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ChemCatChem Dec 2021Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme...
Imine reductases (IREDs) offer biocatalytic routes to chiral amines and have a natural preference for the NADPH cofactor. In previous work, we reported enzyme engineering of the ()-selective IRED from (NADH-IRED-) yielding a NADH-dependent variant with high catalytic efficiency. However, no IRED with NADH specificity and ()-selectivity in asymmetric reductions has yet been reported. Herein, we applied semi-rational enzyme engineering to switch the selectivity of NADH-IRED-. The quintuple variant A241V/H242Y/N243D/V244Y/A245L showed reverse stereopreference in the reduction of the cyclic imine 2-methylpyrroline compared to the wild-type and afforded the ()-amine product with >99 % conversion and 91 % enantiomeric excess. We also report the crystal-structures of the NADPH-dependent ()-IRED- wild-type enzyme and the NADH-dependent NADH-IRED- variant and molecular dynamics (MD) simulations to rationalize the inverted stereoselectivity of the quintuple variant.
PubMed: 35873105
DOI: 10.1002/cctc.202101057 -
Applied and Environmental Microbiology Aug 2021Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase...
Bacteria have two pathways to restart stalled replication forks caused by environmental stresses, error-prone translesion DNA synthesis (TLS) catalyzed by TLS polymerase and error-free template switching catalyzed by RecA, and their competition on the arrested fork affects bacterial SOS mutagenesis. DnaE2 is an error-prone TLS polymerase, and its functions require ImuA and ImuB. Here, we investigated the transcription of , , and in UV-C-irradiated Myxococcus xanthus and found that the induction of occurred significantly earlier than that of the other two genes. Mutant analysis showed that unlike that of or , the deletion of significantly delayed bacterial regrowth and slightly reduced the bacterial mutation frequency and UV resistance. Transcriptomic analysis revealed that the absence of ImuA released the expression of some known SOS genes, including , , , and . Yeast two-hybrid and pulldown analyses proved that ImuA interacts physically with RecA1 besides ImuB. Protein activity analysis indicated that ImuA had no DNA-binding activity but inhibited the DNA-binding and recombinase activity of RecA1. These findings indicate the new role of ImuA in SOS mutagenesis; that is, ImuA inhibits the recombinase activity of RecA1, thereby facilitating SOS mutagenesis in M. xanthus. DnaE2 is responsible for bacterial SOS mutagenesis in nearly one-third of sequenced bacterial strains. However, its mechanism, especially the function of one of its accessory proteins, ImuA, is still unclear. Here, we report that M. xanthus ImuA could affect SOS mutagenesis by inhibiting the recombinase activity of RecA1, which helps to explain the mechanism of DnaE2-dependent TLS and the selection of the two restart pathways to repair the stalled replication fork.
Topics: Bacterial Proteins; DNA; Mutagenesis; Myxococcus xanthus; Rec A Recombinases; SOS Response, Genetics; Two-Hybrid System Techniques
PubMed: 34190612
DOI: 10.1128/AEM.00919-21 -
Nature Communications Jul 2023During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions varies. In rod-shaped Myxococcus...
During cell migration, front-rear polarity is spatiotemporally regulated; however, the underlying design of regulatory interactions varies. In rod-shaped Myxococcus xanthus cells, a spatial toggle switch dynamically regulates front-rear polarity. The polarity module establishes front-rear polarity by guaranteeing front pole-localization of the small GTPase MglA. Conversely, the Frz chemosensory system, by acting on the polarity module, causes polarity inversions. MglA localization depends on the RomR/RomX GEF and MglB/RomY GAP complexes that localize asymmetrically to the poles by unknown mechanisms. Here, we show that RomR and the MglB and MglC roadblock domain proteins generate a positive feedback by forming a RomR/MglC/MglB complex, thereby establishing the rear pole with high GAP activity that is non-permissive to MglA. MglA at the front engages in negative feedback that breaks the RomR/MglC/MglB positive feedback allosterically, thus ensuring low GAP activity at this pole. These findings unravel the design principles of a system for switchable front-rear polarity.
Topics: Myxococcus xanthus; Bacterial Proteins; Cell Movement; Monomeric GTP-Binding Proteins; Cell Polarity
PubMed: 37422455
DOI: 10.1038/s41467-023-39773-y -
Journal of Bacteriology Dec 2022Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene...
Myxococcus xanthus copes with starvation by producing fruiting bodies filled with dormant and stress-resistant spores. Here, we aimed to better define the gene regulatory network associated with Nla28, a transcriptional activator/enhancer binding protein (EBP) and a key regulator of the early starvation response. Previous work showed that Nla28 directly regulates EBP genes that are important for fruiting body development. However, the Nla28 regulatory network is likely to be much larger because hundreds of starvation-induced genes are downregulated in a mutant strain. To identify candidates for direct Nla28-mediated transcription, we analyzed the downregulated genes using a bioinformatics approach. Nine potential Nla28 target promoters (29 genes) were discovered. The results of promoter binding assays, coupled with and mutational analyses, suggested that the nine promoters along with three previously identified EBP gene promoters were indeed targets of Nla28. These results also suggested that Nla28 used tandem, imperfect repeats of an 8-bp sequence for promoter binding. Interestingly, eight of the new Nla28 target promoters were predicted to be intragenic. Based on mutational analyses, the newly identified Nla28 target loci contained at least one gene that was important for starvation-induced development. Most of these loci contained genes predicted to be involved in metabolic or defense-related functions. Using the consensus Nla28 binding sequence, bioinformatics, and expression profiling, 58 additional promoters and 102 genes were tagged as potential Nla28 targets. Among these putative Nla28 targets, functions, such as regulatory, metabolic, and cell envelope biogenesis, were assigned to many genes. In bacteria, starvation leads to profound changes in behavior and physiology. Some of these changes have economic and health implications because the starvation response has been linked to the formation of biofilms, virulence, and antibiotic resistance. To better understand how starvation contributes to changes in bacterial physiology and resistance, we identified the putative starvation-induced gene regulatory network associated with Nla28, a transcriptional activator from the bacterium Myxoccocus xanthus. We determined the mechanism by which starvation-responsive genes were activated by Nla28 and showed that several of the genes were important for the formation of a highly resistant cell type.
Topics: Gene Regulatory Networks; Gene Expression Regulation, Bacterial; Spores, Bacterial; Transcription Factors; DNA-Binding Proteins; Myxococcus xanthus; Bacterial Proteins
PubMed: 36448789
DOI: 10.1128/jb.00265-22 -
Proceedings of the National Academy of... Jul 2017From colony formation in bacteria to wound healing and embryonic development in multicellular organisms, groups of living cells must often move collectively. Although...
From colony formation in bacteria to wound healing and embryonic development in multicellular organisms, groups of living cells must often move collectively. Although considerable study has probed the biophysical mechanisms of how eukaryotic cells generate forces during migration, little such study has been devoted to bacteria, in particular with regard to the question of how bacteria generate and coordinate forces during collective motion. This question is addressed here using traction force microscopy. We study two distinct motility mechanisms of , namely, twitching and gliding. For twitching, powered by type-IV pilus retraction, we find that individual cells exert local traction in small hotspots with forces on the order of 50 pN. Twitching bacterial groups also produce traction hotspots, but with forces around 100 pN that fluctuate rapidly on timescales of <1.5 min. Gliding, the second motility mechanism, is driven by lateral transport of substrate adhesions. When cells are isolated, gliding produces low average traction on the order of 1 Pa. However, traction is amplified approximately fivefold in groups. Advancing protrusions of gliding cells push, on average, in the direction of motion. Together, these results show that the forces generated during twitching and gliding have complementary characters, and both forces have higher values when cells are in groups.
Topics: Bacterial Adhesion; Bacterial Proteins; Biofilms; Fimbriae, Bacterial; Microscopy; Models, Biological; Motion; Movement; Myxococcus xanthus; Pressure; Stress, Mechanical; Time Factors
PubMed: 28655845
DOI: 10.1073/pnas.1621469114 -
Frontiers in Microbiology 2022Myxobacteria exhibit a variety of complex social behaviors that all depend on coordinated movement of cells on solid surfaces. The cooperative nature of cell movements...
Myxobacteria exhibit a variety of complex social behaviors that all depend on coordinated movement of cells on solid surfaces. The cooperative nature of cell movements is known as social (S)-motility. This system is powered by cycles of type IV pili (Tfp) extension and retraction. Exopolysaccharide (EPS) also serves as a matrix to hold cells together. Here, we characterized a new S-motility gene in . This mutant is temperature-sensitive (Ts) for S-motility; however, Tfp and EPS are made. A 1 bp deletion was mapped to the MXAN_4099 locus and the gene was named . Null mutations in exhibit a synthetic enhanced phenotype with a null mutation, a previously characterized S-motility gene that exhibits a similar Ts phenotype. Our results suggest that SglS and SglT contribute toward Tfp function at high temperatures in redundant pathways. However, at low temperatures only one pathway is necessary for wild-type S-motility, while in the double mutant, motility is nearly abolished at low temperatures. Interestingly, the few cells that do move do so with a high reversal frequency. We suggest SglS and SglT play conditional roles facilitating Tfp retraction and hence motility in .
PubMed: 35586861
DOI: 10.3389/fmicb.2022.879090 -
Current Opinion in Microbiology Jun 2024Myxococcus relies on motility to efficiently invade and predate a prey colony. Upon contact with prey, Myxococcus temporarily halts its motility and initiates prey cell... (Review)
Review
Myxococcus relies on motility to efficiently invade and predate a prey colony. Upon contact with prey, Myxococcus temporarily halts its motility and initiates prey cell lysis, which involves two contact-dependent predatory machineries, the Kil system and the needleless T3SS*. Predatory cells grow as they invade and feed on prey cells. When dividing, Myxococcus cells systematically pause their movements before division. This highlights a high level of co-ordination between motility and contact-dependent killing but also with cell division. In this review, we give an overview of the different nanomachines used by Myxococcus to move on surfaces, kill by contact, and divide, and we discuss the potential regulatory mechanisms at play during these different processes.
PubMed: 38843560
DOI: 10.1016/j.mib.2024.102492 -
Frontiers in Microbiology 2024This study aimed to study the characterization and the potential lipid-lowering effects of new isolated lactic acid bacteria from the feces of healthy adult cats.
INTRODUCTION
This study aimed to study the characterization and the potential lipid-lowering effects of new isolated lactic acid bacteria from the feces of healthy adult cats.
METHODS
We collected 85 cat fecal samples, isolated, screening lactic acid bacteria strains from samples, and investigated their and biological properties.
RESULTS
A total of 221 lactic acid bacteria strains were isolated from 85 cat fecal samples. Sixteen strains with calcium dissolution rings greater than 1 mm were identified and selected for further characterization. Three lactic acid bacteria strains, L-27-2, L-14-1, and , were identified as showing the most promising rates of cholesterol degradation (greater than 20%) and bacteriostatic radius (over 15 mm). These three strains exhibited robust growth and adherence to epithelial cells, along with adaptability to low pH (greater than 70%) and high bile salt conditions (greater than 60%), and remarkable cholesterol degradation and anti-pathogen activity. Sixteen mice were fed a high-fat diet (HFD) from 4 to 8 weeks of age, while a control group of the same size received a normal diet (ND). At 8 weeks of age, serum, feces and adipose tissue were collected. The results showed that, compared with mice fed an HFD diet alone, all mice fed an HFD diet plus lactic acid bacteria could decrease weight gain. < 0.05 and the pathological changes of adipose tissue were alleviated. In addition, mice fed L-14-1 and F203 showed abdominal fat accumulation decreased ( < 0.05). Mice fed L-27-2 showed serum and liver triglyceride (TG) decreased ( < 0.05) and mice fed F203 showed serum high density lipoprotein cholesterol (HDL-C) increased ( < 0.01). mice fed L-27-2 and L-14-1 showed inflammatory cytokines (IL-6) was decreased ( < 0.01) Analysis of the fecal microbiota of mice fed these three lactic acid bacteria strains revealed alterations in the gut microbial community. There were common changes in intestinal microbes in mice fed these three lactic acid bacteria: (1) decreased; (2) increased; (3) decreased. The microbes mentioned are all part of the core intestinal flora.
DISCUSSION
This study provided three potential lactic acid bacteria for alleviating animal obesity and inflammation.
PubMed: 38721604
DOI: 10.3389/fmicb.2024.1392864 -
Frontiers in Microbiology 2023Myxobacteria are part of the phylum Myxococcota, encompassing four orders. Most of them display complex lifestyles and broad predation profiles. However, metabolic...
Myxobacteria are part of the phylum Myxococcota, encompassing four orders. Most of them display complex lifestyles and broad predation profiles. However, metabolic potential and predation mechanisms of different myxobacteria remains poorly understood. Herein, we used comparative genomics and transcriptomics to analyze metabolic potentials and differentially expressed gene (DEG) profiles of monoculture (Mx) compared to coculture with (MxE) and (MxM) prey. The results showed that myxobacteria had conspicuous metabolic deficiencies, various protein secretion systems (PSSs) and the common type II secretion system (T2SS). RNA-seq data demonstrated that overexpressed the potential predation DEGs, particularly those encoding T2SS, the tight adherence (Tad) pilus, different secondary metabolites (myxochelin A/B, myxoprincomide, myxovirescin A1, geosmin and myxalamide), glycosyl transferases and peptidase during predation. Furthermore, the myxalamide biosynthesis gene clusters, two hypothetical gene clusters and one arginine biosynthesis clusters were highly differential expressed in MxE versus MxM. Additionally, homologue proteins of the Tad (kil) system and five secondary metabolites were in different obligate or facultative predators. Finally, we provided a working model for exhibiting multiple predatory strategies when prey on and . These results might spur application-oriented research on the development of novel antibacterial strategies.
PubMed: 37213496
DOI: 10.3389/fmicb.2023.1146523 -
Malaria Journal Feb 2021Insecticide resistance poses a growing challenge to malaria vector control in Kenya and around the world. Following evidence of associations between the mosquito...
BACKGROUND
Insecticide resistance poses a growing challenge to malaria vector control in Kenya and around the world. Following evidence of associations between the mosquito microbiota and insecticide resistance, the microbiota of Anopheles gambiae sensu stricto (s.s.) from Tulukuyi village, Bungoma, Kenya, with differing permethrin resistance profiles were comparatively characterized.
METHODS
Using the CDC bottle bioassay, 133 2-3 day-old, virgin, non-blood fed female F progeny of field-caught An. gambiae s.s. were exposed to five times (107.5 µg/ml) the discriminating dose of permethrin. Post bioassay, 50 resistant and 50 susceptible mosquitoes were subsequently screened for kdr East and West mutations, and individually processed for microbial analysis using high throughput sequencing targeting the universal bacterial and archaeal 16S rRNA gene.
RESULTS
47 % of the samples tested (n = 133) were resistant, and of the 100 selected for further processing, 99 % were positive for kdr East and 1 % for kdr West. Overall, 84 bacterial taxa were detected across all mosquito samples, with 36 of these shared between resistant and susceptible mosquitoes. A total of 20 bacterial taxa were unique to the resistant mosquitoes and 28 were unique to the susceptible mosquitoes. There were significant differences in bacterial composition between resistant and susceptible individuals (PERMANOVA, pseudo-F = 2.33, P = 0.001), with presence of Sphingobacterium, Lysinibacillus and Streptococcus (all known pyrethroid-degrading taxa), and the radiotolerant Rubrobacter, being significantly associated with resistant mosquitoes. On the other hand, the presence of Myxococcus, was significantly associated with susceptible mosquitoes.
CONCLUSIONS
This is the first report of distinct microbiota in An. gambiae s.s. associated with intense pyrethroid resistance. The findings highlight differentially abundant bacterial taxa between resistant and susceptible mosquitoes, and further suggest a microbe-mediated mechanism of insecticide resistance in mosquitoes. These results also indicate fixation of the kdr East mutation in this mosquito population, precluding further analysis of its associations with the mosquito microbiota, but presenting the hypothesis that any microbe-mediated mechanism of insecticide resistance would be likely of a metabolic nature. Overall, this study lays initial groundwork for understanding microbe-mediated mechanisms of insecticide resistance in African mosquito vectors of malaria, and potentially identifying novel microbial markers of insecticide resistance that could supplement existing vector surveillance tools.
Topics: Animals; Anopheles; Female; Insecticide Resistance; Insecticides; Kenya; Microbiota; Mosquito Control; Mosquito Vectors; Permethrin
PubMed: 33557825
DOI: 10.1186/s12936-021-03606-4