are sequestered from solvent by a pair of loops from adjacent protomers that bury the bound ligand via polar contacts to several threonine residues. Binding of the Trp ligands occurs cooperatively, such that successive binding events occur with higher apparent affinity but the structural basis for this cooperativity is poorly understood. We used solution methyl-TROSY NMR relaxation experiments focused on threonine and isoleucine sidechains, as well as magic angle spinning solid-state NMR 13 C- 13 C and 15 N- 13 C chemical shift correlation spectra on uniformly labeled samples recorded at 800 and 1200 MHz, to characterize the structure and dynamics of the protein. Methyl 13 C relaxation dispersion experiments on ligand-free apo TRAP revealed concerted exchange dynamics on the µs-ms time scale, consistent with transient sampling of conformations that could allow ligand binding. Cross-correlated relaxation experiments revealed widespread disorder on fast timescales. Chemical shifts for methyl-bearing side chains in apo- and Trp-bound TRAP revealed subtle changes in the distribution of sampled sidechain rotameric states. These observations reveal a pathway and mechanism for induced conformational changes to generate homotropic Trp-Trp binding cooperativity., 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 Ltd. All rights reserved.)

ABC transporters are ancient and ubiquitous nutrient transport systems in bacteria and play a central role in defining lifestyles. Periplasmic solute-binding proteins (SBPs) are components that deliver ligands to their translocation machinery. SBPs have diversified to bind a wide range of ligands with high specificity and affinity. However, accurate assignment of cognate ligands remains a challenging problem in SBPs. Urea metabolism plays an important role in the nitrogen cycle; anthropogenic sources account for more than half of global nitrogen fertilizer. We report identification of urea-binding proteins within a large SBP sequence family that encodes diverse functions. By combining genetic linkage between SBPs, ABC transporter components, enzymes or transcription factors, we accurately identified cognate ligands, as we verified experimentally by biophysical characterization of ligand binding and crystallographic determination of the urea complex of a thermostable urea-binding homolog. Using three-dimensional structure information, these functional assignments were extrapolated to other members in the sequence family lacking genetic linkage information, which revealed that only a fraction bind urea. Using the same combined approaches, we also inferred that other family members bind various short-chain amides, aliphatic amino acids (leucine, isoleucine, valine), γ-aminobutyrate, and as yet unknown ligands. Comparative structural analysis revealed structural adaptations that encode diversification in these SBPs. Systematic assignment of ligands to SBP sequence families is key to understanding bacterial lifestyles, and also provides a rich source of biosensors for clinical and environmental analysis, such as the thermostable urea-binding protein identified here., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: ‘Homme W. Hellinga and Malin J. Allert are co-inventors on a patent “Urea biosensors and uses thereof” US 11,906,524.’., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cyclic diguanosine monophosphate (c-di-GMP) is a second messenger in bacteria that regulates multiple biological functions, including biofilm formation, virulence, and intercellular communication. However, c-di-GMP signaling is virtually unknown in economically important filamentous cyanobacteria, Arthrospira. In this study, we predicted 31 genes encoding GGDEF-domain proteins from A. platensis NIES39 as potential diguanylate cyclases (DGCs). Phylogenetic distribution analysis showed five genes (RS09460, RS04865, RS26155, M01840, and E02220) with highly conserved distribution across 25 Arthrospira strains. Adc1 encoded by RS09460 was further characterized as a typical DGC. By establishing the genetic transformation system of Arthrospira, we demonstrated that the overexpression of Adc1 promoted the production of extracellular polymeric substances (EPS), which in turn caused the aggregation of filaments. We also confirmed that RS04865 and RS26155 may encode active DGCs, while enzymatic activity assays showed that proteins encoded by M01840 and E02220 have phosphodiesterase (PDE) activity. Meta-analysis revealed that the expression profiles of RS09460 and RS04865 were unaffected under 31 conditions, suggesting that they may function as conserved genes in maintaining the basal level of c-di-GMP in Arthrospira. In summary, this report will provide the basis for further studies of c-di-GMP signal in Arthrospira., 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.)

Background: Transgenic insect-resistant rice offers an environmentally friendly approach to mitigate yield losses caused by lepidopteran pests, such as stem borers. Bt (Bacillus thuringiensis) genes encode insecticidal proteins and are widely used to confer insect resistance to genetically modified crops. This study investigated the integration, inheritance, and expression characteristics of codon-optimised synthetic Bt genes, cry1C* and cry2A*, in transgenic early japonica rice lines., Methods: The early japonica rice cultivar, Songgeng 9 (Oryza sativa), was transformed with cry1C* or cry2A*, which are driven by the ubi promoter via Agrobacterium tumefaciens-mediated transformation. Molecular analyses, including quantitative PCR (qPCR), enzyme-linked immunosorbent assay (ELISA), and Southern blot analysis were performed to confirm transgene integration, inheritance, transcriptional levels, and protein expression patterns across different tissues and developmental stages., Results: Stable transgenic early japonica lines exhibiting single-copy transgene integration were established. Transcriptional analysis revealed variations in Bt gene expression among lines, tissues, and growth stages, with higher expression levels observed in leaves than in other organs. Notably, cry2A* exhibited consistently higher mRNA and protein levels than cry1C* across all examined tissues and developmental time points. Bt protein accumulation followed the trend of leaves > stem sheaths > young panicles > brown rice, with peak expression during the filling stage in the vegetative tissues., Conclusions: Synthetic cry2A* displayed markedly elevated transcription and translation compared to cry1C* in the transgenic early japonica rice lines examined. Distinct spatiotemporal patterns of Bt gene expression were elucidated, providing insights into the potential insect resistance conferred by these genes in rice. These findings will contribute to the development of insect-resistant japonica rice varieties and facilitate the rational deployment of Bt crops., 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.)

The gut microbiota is a treasure trove of carbohydrate-active enzymes (CAZymes). To explore novel and efficient CAZymes, we analyzed the 4,142 metagenome-assembled genomes (MAGs) of the horse gut microbiota and found the MAG117.bin13 genome (Bacteroides fragilis) contains the highest number of polysaccharide utilisation loci sites (PULs), indicating its high capability for carbohydrate degradation. Bioinformatics analysis indicate that the PULs region of the MAG117.bin13 genome encodes many hypothetical proteins, which are important sources for exploring novel CAZymes. Interestingly, we discovered a hypothetical protein (595 amino acids). This protein exhibits potential CAZymes activity and has a lower similarity to CAZymes, we named it BfLac2275. We purified the protein using prokaryotic expression technology and studied its enzymatic function. The hydrolysis experiment of the polysaccharide substrate showed that the BfLac2275 protein has the ability to degrade α-lactose (156.94 U/mg), maltose (92.59 U/mg), raffinose (86.81 U/mg), and hyaluronic acid (5.71 U/mg). The enzyme activity is optimal at pH 5.0 and 30 ℃, indicating that the hypothetical protein BfLac2275 is a novel and multifunctional CAZymes in the glycoside hydrolases (GHs). These properties indicate that BfLac2275 has broad application prospects in many fields such as plant polysaccharide decomposition, food industry, animal feed additives and enzyme preparations. This study not only serves as a reference for exploring novel CAZymes encoded by gut microbiota but also provides an example for further studying the functional annotation of hypothetical genes in metagenomic assembly genomes., 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.)

Detection methods based on CRISPR/Cas12a have been widely developed in the application of pathogenic microorganisms to guarantee food safety and public health. For sensitive detection, the CRISPR-based strategies are often in tandem with amplification methods. However, that may increase the detection time and the process may introduce nucleic acid contamination resulting in non-specific amplification. Herein, we established a sensitive S. aureus detection strategy based on the CRISPR/Cas12a system combined with DNAzyme. The activity of Cas12a is blocked by extending the spacer of crRNA (bcrRNA) and can be reactivated by Mn 2+ . NH 2 -modified S. aureus-specific aptamer was loaded on the surface of Fe 3 O 4 MNPs (apt-Fe 3 O 4 MNPs) and MnO 2 NPs (apt-MnO 2 NPs) by EDC/NHS chemistry. The S. aureus was captured to form apt-Fe 3 O 4 MNPs/S. aureus/apt-MnO 2 NPs complex and then MnO 2 NPs were etched to release Mn 2+ to activate DNAzyme. The active DNAzyme can cleave the hairpin structure in bcrRNA to recover the activity of the CRISPR/Cas system. By initiating the whole detection process by generating Mn 2+ through nanoparticle etching, we established a rapid detection assay without nucleic acid extraction and amplification process. The proposed strategy has been applied in the ultrasensitive quantitative detection of S. aureus and has shown good performance with an LOD of 5 CFU/mL in 29 min. Besides, the proposed method can potentially be applied to other targets by simply changing the recognition element and has the prospect of developing a universal detection strategy., 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.)

Pseudomonas aeruginosa PA2196 is a TetR family transcriptional repressor. In this study, the deletion of the PA2196 gene caused increased expression of the downstream gene curA (PA2197), which encodes for a NADPH-dependent curcumin/dihydrocurcumin reductase. The PA2196 gene was then identified as curR, and a DNA footprinting assay showed that CurR directly bound to the curA promoter at an imperfect 15-bp inverted repeat, 5'-TAGTTGA-C-TGGTCTA-3'. A curA promoter-lacZ fusion assay and site-directed mutagenesis further demonstrated that the identified CurR binding site plays a crucial role in curA repression by CurR. curA transcription was inducible by sodium hypochlorite (NaOCl) and N-ethylmaleimide (NEM) but not by hydrogen peroxide, organic hydroperoxide, or curcumin. The oxidation and alkylation of CurR by NaOCl and NEM, respectively, resulted in the inactivation of its DNA-binding activity, which induced curA expression. Under the tested conditions, the deletion of either curR or curA did not affect the survival of P. aeruginosa under NaOCl stress in the absence or presence of curcumin., 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.)

Antiviral STANDs (Avs) are bacterial anti-phage proteins evolutionarily related to immune pattern recognition receptors of the NLR family. Type 2 Avs proteins (Avs2) were suggested to recognize the phage large terminase subunit as a signature of phage infection. Here, we show that Avs2 from Klebsiella pneumoniae (KpAvs2) can recognize several different phage proteins as signature for infection. While KpAvs2 recognizes the large terminase subunit of Seuratvirus phages, we find that to protect against Dhillonvirus phages, KpAvs2 recognizes a different phage protein named KpAvs2-stimulating protein 1 (Ksap1). KpAvs2 directly binds Ksap1 to become activated, and phages mutated in Ksap1 escape KpAvs2 defense despite encoding an intact terminase. We further show that KpAvs2 protects against a third group of phages by recognizing another protein, Ksap2. Our results exemplify the evolutionary diversification of molecular pattern recognition in bacterial Avs2, and show that a single pattern recognition receptor evolved to recognize different phage-encoded proteins., Competing Interests: Competing interests R.S. is a scientific cofounder and advisor of BiomX and Ecophage. The other authors declare no competing interests., (© 2024. The Author(s).)

Carbapenem-resistant bacteria (CRB) present a significant global public health concern. Sub-Saharan Africa has borne a heavy burden of CRB with a reported prevalence of up to 60% in some patient populations. es in Africa focus on clinical CRB isolates, with limited data on their spread in the natural environment. Therefore, the purpose of this study was to report the recovery of CRB from Nairobi River surface waters and nearby anthropogenic and zoonotic sources in Nairobi County, Kenya. A total of 336 CRB were recovered from 336 (250 mL) samples, with 230 of the samples (68.5%) producing one or more CRB isolates. CRB were recovered most commonly from untreated sewage influent (100% of 36 samples; 79 total isolates), treated effluent (93% of 118 samples; 116 total isolates), Nairobi River surface waters upstream (100% of 36 samples; 57 total isolates), downstream (100% of 36 samples; 45 total isolates), and way downstream from the wastewater treatment plant (73% of 11 samples; 19 total isolates), slaughterhouse effluent discharges 1.5%, (5/336), animal contact areas 0.9%, (3/336), a manhole sewer from the affluent neighborhood of Karen at 2.7%, (9/336) respectively. The CRB included Escherichia coli (158, 47%), Klebsiella pneumoniae (74, 22%), and Enterobacter spp (43, 13%). Aeromonas spp (29, 9%) Acinetobacter baumannii (12, 3.6%), Citrobacter freundii (7, 2.1%), Pseudomonas aeruginosa (5, 1.5%) and other species (8, 2.4%). CRB genotypes included blaNDM (246, 73.2%), blaKPC (40, 12%), blaVIM (51, 15.2%), blaOXA-48-like (65, 19.3%), blaIMP (15, 4.5%), and blaGES (7, 2.1%). Sixty-nine of the CRB isolates (20.5%) harbored multiple carbapenemase-encoding genes. Our results indicate that clinically important CRB are commonly present in Nairobi River surface water and from nearby wastewater and livestock sources. These pose an important public health threat that requires urgent intervention strategies and additional investigation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Background: 2-O-α-D-glucosyl glycerol (2-αGG) is a valuable ingredient in cosmetics, health-care and food fields. Sucrose phosphorylase (SPase) is a favorable choice for biosynthesis of 2-αGG, while its glucosyl-acceptor affinity and thermodynamic feature remain largely unknown, limiting 2-αGG manufacturing., Results: Here, three SPases were obtained from lactobacilli and bifidobacteria, and the one encoded by Lb. reuteri SDMCC050455 (LrSP) had the best transglucosylation ability, with 2-αGG accounting for 86.01% in the total product. However, the LrSP exhibited an initial forward reaction rate of 11.83/s and reached equilibrium of 56.90% at 110 h, indicating low glycerol affinity and conversion rate. To improve catalytic efficiency, the LrSP was overexpressed in Lb. paracasei BL-SP, of which the intracellular SPase activity increased by 6.67-fold compared with Lb. reuteri SDMCC050455. After chemically permeabilized with Triton X-100, the whole-cell biocatalysis of Lb. paracasei BL-SP was prepared and showed the highest activity, with the initial forward reaction rate improved to 50.17/s and conversion rate risen to 80.79% within 17 h. Using the whole-cell biocatalyst, the final yield of 2-αGG was 203.21 g/L from 1 M sucrose and 1 M glycerol., Conclusion: The food grade strain Lb. paracasei was used for the first time as cell factory to recombinantly express the LrSP and construct a whole-cell biocatalyst for the production of 2-αGG. After condition optimization and cell permeabilization, the whole-cell biocatalyst exhibited 23.89% higher equilibrium conversion and 9.10-fold of productivity compared with the pure enzyme catalytic system. This work would provide a reference for large-scale bioprocess of 2-αGG., Competing Interests: Declarations Ethics approval and consent to participate This article did not involve any experiment on human participants or animals. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

The multifunctional proteins of class 2 CRISPR systems such as Cas9, have been employed to activate cryptic biosynthetic gene clusters (BGCs) in Streptomyces, which represent a large and hidden reservoir of natural products. However, such approaches are not applicable to most Streptomyces strains with reasons to be comprehended. Inspired by the prevalence of the class 1 subtype especially the type I-E CRISPR system in Streptomyces, here we report the development of the type I-E CRISPR system into a series of transcriptional regulation tools. We further demonstrate the effectiveness of such activators in nine phylogenetically distant Streptomyces strains. Using these tools, we successfully activate 13 out of 21 BGCs and lead to the identification and characterization of one polyketide, one Ripp and three alkaloid products. Our work is expected to have a profound impact and to facilitate the discovery of numerous structurally diverse compounds from Streptomyces., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Multidrug-resistant tuberculosis (MDR-TB), defined as resistance to the first-line drugs isoniazid and rifampin, is a growing source of global mortality and threatens global control of tuberculosis disease. The diarylquinoline bedaquiline has recently emerged as a highly efficacious drug against MDR-TB and kills Mycobacterium tuberculosis by inhibiting mycobacterial ATP synthase. However, the mechanisms underlying bedaquiline's efficacy against MDR-TB remain unknown. Here we investigate bedaquiline hyper-susceptibility in drug-resistant Mycobacterium tuberculosis using systems biology approaches. We discovered that MDR clinical isolates are commonly sensitized to bedaquiline. This hypersensitization is caused by several physiological changes induced by deficient catalase activity. These include enhanced accumulation of reactive oxygen species, increased susceptibility to DNA damage, induction of sensitizing transcriptional programs, and metabolic repression of several biosynthetic pathways. In this work we demonstrate how resistance-associated changes in bacterial physiology can mechanistically induce collateral antimicrobial drug sensitivity and reveal druggable vulnerabilities in antimicrobial resistant pathogens., Competing Interests: Competing interests J.J.C. is a co-founder and board member of Phare Bio, a nonprofit venture focused on antibiotic drug development. P.C.B. is a consultant to or holds equity in 10X Genomics, General Automation Lab Technologies/Isolation Bio, Celsius Therapeutics, Next Gen Diagnostics, Cache DNA, Concerto Biosciences, Stately Bio, Ramona Optics, Bifrost Biosystems, and Amber Bio. His laboratory has received research funding from Calico Life Sciences, Merck, and Genentech for unrelated work. None of these interests are connected to this study. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Drug-resistant strains of Mycobacterium tuberculosis are a major global health problem. Resistance to the front-line antibiotic isoniazid is often associated with mutations in the katG-encoded bifunctional catalase-peroxidase. We hypothesise that perturbed KatG activity would generate collateral vulnerabilities in isoniazid-resistant katG mutants, providing potential pathway targets to combat isoniazid resistance. Whole genome CRISPRi screens, transcriptomics, and metabolomics were used to generate a genome-wide map of cellular vulnerabilities in an isoniazid-resistant katG mutant strain of M. tuberculosis. Here, we show that metabolic and transcriptional remodelling compensates for the loss of KatG but in doing so generates vulnerabilities in respiration, ribosome biogenesis, and nucleotide and amino acid metabolism. Importantly, these vulnerabilities are more sensitive to inhibition in an isoniazid-resistant katG mutant and translated to clinical isolates. This work highlights how changes in the physiology of drug-resistant strains generates druggable vulnerabilities that can be exploited to improve clinical outcomes., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Diverse post-translational modifications have been shown to play important roles in regulating protein function in eukaryotes. By contrast, the roles of post-translational modifications in bacteria are not so well understood, particularly as they relate to pathogenesis. Here, we demonstrate post-translational protein modification by covalent addition of lactate to lysine residues (lactylation) in the human pathogen Staphylococcus aureus. Lactylation is dependent on lactate concentration and specifically affects alpha-toxin, in which a single lactylated lysine is required for full activity and virulence in infection models. Given that lactate levels typically increase during infection, our results suggest that the pathogen can use protein lactylation as a mechanism to increase toxin-mediated virulence during infection., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The non-pathogenic Mycoplasma pneumoniae engineered chassis (Mycochassis) has demonstrated the ability to express therapeutic molecules in vitro and to be effective for treatment of lung infectious diseases in in vivo mouse models. However, the expression of heterologous molecules, whether secreted or exposed on the bacterial membrane has not been optimized to ensure sufficient secretion and/or exposure levels to exert a maximum in vivo biological effect. Here, we have improved the currently used secretion signal from MPN142 protein. We found that mutations at P1' position of the signal peptide cleavage site do not abrogate secretion but affect it. Increasing hydrophobicity and mutations at the C-terminal of the signal peptide increases secretion. We tested different lipoprotein signal peptides as possible N-terminal protein anchoring motifs on the Mpn cell surface. Unexpectedly we found that these peptides exhibit variable retention and secretion rates of the protein, with some sequences behaving as full secretion motifs. This raises the question of the biological role of the lipobox motif traditionally thought to anchor membrane proteins without a helical transmembrane domain. These results altogether represent a step forward in chassis optimization, offering different sequences for secretion or membrane retention, which could be used to improve Mycochassis as a delivery vector, and broadening its therapeutic possibilities., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)