rRNA operon improves species-level classification of bacteria and microbial community analysis compared to 16S rRNA.

Precise identification of species is fundamental in microbial genomics and is crucial for understanding the microbial communities. While the 16S rRNA gene, particularly its V3-V4 regions, has been extensively employed for microbial identification, however has limitations in achieving species-level resolution. Advancements in long-read sequencing technologies have highlighted the rRNA operon as a more accurate marker for microbial classification and analysis than the 16S rRNA gene. This study aims to compare the accuracy of species classification and microbial community analysis using the rRNA operon versus the 16S rRNA gene. We evaluated the species classification accuracy of the rRNA operon,16S rRNA gene, and 16S rRNA V3-V4 regions using a BLAST-based method and a k-mer matching-based method with public data available from NCBI. We further performed simulations to model microbial community analysis. We accessed the performance using each marker in community composition estimation and differential abundance analysis. Our findings demonstrate that the rRNA operon offers an advantage over the 16S rRNA gene and its V3-V4 regions for species-level classification within the genus. When applied to microbial community analysis, the rRNA operon enables a more accurate determination of composition. Using the rRNA operon yielded more reliable results in differential abundance analysis as well. IMPORTANCE: We quantitatively demonstrated that the rRNA operon outperformed the 16S rRNA and its V3-V4 regions in accuracy for both individual species identification and species-level microbial community analysis. Our findings can provide guidelines for selecting appropriate markers in the field of microbial research.

Fatty acid synthesis promoted by PA1895-1897 operon delays quorum sensing activation in Pseudomonas aeruginosa.

PA1895-1897 is a quorum sensing (QS) operon regulated by the anti-activator LuxR homologue QscR in Pseudomonas aeruginosa. We aimed to investigate its impact on bacterial metabolism, and whether it contributes to the delayed QS activation. We performed liquid chromatograph-mass spectrometer-based metabolomics using wildtype PAO1, PA1895-1897-knockout mutant, and mutant with pJN105.PA1895-1897 overexpression vector. The impact of metabolites on QS signaling molecule (3OC12-HSL and C4-HSL) concentrations, pyocyanin production, and QS gene (lasR, lasI, rhlR, and rhlI) expression was examined. Metabolomics analysis found that fatty acid biosynthesis had the highest fold enrichment among all metabolic pathways in PA1895-1897-overexpressed mutants. Among these enriched fatty acids, palmitoleic acid and acetic acid were the predominantly abundant ones that significantly affected by PA1895-1897 operon. When different doses of exogenous palmitoleic acid or acetic acid were added to the cultures of PA1895-1897 knockout mutants, their levels of 3OC12-HSL, C4-HSL, and pyocyanin were decreased in a dose-dependent manner. High doses of palmitoleic acid and acetic acid suppressed the mRNA expression of lasR, rhlR, and rhlI. Inhibition of fatty acid biosynthesis increased the production of 3OC12-HSL, C4-HSL, and pyocyanin in PA1895-1897-overexpressed cultures. Our data suggest that fatty acid synthesis is promoted by PA1895-1897 operon, and contributes the delayed expression of QS phenotypes, furthering the understanding about the regulation of bacterial QS activation.

Double-Wing Motif Protein is a Novel Biofilm Regulatory Factor of the Plant Disease Biocontrol Agent, Bacillus subtilis.

Transposon mutagenesis screening of Bacillus subtilis YB-1471, a novel rhizosphere biocontrol agent of Fusarium crown rot (FCR) of wheat, resulted in the identification of orf04391, linked to reduced biofilm formation. The gene encodes a protein possessing a putative tertiary structure of a "double-wing" DNA-binding domain. Expression of orf04391 increased during biofilm development in stationary cultures and during rapid growth in shaking cultures. An orf04391 deletion strain showed reduced biofilm production related to lower levels of the extracellular matrix, and the mutant also had reduced sporulation, adhesion, root colonization, and FCR biocontrol efficiency. Transcriptome analysis of YB-1471 and Δorf04391 in stationary culture showed that the loss of orf04391 resulted in altered expression of numerous genes, including sinI, an initiator of biofilm formation. DNA binding was shown with his-tagged Orf04391 binding to the sinIR operon in vivo and in vitro. Orf04391 appears to be a transcriptional regulator of biofilm formation in B. subtilis through the Spo0A-SinI/SinR pathway.

Blending of selected yeast extract and peptone for inducible and constitutive protein production in Escherichia coli using the pET system.

pET vectors allow inducible expression of recombinant proteins in Escherichia coli. In this system, isopropyl ß-d-1-thiogalactopyranoside (IPTG) drives lacUV5 promoter to produce T7 RNA polymerase, simultaneously releasing the suppression of T7lac promoter. T7 RNA polymerase then strongly transcribes the target gene. A lac repressor encoded by lacI in the vector represses the promoters. Despite stringent repression and inducible expression achieved with the pET system, unexpected leaky expression can occur without IPTG induction. Here, by evaluating leaky expression in recombinant cells cultured in various Luria-Bertani (LB) media, prepared using yeast extract and peptone from different suppliers, as well as in five commercial premix-LB media, we confirmed the presence of unknown lac inducers in LB. To explore these inducers, we examined E. coli growth in media comprising yeast extract or peptone. At 4% concentration, five commercial yeast extract and six peptone samples individually allowed E. coli growth equivalent to that in LB medium. We determined the luciferase activity of the luxCDABE operon in the pET vector under these conditions. The presence of different concentrations of inducers was detected in both the yeast extract and peptone. Furthermore, we blended yeast extract and peptone with low or high concentrations of lac inducers. The low-expression blend, used as a basal medium before IPTG addition, allowed leak-free, tightly controlled expression. The high-expression blend was used for constitutive high-expression and pET induction with the basal medium, in lieu of IPTG. These blended media can be used for well-controlled inducible and constitutive expression using the pET system.

Exploring Levansucrase Operon Regulating Levan-Type Fructooligosaccharides Production in Priestia koreensis HL12.

Levan biopolymer and levan-type fructooligosaccharides (L-FOSs) are ß-2,6-linked fructans that have been used as non-digestible dietary fiber and prebiotic oligosaccharides in food and cosmeceutical applications. In this study, we explore the operon responsible for levan and lL-FOSs production in Priestia koreensis HL12. Presented is the first genomic perspective on sucrose utilization and the levan biosynthesis pathway in this bacterium. Regarding sequence annotation, the putative levansucrase operon responsible for ß-2,6-linked fructan was identified in the genome of strain HL12, and comprises sacB levansucrase gene belonging to GH68, located adjacent to levB endo-levanase gene, which belongs to GH32. Importantly, sugars related with the levan biosynthesis pathway are proposed to be transported via 3 types of transportation systems, including multiple ABCSugar and glucose/H+ transporters, as well as glucoseand fructose-specific PTS systems. Based on product profile analysis, the HL12 strain exhibited high efficiency in levan production from high sucrose concentration (300 g/l), achieving the highest yield of 127 g/l (equivalent to 55% conversion based on sucrose consumption), together with short-chain L-FOSs (DP3-5) and long-chain L-FOSs with respective size larger than DP6 after 48 h incubation. These findings highlight the potential of P. koreensis HL12 as a whole-cell biocatalyst for producing levan and L-FOSs, and underscore its novelty in converting sugars into high-value-added products for diverse commercial and industrial applications.

Temporal changes in the mouse gut bacteriota influenced by host sex, diet, and exercise.

The gut microbiota plays an important role in host physiology. However, the effects of host sex, lifestyle, and temporal influences on the bacterial community within the gut remain ill-defined. To address this gap, we evaluated 56 male and female mice over a 10-week study to assess the effects of sex, diet, and exercise on gut community dynamics. Mice were randomly assigned to high-fat or control diet feeding and had free access to running wheels or remained sedentary throughout the study period. The fecal bacterial community was characterized by rRNA operon amplicon profiling via nanopore sequencing. Differential abundance testing indicated that ~200 bacterial taxa were significantly influenced by sex, diet, or exercise (4.2% of total community), which also changed over time (82 taxa, 1.7% of total community). Phylogenetic analysis of taxa closely related to Dysosmobacter welbionis and several members of the family Muribaculaceae were examined more closely and demonstrated distinct species/strain-level sub-clustering by host sex, diet, and exercise. Collectively, this data suggests that sex and lifestyle can alter the gut bacteriota at the species/strain-level which may play a role in host health. These results also highlight the need for improved characterization methods to survey microbial communities at finer taxonomic resolution.

AraC Functional Suppressors of Mutations in the C-Terminal Domain of the RpoA Subunit of the Escherichia coli RNA Polymerase.

In E. coli, transcriptional activation is often mediated by the C-terminal domain of RpoA, the α subunit of RNA polymerase. Random mutations that prevent activation of the arabinose PBAD promoter are clustered in the RpoA C-terminal domain (α-CTD). We have isolated functional suppressors of rpoA α-CTD mutations that map to araC, the main transcriptional regulator of ara genes, or to the PBAD promoter. No mutation was found in the DNA regulatory region between araC and PBAD. Most suppressors that improve PBAD transcription are localized to the N-terminal domain of AraC. One class of araC mutations generates substitutions in the core of the N-terminal domain, suggesting that they affect its conformation. Other suppressors localize to the flexible N-terminal arm of AraC. Some, but not all, suppressors confer an arabinose constitutive phenotype. Suppression by both classes of araC mutations requires the α-CTD to stimulate expression from PBAD. Surprisingly, in rpoA+ strains lacking Crp, the cAMP receptor protein, these araC mutations largely restore arabinose gene expression and can essentially bypass Crp activation. Thus, the N-terminal domain of AraC exhibits at least three distinct activities: dimerization, arabinose binding, and transcriptional activation. Finally, one mutation maps to the AraC C-terminal domain and can synergize with AraC mutations in the N-terminal domain.

Pseudomonas aeruginosa mucinous phenotypes and algUmucABD operon mutant characteristics obtained from inpatients with bronchiectasis and their correlation with acute aggravation.

Objective: Although the mechanism is unclear, Pseudomonas aeruginosa (PA) infection directly affects the frequency of acute exacerbations in patients with bronchiectasis. The aims of this article are to analyze the genetic mutation characteristics of the algUmucABD operon in PA, isolated from hospitalized patients with bronchiectasis, and to explore independent risk factors for frequent acute exacerbations of bronchiectasis. Methods: Based on the number of acute exacerbations that occurred in the past year, these patients with bronchiectasis were divided into those with frequent acute exacerbations (Group A) and those with non-frequent acute exacerbations (Group B). We identified the distribution of mucoid phenotypes (MPs) and alginate morphotypes (AMs) in PA, and classified them into I-IV categories based on their different AMs; otherwise, the gene mutation types (GMTs) of the algUmucABD operon were tested. Subsequently, the relationship between GMT, MP, and AM and the independent risk factors for frequent acute exacerbations in patients with bronchiectasis were explored. Results: A total of 93 patients and 75 PA strains, from January 2019 to August 2023, were included in this study. The MP and AM distributions of PA were as follows: 64 strains (85.33%) of mucoid (the AMs were 38 strains of type I, 3 strains of type II, and 23 strains of type IV) and 11 strains of non-mucoid (the AM was type III only). Mucoid PA with algU, mucA, mucB, and mucD mutations accounted for 19.61%, 74.51%, 31.37%, and 50.98%, respectively. GMT was divided into the following: mucA mutations only, mucA combined with other gene mutations, other gene mutations without mucA mutations, and without gene mutations. In 91.7% of PA with type I of AM, only mucA mutations occurred, and in both separate MP and AM, the GMT differences were statistically significant. Lastly, the number of lung lobes with bronchiectasis and the number of PA with mucA mutations only were the independent risk factors for frequent acute exacerbations. Conclusion: The mucA mutation was primarily responsible for the mucoid of MP and type I of AM in PA, and it was also an independent risk factor for frequent exacerbations of bronchiectasis.

Role of glycogen metabolism in Clostridioides difficile virulence.

Glycogen plays a vital role as an energy reserve in various bacterial and fungal species. Clostridioides difficile possesses a glycogen metabolism operon that contains genes for both glycogen synthesis and utilization. In our investigation, we focused on understanding the significance of glycogen metabolism in the physiology and pathogenesis of C. difficile. To explore this, we engineered a C. difficile JIR8094 strain lacking glycogen synthesis capability by introducing a group II intron into the glgC gene, the operon's first component. Quantification of intracellular glycogen levels validated the impact of this modification. Interestingly, the mutant strain exhibited a 1.5-fold increase in toxin production compared with the parental strain, without significant changes in the sporulation rate. Our analysis also revealed that wild-type C. difficile spores contained glycogen, whereas spores from the mutant strain lacking stored glycogen showed increased sensitivity to physical and chemical treatments and had a shorter storage life. By suppressing glgP expression, the gene coding for glycogen-phosphorylase, via CRISPRi, we demonstrated that glycogen accumulation but not the utilization is needed for spore resilience in C. difficile. Transmission electron microscopy analysis revealed a significantly lower core/cortex ratio in glgC mutant strain spores. In hamster challenge experiments, both the parental and glgC mutant strains colonized hosts similarly; however, the mutant strain failed to induce infection relapse after antibiotic treatment cessation. These findings highlight the importance of glycogen metabolism in C. difficile spore resilience and suggest its role in disease relapse.IMPORTANCEThis study on the role of glycogen metabolism in Clostridioides difficile highlights its critical involvement in the pathogen's energy management, its pathogenicity, and its resilience. Our results also revealed that glycogen presence in spores is pivotal for their structural integrity and resistance to adverse conditions, which is essential for their longevity and infectivity. Importantly, the inability of the mutant strain to cause infection relapse in hamsters post-antibiotic treatment pinpoints a potential target for therapeutic interventions, highlighting the importance of glycogen in disease dynamics. This research thus significantly advances our understanding of C. difficile physiology and pathogenesis, offering new avenues for combating its persistence and recurrence.

The MarR family regulator RmaH mediates acid tolerance of Lactococcus lactis through regulating peptidoglycan modification genes.

Lactococcus lactis, widely used in the food fermentation industry, has developed various ways to regulate acid adaptation in the process of evolution. The investigation into how peptidoglycan (PG) senses and responds to acid stress is an expanding field. Here, we addressed the regulation of murT-gatD genes which are responsible for the amidation of PG D-Glu. We found that lactic acid stress reduced murT-gatD expression, and overexpressing these genes notably decreased acid tolerance of L. lactis NZ9000, possibly due to a reduction in PG's negative charge, facilitating the influx of extracellular protons into the cell. Subsequently, using a combination of DNA pull-down assay and electrophoretic mobility shift assay (EMSA), we identified a novel MarR family regulator, RmaH, as an activator of murT-gatD transcription. Further MEME motif prediction, EMSA verification and fluorescent protein reporter assay showed that RmaH directly bound to the DNA motif 5'-KGVAWWTTTTGCT-3' located in the upstream region of murT-gatD. Beyond the mechanistic investigation of RmaH activation of murT-gatD, this study provides new insight into how peptidoglycan modification is regulated and responds to lactic acid stress.

Design of Thermoresponsive Genetic Controls with Minimal Heat-Shock Response.

As temperature serves as a versatile input signal, thermoresponsive genetic controls have gained significant interest for recombinant protein production and metabolic engineering applications. The conventional thermoresponsive systems normally require the continuous exposure of heat stimuli to trigger the prolonged expression of targeted genes, and the accompanied heat-shock response is detrimental to the bioproduction process. In this study, we present the design of thermoresponsive quorum-sensing (ThermoQS) circuits to make Escherichia coli record transient heat stimuli. By conversion of the heat input into the accumulation of quorum-sensing molecules such as acyl-homoserine lactone derived from Pseudomonas aeruginosa, sustained gene expressions were achieved by a minimal heat stimulus. Moreover, we also demonstrated that we reprogrammed the E. coli Lac operon to make it respond to heat stimuli with an impressive signal-to-noise ratio (S/N) of 15.3. Taken together, we envision that the ThermoQS systems reported in this study are expected to remarkably diminish both design and experimental expenditures for future metabolic engineering applications.

Bioremediation of heavy metals by an unexplored bacterium, Pseudoxanthomonas mexicana strain GTZY isolated from aerobic-biofilm wastewater system.

We prompted to characterize a wastewater bacterium, Pseudoxanthomonas mexicana GTZY, that efficiently transforms toxic mercury and arsenic, explores its bioremediation capability, and reveals their relevant gene resistance operons. The isolated strain was characterized by its phylogenetic, biochemical, and phenotypic properties. The strain GTZY potentially removed 84.3% of mercury and their mercury volatilization (Hg(II) to Hg(0)) was confirmed using the X-ray film method, and its respective merA gene was PCR amplified. In addition, strain GTZY efficiently removed arsenate (68.5%) and arsenite (63.2%), and showed resistance up to > 175 and > 55 mM, respectively. Their genomic annotations disclosed the linkage of Tn2-transposon and int1 in both ends of mer operon (merAPTR). The co-existence of arsP and arsH proteins in its intrinsic ars operon (arsCPRH) was extremely diverse from its ancestral species. We believe that the mercury resistance-conferring mer operon of P. mexicana GTZY presumably derived horizontally from other species in the reactor, while the arsenic resistance-conferring intrinsic ars operon was highly diversified and evolved from its ancestral species. By considering the potential of the strain GTZY to transform heavy metals, this can be used to recover contaminated sites.

A small intestinal bile acid modulates the gut microbiome to improve host metabolic phenotypes following bariatric surgery.

Bariatric surgical procedures such as sleeve gastrectomy (SG) provide effective type 2 diabetes (T2D) remission in human patients. Previous work demonstrated that gastrointestinal levels of the bacterial metabolite lithocholic acid (LCA) are decreased after SG in mice and humans. Here, we show that LCA worsens glucose tolerance and impairs whole-body metabolism. We also show that taurodeoxycholic acid (TDCA), which is the only bile acid whose concentration increases in the murine small intestine post-SG, suppresses the bacterial bile acid-inducible (bai) operon and production of LCA both in vitro and in vivo. Treatment of diet-induced obese mice with TDCA reduces LCA levels and leads to microbiome-dependent improvements in glucose handling. Moreover, TDCA abundance is decreased in small intestinal tissue from T2D patients. This work reveals that TDCA is an endogenous inhibitor of LCA production and suggests that TDCA may contribute to the glucoregulatory effects of bariatric surgery.

BlpU is a broad-spectrum bacteriocin in Streptococcus thermophilus.

Streptococcus thermophilus strain B59671 naturally produces thermophilin 110, a broad-spectrum bacteriocin encoded within the bacteriocin-like peptide (blp) gene cluster, and thermophilin 13 from a separate chromosomal locus. Analysis of the blp gene cluster revealed two genes, blpU and blpK, as potentially encoding bacteriocins. Deletion of blpK from the B59671 chromosome did not result in a loss of antimicrobial activity against either S. thermophilus ST113 or Pediococcus acidilactici F. A deletion mutant of blpU could not be generated in B59671, but the mature BlpU peptide obtained through overexpression in E. coli BL21 or chemical synthesis inhibited the growth of S. thermophilus strains, Streptococcus mutans UA159, P. acidilactici F, and Listeria innocua GV9 L-S, evidencing as a broad-spectrum bacteriocin that does not require modification for activity. This study also showed that the transcription of blpU was approximately 16-fold higher in B59671 than in an induced culture of S. thermophilus LMD-9, which produces a blp-encoded bacteriocin. The increased expression of BlpU in B59671 may explain the unique antimicrobial spectrum associated with this strain. Additionally, it was shown that a blpC deletion mutant of B59671, which prevents expression of BlpU and BlpK, inhibited the growth of other S. thermophilus strains and Bacillus cereus, suggesting that thermophilin 13 produced by B59671 possessed both intra- and interspecies antimicrobial activity. While this study confirmed that BlpU can function as an independent antimicrobial peptide, further studies are required to determine if BlpK can function independently as a broad-spectrum antimicrobial.

Integrative in vivo analysis of the ethanolamine utilization bacterial microcompartment in Escherichia coli.

Bacterial microcompartments (BMCs) are self-assembling protein megacomplexes that encapsulate metabolic pathways. Although approximately 20% of sequenced bacterial genomes contain operons encoding putative BMCs, few have been thoroughly characterized, nor any in the most studied Escherichia coli strains. We used an interdisciplinary approach to gain deep molecular and functional insights into the ethanolamine utilization (Eut) BMC system encoded by the eut operon in E. coli K-12. The eut genotype was linked with the ethanolamine utilization phenotype using deletion and overexpression mutants. The subcellular dynamics and morphology of the E. coli Eut BMCs were characterized in cellula by fluorescence microscopy and electron (cryo)microscopy. The minimal proteome reorganization required for ethanolamine utilization and the in vivo stoichiometric composition of the Eut BMC were determined by quantitative proteomics. Finally, the first flux map connecting the Eut BMC with central metabolism in cellula was obtained by genome-scale modeling and 13C-fluxomics. Our results reveal that contrary to previous suggestions, ethanolamine serves both as a nitrogen and a carbon source in E. coli K-12, while also contributing to significant metabolic overflow. Overall, this study provides a quantitative molecular and functional understanding of the BMCs involved in ethanolamine assimilation by E. coli.IMPORTANCEThe properties of bacterial microcompartments make them an ideal tool for building orthogonal network structures with minimal interactions with native metabolic and regulatory networks. However, this requires an understanding of how BMCs work natively. In this study, we combined genetic manipulation, multi-omics, modeling, and microscopy to address this issue for Eut BMCs. We show that the Eut BMC in Escherichia coli turns ethanolamine into usable carbon and nitrogen substrates to sustain growth. These results improve our understanding of compartmentalization in a widely used bacterial chassis.

Horizontal gene transfer of the Mer operon is associated with large effects on the transcriptome and increased tolerance to mercury in nitrogen-fixing bacteria.

BACKGROUND: Mercury (Hg) is highly toxic and has the potential to cause severe health problems for humans and foraging animals when transported into edible plant parts. Soil rhizobia that form symbiosis with legumes may possess mechanisms to prevent heavy metal translocation from roots to shoots in plants by exporting metals from nodules or compartmentalizing metal ions inside nodules. Horizontal gene transfer has potential to confer immediate de novo adaptations to stress. We used comparative genomics of high quality de novo assemblies to identify structural differences in the genomes of nitrogen-fixing rhizobia that were isolated from a mercury (Hg) mine site that show high variation in their tolerance to Hg. RESULTS: Our analyses identified multiple structurally conserved merA homologs in the genomes of Sinorhizobium medicae and Rhizobium leguminosarum but only the strains that possessed a Mer operon exhibited 10-fold increased tolerance to Hg. RNAseq analysis revealed nearly all genes in the Mer operon were significantly up-regulated in response to Hg stress in free-living conditions and in nodules. In both free-living and nodule environments, we found the Hg-tolerant strains with a Mer operon exhibited the fewest number of differentially expressed genes (DEGs) in the genome, indicating a rapid and efficient detoxification of Hg from the cells that reduced general stress responses to the Hg-treatment. Expression changes in S. medicae while in bacteroids showed that both rhizobia strain and host-plant tolerance affected the number of DEGs. Aside from Mer operon genes, nif genes which are involved in nitrogenase activity in S. medicae showed significant up-regulation in the most Hg-tolerant strain while inside the most Hg-accumulating host-plant. Transfer of a plasmid containing the Mer operon from the most tolerant strain to low-tolerant strains resulted in an immediate increase in Hg tolerance, indicating that the Mer operon is able to confer hyper tolerance to Hg. CONCLUSIONS: Mer operons have not been previously reported in nitrogen-fixing rhizobia. This study demonstrates a pivotal role of the Mer operon in effective mercury detoxification and hypertolerance in nitrogen-fixing rhizobia. This finding has major implications not only for soil bioremediation, but also host plants growing in mercury contaminated soils.

Using origami and Shrinky Dinks to create active learning activities to tackle two microbiology concepts: cell structure differences and operon regulation.

This paper presents two low-cost hands-on activities designed to enhance student understanding and address the pedagogical challenges faced by microbiology professors in teaching concepts related to cell structure and gene regulation. In the first activity, we used Shrinky Dinks and Jeopardy-style game questions to explore the differences between prokaryotic and eukaryotic cells. Students have to collect pieces and physically build their cell models. The second activity uses origami organelles sets from Edvotek to illustrate the regulation of gene expression in the lac and trp operons, incorporating mutation scenarios for analysis. The intended audience comprises undergraduate students in microbiology, including biology, pre-medical studies, and health profession majors. The activities were deployed in three microbiology lectures, and students were surveyed. Students' feedback highlights the efficacy of the hands-on approach and increased class participation, as two of the recurring words in the students' survey were "helpful" and "fun."

IS1-mediated chromosomal amplification of the arn operon leads to polymyxin B resistance in Escherichia coli B strains.

Polymyxins [colistin and polymyxin B (PMB)] comprise an important class of natural product lipopeptide antibiotics used to treat multidrug-resistant Gram-negative bacterial infections. These positively charged lipopeptides interact with lipopolysaccharide (LPS) located in the outer membrane and disrupt the permeability barrier, leading to increased uptake and bacterial cell death. Many bacteria counter polymyxins by upregulating genes involved in the biosynthesis and transfer of amine-containing moieties to increase positively charged residues on LPS. Although 4-deoxy-l-aminoarabinose (Ara4N) and phosphoethanolamine (PEtN) are highly conserved LPS modifications in Escherichia coli, different lineages exhibit variable PMB susceptibilities and frequencies of resistance for reasons that are poorly understood. Herein, we describe a mechanism prevalent in E. coli B strains that depends on specific insertion sequence 1 (IS1) elements that flank genes involved in the biosynthesis and transfer of Ara4N to LPS. Spontaneous and transient chromosomal amplifications mediated by IS1 raise the frequency of PMB resistance by 10- to 100-fold in comparison to strains where a single IS1 element located 90 kb away from the end of the arn operon has been deleted. Amplification involving IS1 becomes the dominant resistance mechanism in the absence of PEtN modification. Isolates with amplified arn operons gradually lose their PMB-resistant phenotype with passaging, consistent with classical PMB heteroresistance behavior. Analysis of the whole genome transcriptome profile showed altered expression of genes residing both within and outside of the duplicated chromosomal segment, suggesting complex phenotypes including PMB resistance can result from tandem amplification events.IMPORTANCEPhenotypic variation in susceptibility and the emergence of resistant subpopulations are major challenges to the clinical use of polymyxins. While a large database of genes and alleles that can confer polymyxin resistance has been compiled, this report demonstrates that the chromosomal insertion sequence (IS) content and distribution warrant consideration as well. Amplification of large chromosomal segments containing the arn operon by IS1 increases the Ara4N content of the lipopolysaccharide layer in Escherichia coli B lineages using a mechanism that is orthogonal to transcriptional upregulation through two-component regulatory systems. Altogether, our work highlights the importance of IS elements in modulating gene expression and generating diverse subpopulations that can contribute to phenotypic polymyxin B heteroresistance.

Evidence for multiple forms of heritable RNA silencing.

Heritable gene silencing has been proposed to rely on DNA methylation, histone modifications, and/or non-coding RNAs in different organisms. Here we demonstrate that multiple RNA-mediated mechanisms with distinct and easily detectable molecular signatures can underlie heritable silencing of the same open-reading frame in the nematode C. elegans. Using two-gene operons, we reveal three cases of gene-selective silencing that provide support for the transmission of heritable epigenetic changes through different mechanisms of RNA silencing independent of changes in chromatin that would affect all genes of an operon equally. Different heritable epigenetic states of a gene were associated with distinct populations of stabilized mRNA fragments with untemplated poly-UG (pUG) tails, which are known intermediates of RNA silencing. These 'pUG signatures' provide a way to distinguish the multiple mechanisms that can drive heritable RNA silencing of a single gene.

Enhancing the Antibacterial Impact of Lipopeptide Extracted from Bacillus licheniformis as a Probiotic against MDR Acinetobacter baumannii.

BACKGROUND: The antibiotic resistance of microorganisms is escalating rapidly. Infections caused by opportunistic pathogens in immunocompromised individuals have prompted researchers to seek for potent and safe antibacterial agents. The purpose of this investigation was to explore the suppression of virulence gene expression, specifically the pga operon genes responsible in biofilm formation in Acinetobacter baumannii, through the utilization of metabolites obtained from probiotic bacteria. METHODS: To assess the antimicrobial properties, standard strains of five probiotic bacteria were tested against a standard strain of multidrug-resistant (MDR) A. baumannii employing the agar gel diffusion technique. Following the identification of the most potent probiotic strain (Bacillus licheniformis), the existence of its LanA and LanM genes was confirmed using the polymerase chain reaction (PCR) test. High-performance liquid chromatography (HPLC) and fourier-transform infrared spectroscopy (FTIR) techniques were employed to identify the intended metabolite, which was found to be a lipopeptide nature. The minimum inhibitory concentration (MIC) values and anti-biofilm activity of the targeted metabolite were determined using a dilution method in 96-well microplates and field emission scanning electron microscopy (FE-SEM). Real-time PCR (qPCR) was utilized for comparing the expression of pga operon genes, including pgaABCD, in A. baumannii pre- and post-exposure to the derived lipopeptide. RESULTS: The MIC results indicated that the probiotic product inhibited the growth of A. baumannii at concentrations lower than those needed for conventional antibiotics. Furthermore, it was observed that the desired genes' expression decreased due to the effect of this substance. CONCLUSIONS: This research concludes that the B. licheniformis probiotic product could be a viable alternative for combating drug resistance in A. baumannii.