Manipulation of natural transformation by AbaR-type islands promotes fixation of antibiotic resistance in Acinetobacter baumannii.

The opportunistic pathogen Acinetobacter baumannii, carries variants of A. baumannii resistance islands (AbaR)-type genomic islands conferring multidrug resistance. Their pervasiveness in the species has remained enigmatic. The dissemination of AbaRs is intricately linked to their horizontal transfer via natural transformation, a process through which bacteria can import and recombine exogenous DNA, effecting allelic recombination, genetic acquisition, and deletion. In experimental populations of the closely related pathogenic Acinetobacter nosocomialis, we quantified the rates at which these natural transformation events occur between individuals. When integrated into a model of population dynamics, they lead to the swift removal of AbaRs from the population, contrasting with the high prevalence of AbaRs in genomes. Yet, genomic analyses show that nearly all AbaRs specifically disrupt comM, a gene encoding a helicase critical for natural transformation. We found that such disruption impedes gene acquisition, and deletion, while moderately impacting acquisition of single nucleotide polymorphism. A mathematical evolutionary model demonstrates that AbaRs inserted into comM gain a selective advantage over AbaRs inserted in sites that do not inhibit or completely inhibit transformation, in line with the genomic observations. The persistence of AbaRs can be ascribed to their integration into a specific gene, diminishing the likelihood of their removal from the bacterial genome. This integration preserves the acquisition and elimination of alleles, enabling the host bacterium-and thus its AbaR-to adapt to unpredictable environments and persist over the long term. This work underscores how manipulation of natural transformation by mobile genetic elements can drive the prevalence of multidrug resistance.

Genetic Manipulation of Neisseria gonorrhoeae and Commensal Neisseria Species.

The sexually transmitted pathogen, Neisseria gonorrhoeae, undergoes natural transformation at high frequency. This property has led to the rapid dissemination of antibiotic resistance markers and the panmictic structure of the gonococcal population. However, high-frequency transformation also makes N. gonorrhoeae one of the easiest bacterial species to manipulate genetically in the laboratory. Techniques have been developed that result in transformation frequencies >50%, allowing the identification of mutants by screening and without selection. Constructs have been created to take advantage of this high-frequency transformation, facilitating genetic mutation, complementation, and heterologous gene expression. Similar methods have been developed for N. meningitidis and nonpathogenic Neisseria including N. mucosa and N. musculi. Techniques are described for genetic manipulation of N. gonorrhoeae and commensal Neisseria species, as well as for growth of these fastidious organisms. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Spot transformation of Neisseria gonorrhoeae on agar plates Basic Protocol 2: Spot transformation of commensal Neisseria on agar plates Basic Protocol 3: Transformation of Neisseria gonorrhoeae in liquid culture Basic Protocol 4: Electroporation of Neisseria gonorrhoeae Basic Protocol 5: Creation of unmarked mutations using a positive and negative selection cassette Basic Protocol 6: In vitro mutagenesis of Neisseria gonorrhoeae chromosomal DNA using EZ-Tn5 Basic Protocol 7: Chemical mutagenesis Basic Protocol 8: Complementation on the Neisseria gonorrhoeae chromosome Alternate Protocol 1: Complementation with replicating plasmids Alternate Protocol 2: Complementation on the Neisseria musculi or Neisseria mucosa chromosome Basic Protocol 9: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown on solid medium Alternate Protocol 3: Preparation of chromosomal DNA from Neisseria gonorrhoeae grown in broth Support Protocol: Preparing PCR templates from Neisseria gonorrhoeae colonies.

BsuMI regulates DNA transformation in Bacillus subtilis besides the defense system and the constructed strain with BsuMI-absence is applicable as a universal transformation platform for wild-type Bacillus.

BACKGROUND: To effectively introduce plasmids into Bacillus species and conduct genetic manipulations in Bacillus chassis strains, it is essential to optimize transformation methods. These methods aim to extend the period of competence and enhance the permeability of the cell membrane to facilitate the entry of exogenous DNA. Although various strategies have been explored, few studies have delved into identifying metabolites and pathways associated with enhanced competence. Additionally, derivative Bacillus strains with non-functional restriction-modification systems have demonstrated superior efficiency in transforming exogenous DNA, lacking more explorations in the regulation conducted by the restriction-modification system to transformation process. RESULTS: Transcriptomic comparisons were performed to discover the competence forming mechanism and the regulation pathway conducted by the BsuMI methylation modification group in Bacillus. subtilis 168 under the Spizizen transformation condition, which were speculated to be the preferential selection of carbon sources by the cells and the preference for specific metabolic pathway when utilizing the carbon source. The cells were found to utilize the glycolysis pathway to exploit environmental glucose while reducing the demand for other phosphorylated precursors in this pathway. The weakening of these ATP-substrate competitive metabolic pathways allowed more ATP substrates to be distributed into the auto-phosphorylation of the signal transduction factor ComP during competence formation, thereby increasing the expression level of the key regulatory protein ComK. The expression of ComK upregulated the expression of the negative regulator SacX of starch and sucrose in host cells, reinforcing the preference for glucose as the primary carbon source. The methylation modification group of the primary protein BsuMI in the restriction-modification system was associated with the functional modification of key enzymes in the oxidative phosphorylation pathway. The absence of the BsuMI methylation modification group resulted in a decrease in the expression of subunits of cytochrome oxidase, leading to a weakening of the oxidative phosphorylation pathway, which promoted the glycolytic rate of cells and subsequently improved the distribution of ATP molecules into competence formation. A genetic transformation platform for wild-type Bacillus strains was successfully established based on the constructed strain B. subtilis 168-R-M- without its native restriction-modification system. With this platform, high plasmids transformation efficiencies were achieved with a remarkable 63-fold improvement compared to the control group and an increased universality in Bacillus species was also obtained. CONCLUSIONS: The enhanced competence formation mechanism and the regulation pathway conducted by the functional protein BsuMI of the restriction-modification system were concluded, providing a reference for further investigation. An effective transformation platform was established to overcome the obstacles in DNA transformations in wild-type Bacillus strains.

Streamlining marker-less allelic replacement in Streptococcus pneumoniae through a single transformation step strategy: easyJanus.

The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. Since the 2000s, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic exchange predominantly using the kanR-rpsL cassette known as "Janus." The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette and the target gene's flanking DNA. We present an innovative strategy to achieve marker-less allelic replacement through a single transformation step. Our strategy involves integrating an additional copy of the target's downstream region before the Janus cassette, leading to a modified genetic arrangement. This single modification reduced the number of required PCR fragments from five to four, lowered the number of assembly reactions from two to one, and simplified the transformation process to a single step. To validate the efficacy of our approach, we implemented this strategy to delete in Spn serotype 4 strain TIGR4 the virulence gene pspA, the entire capsular polysaccharide synthesis locus cps4, and to introduce a single-nucleotide replacement into the chromosome. Notably, beyond streamlining the procedure, our method markedly reduced false positives typically encountered during negative selection with streptomycin when employing the traditional Janus protocol. Furthermore, and as consequence of reducing the amount of exogenous DNA required for construct synthesis, we show that our new method is amendable to the use of commercially available synthetic DNA for construct creation, further reducing the work needed to obtain a mutant. Our streamlined strategy, termed easyJanus, substantially expedites the genetic manipulation of Spn facilitating future research endeavors. IMPORTANCE: We introduce a new strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections. Our approach involves a modified genetic arrangement of the Janus cassette to facilitate self-excision during the segregation step. Since this new method reduces the amount of exogenous DNA required, it is highly amendable to the use of synthetic DNA for construction of the mutagenic construct. Our streamlined strategy, called easyJanus, offers significant time and cost savings while concurrently enhancing the efficiency of obtaining marker-less allelic replacement in S. pneumoniae.

Generation of tools for expression and purification of the phage-encoded Type I restriction enzyme inhibitor, Ocr.

DNA manipulation is an essential tool in molecular microbiology research that is dependent on the ability of bacteria to take up and preserve foreign DNA by horizontal gene transfer. This process can be significantly impaired by the activity of bacterial restriction modification systems; bacterial operons comprising paired enzymatic activities that protectively methylate host DNA, while cleaving incoming unmodified foreign DNA. Ocr is a phage-encoded protein that inhibits Type I restriction modification systems, the addition of which significantly improves bacterial transformation efficiency. We recently established an improved and highly efficient transformation protocol for the important human pathogen group A Streptococcus using commercially available recombinant Ocr protein, manufacture of which has since been discontinued. In order to ensure the continued availability of Ocr protein within the research community, we have generated tools and methods for in-house Ocr production and validated the activity of the purified recombinant protein.

Optimizing CaCl2-mediated transformation of Pseudomonas aeruginosa SDK-6 with pJN105 using OFAT: A novel and efficient cloning approach.

Cloning and expression of a gene in the desired host is required for optimum production in recombinant strains. The present research is the first attempt to optimize the physiological conditions for the transformation of Pseudomonas aeruginosa SDK-6 with pJN105. Different factors, such as inoculum size, incubation period, heat shock temperature, and heat shock time were optimized using one factor at a time (OFAT) followed by the selection of transformants using gentamicin resistance marker. The maximum number of transformants (2.002 ± 0.077 × 105 cfu/ µg of plasmid DNA) were reported with 0.5% (v/v) inoculum, an incubation period of 3 h, and heat shock treatment at 50 °C for 1 min. An overall 12-fold increase in transformation efficiency was observed. The presence of a 6055 bp band on agarose gel confirmed the transformation of Pseudomonas aeruginosa with the vector pJN105.

Unveiling the regulatory network controlling natural transformation in lactococci.

Lactococcus lactis is a lactic acid bacterium of major importance for food fermentation and biotechnological applications. The ability to manipulate its genome quickly and easily through competence for DNA transformation would accelerate its general use as a platform for a variety of applications. Natural transformation in this species requires the activation of the master regulator ComX. However, the growth conditions that lead to spontaneous transformation, as well as the regulators that control ComX production, are unknown. Here, we identified the carbon source, nitrogen supply, and pH as key factors controlling competence development in this species. Notably, we showed that these conditions are sensed by three global regulators (i.e., CcpA, CodY, and CovR), which repress comX transcription directly. Furthermore, our systematic inactivation of known signaling systems suggests that classical pheromone-sensing regulators are not involved. Finally, we revealed that the ComX-degrading MecA-ClpCP machinery plays a predominant role based on the identification of a single amino-acid substitution in the adaptor protein MecA of a highly transformable strain. Contrasting with closely-related streptococci, the master competence regulator in L. lactis is regulated both proximally by general sensors and distantly by the Clp degradation machinery. This study not only highlights the diversity of regulatory networks for competence control in Gram-positive bacteria, but it also paves the way for the use of natural transformation as a tool to manipulate this biotechnologically important bacterium.

Pneumococcal competence is a populational health sensor driving multilevel heterogeneity in response to antibiotics.

Competence for natural transformation is a central driver of genetic diversity in bacteria. In the human pathogen Streptococcus pneumoniae, competence exhibits a populational character mediated by the stress-induced ComABCDE quorum-sensing (QS) system. Here, we explore how this cell-to-cell communication mechanism proceeds and the functional properties acquired by competent cells grown under lethal stress. We show that populational competence development depends on self-induced cells stochastically emerging in response to stresses, including antibiotics. Competence then propagates through the population from a low threshold density of self-induced cells, defining a biphasic Self-Induction and Propagation (SI&P) QS mechanism. We also reveal that a competent population displays either increased sensitivity or improved tolerance to lethal doses of antibiotics, dependent in the latter case on the competence-induced ComM division inhibitor. Remarkably, these surviving competent cells also display an altered transformation potential. Thus, the unveiled SI&P QS mechanism shapes pneumococcal competence as a health sensor of the clonal population, promoting a bet-hedging strategy that both responds to and drives cells towards heterogeneity.

Role of cspA on the Preparation of Escherichia coli Competent Cells by Calcium Chloride Method.

One of the fundamental techniques in genetic engineering is the creation of Escherichia coli competent cells using the CaCl2 method. However, little is known about the mechanism of E. coli competence formation. We have previously found that the cspA gene may play an indispensable role in the preparation of E. coli DH5α competent cells through multiomics analysis. In the present study, the cellular localization, physicochemical properties, and function of the protein expressed by the cspA gene were analyzed. To investigate the role of the cspA gene in E. coli transformation, cspA-deficient mutant was constructed by red homologous recombination. The growth, transformation efficiency, and cell morphology of the cspA-deficient strain and E. coli were compared. It was found that there were no noticeable differences in growth and morphology between E. coli and the cspA-deficient strain cultured at 37°C, but the mutant exhibited increased transformation efficiencies compared to E. coli DH5α for plasmids pUC19, pET-32a, and p1304, with enhancements of 2.23, 2.24, and 3.46 times, respectively. It was proved that cspA gene is an important negative regulatory gene in the CaCl2 preparation of competent cells.

ComI inhibits transformation in Bacillus subtilis by selectively killing competent cells.

Many bacteria build elaborate molecular machines to import DNA via natural competence, yet this activity is often not identified until strains have been handled and domesticated in laboratory settings. For example, one of the best studied Gram-positive model organisms, Bacillus subtilis, has a poorly transformable ancestor. Transformation in the ancestral strain is inhibited by a transmembrane peptide, ComI, which is encoded on an extrachromosomal plasmid. Although ComI was shown to be necessary and sufficient to inhibit transformation when produced at high levels under an inducible promoter, the mechanism by which ComI inhibits transformation is unknown. Here, we examine the native regulation and mechanism of transformation inhibition by ComI. We find that under native regulation, ComI expression is restricted in the absence of the plasmid. In the presence of the plasmid, we find that ComI is expressed at higher levels in cells that are differentiating into a competent state. The subcellular localization of ComI, however, does not depend on any other competence proteins, and permeabilization activity is concentration-dependent. Time-lapse microscopy reveals that competent cells producing ComI are first permeabilized and then die. Based on these observations, we propose a new model for the mechanism of ComI in which response to competence activation leads to selective elimination of the competent subpopulation. IMPORTANCE: Natural transformation mechanisms have been studied across several bacterial systems, but few examples of inhibition exist. This work investigates the mechanism of action of a plasmid-encoded transmembrane inhibitor of natural transformation. The data reveal that the peptide can cause cell permeabilization. Permeabilization is synergistic with entry of Bacillus subtilis into the "competent" state, such that cells with the ability to be transformed are preferentially killed. These findings reveal a self-preservation mechanism coupled to the physiological state of the cells that ensures that the population can maintain an unaltered plasmid and its predicted prophage.

A cell-free transcription-translation pipeline for recreating methylation patterns boosts DNA transformation in bacteria.

The bacterial world offers diverse strains for understanding medical and environmental processes and for engineering synthetic biological chassis. However, genetically manipulating these strains has faced a long-standing bottleneck: how to efficiently transform DNA. Here, we report imitating methylation patterns rapidly in TXTL (IMPRINT), a generalized, rapid, and scalable approach based on cell-free transcription-translation (TXTL) to overcome DNA restriction, a prominent barrier to transformation. IMPRINT utilizes TXTL to express DNA methyltransferases from a bacterium's restriction-modification systems. The expressed methyltransferases then methylate DNA in vitro to match the bacterium's DNA methylation pattern, circumventing restriction and enhancing transformation. With IMPRINT, we efficiently multiplex methylation by diverse DNA methyltransferases and enhance plasmid transformation in gram-negative and gram-positive bacteria. We also develop a high-throughput pipeline that identifies the most consequential methyltransferases, and we apply IMPRINT to screen a ribosome-binding site library in a hard-to-transform Bifidobacterium. Overall, IMPRINT can enhance DNA transformation, enabling the use of sophisticated genetic manipulation tools across the bacterial world.

Genetic modification of Streptococcus dysgalactiae by natural transformation.

Streptococcus dysgalactiae is an emerging human and animal pathogen. Functional studies of genes involved in virulence of S. dysgalactiae and other pyogenic group streptococci are often hampered by limited genetic tractability. It is known that pyogenic streptococci carry genes required for competence for natural transformation; however, in contrast to other streptococcal subgroups, there is limited evidence for gene transfer by natural transformation in these bacteria. In this study, we systematically assessed the genomes of 179 S. dysgalactiae strains of both human and animal origins (subsp. equisimilis and dysgalactiae, respectively) for the presence of genes required for natural transformation. While a considerable fraction of the strains contained inactive genes, the majority (64.2%) of the strains had an intact gene set. In selected strains, we examined the dynamics of competence activation after addition of competence-inducing pheromones using transcriptional reporter assays and exploratory RNA-seq. Based on these findings, we were able to establish a protocol allowing us to utilize natural transformation to construct deletion mutants by allelic exchange in several S. dysgalactiae strains of both subspecies. As part of the work, we deleted putative lactose utilization genes to study their role in growth on lactose. The data presented here provide new knowledge on the potential of horizonal gene transfer by natural transformation in S. dysgalactiae and, importantly, demonstrates the possibility to exploit natural transformation for genetic engineering in these bacteria. IMPORTANCE: Numerous Streptococcus spp. exchange genes horizontally through natural transformation, which also facilitates efficient genetic engineering in these organisms. However, for the pyogenic group of streptococci, including the emerging pathogen Streptococcus dysgalactiae, there is limited experimental evidence for natural transformation. In this study, we demonstrate that natural transformation in vitro indeed is possible in S. dysgalactiae strains under optimal conditions. We utilized this method to perform gene deletion through allelic exchange in several strains, thereby paving the way for more efficient gene engineering methods in pyogenic streptococci.

Transcriptional regulation of TacL-mediated lipoteichoic acids biosynthesis by ComE during competence impacts pneumococcal transformation.

Competence development is essential for bacterial transformation since it enables bacteria to take up free DNA from the surrounding environment. The regulation of teichoic acid biosynthesis is tightly controlled during pneumococcal competence; however, the mechanism governing this regulation and its impact on transformation remains poorly understood. We demonstrated that a defect in lipoteichoic acid ligase (TacL)-mediated lipoteichoic acids (LTAs) biosynthesis was associated with impaired pneumococcal transformation. Using a fragment of tacL regulatory probe as bait in a DNA pulldown assay, we successfully identified several regulatory proteins, including ComE. Electrophoretic mobility shift assays revealed that phosphomimetic ComE, but not wild-type ComE, exhibited specific binding to the probe. DNase I footprinting assays revealed the specific binding sequences encompassing around 30 base pairs located 31 base pairs upstream from the start codon of tacL. Expression of tacL was found to be upregulated in the ΔcomE strain, and the addition of exogenous competence-stimulating peptide repressed the tacL transcription in the wild-type strain but not the ΔcomE mutant, indicating that ComE exerted a negative regulatory effect on the transcription of tacL. Mutation in the JH2 region of tacL upstream regulatory sequence led to increased LTAs abundance and displayed higher transformation efficiency. Collectively, our work identified the regulatory mechanisms that control LTAs biosynthesis during competence and thereby unveiled a repression mechanism underlying pneumococcal transformation.

The elimination of two restriction enzyme genes allows for electroporation-based transformation and CRISPR-Cas9-based base editing in the non-competent Gram-negative bacterium Acinetobacter sp. Tol 5.

Environmental isolates are promising candidates for new chassis of synthetic biology because of their inherent capabilities, which include efficiently converting a wide range of substrates into valuable products and resilience to environmental stresses; however, many remain genetically intractable and unamenable to established genetic tools tailored for model bacteria. Acinetobacter sp. Tol 5, an environmentally isolated Gram-negative bacterium, possesses intriguing properties for use in synthetic biology applications. Despite the previous development of genetic tools for the engineering of strain Tol 5, its genetic manipulation has been hindered by low transformation efficiency via electroporation, rendering the process laborious and time-consuming. This study demonstrated the genetic refinement of the Tol 5 strain, achieving efficient transformation via electroporation. We deleted two genes encoding type I and type III restriction enzymes. The resulting mutant strain not only exhibited marked efficiency of electrotransformation but also proved receptive to both in vitro and in vivo DNA assembly technologies, thereby facilitating the construction of recombinant DNA without reliance on intermediate Escherichia coli constructs. In addition, we successfully adapted a CRISPR-Cas9-based base-editing platform developed for other Acinetobacter species. Our findings provide genetic modification strategies that allow for the domestication of environmentally isolated bacteria, streamlining their utilization in synthetic biology applications.IMPORTANCERecent synthetic biology has sought diverse bacterial chassis from environmental sources to circumvent the limitations of laboratory Escherichia coli strains for industrial and environmental applications. One of the critical barriers in cell engineering of bacterial chassis is their inherent resistance to recombinant DNA, propagated either in vitro or within E. coli cells. Environmental bacteria have evolved defense mechanisms against foreign DNA as a response to the constant threat of phage infection. The ubiquity of phages in natural settings accounts for the genetic intractability of environmental isolates. The significance of our research is in demonstrating genetic modification strategies for the cell engineering of such genetically intractable bacteria. This research marks a pivotal step in the domestication of environmentally isolated bacteria, promising candidates for emerging synthetic biology chassis. Our work thus significantly contributes to advancing their applications across industrial, environmental, and biomedical fields.

Current approaches to genetic modification of marine bacteria and considerations for improved transformation efficiency.

Marine bacteria play vital roles in symbiosis, biogeochemical cycles and produce novel bioactive compounds and enzymes of interest for the pharmaceutical, biofuel and biotechnology industries. At present, investigations into marine bacterial functions and their products are primarily based on phenotypic observations, -omic type approaches and heterologous gene expression. To advance our understanding of marine bacteria and harness their full potential for industry application, it is critical that we have the appropriate tools and resources to genetically manipulate them in situ. However, current genetic tools that are largely designed for model organisms such as E. coli, produce low transformation efficiencies or have no transfer ability in marine bacteria. To improve genetic manipulation applications for marine bacteria, we need to improve transformation methods such as conjugation and electroporation in addition to identifying more marine broad host range plasmids. In this review, we aim to outline the reported methods of transformation for marine bacteria and discuss the considerations for each approach in the context of improving efficiency. In addition, we further discuss marine plasmids and future research areas including CRISPR tools and their potential applications for marine bacteria.

Optimisation of DNA electroporation protocols for different plant-associated bacteria.

Electroporation is a vital process that facilitates the use of modern recombineering and other high-throughput techniques in a wide array of microorganisms, including non-model bacteria like plant growth-promoting bacteria (PGPB). These microorganisms play a significant role in plant health by colonizing plants and promoting growth through nutrient exchange and hormonal regulation. In this study, we introduce a sequential Design of Experiments (DOE) approach to obtain highly competent cells swiftly and reliably for electroporation. Our method focuses on optimizing the three stages of the electroporation procedure-preparing competent cells, applying the electric pulse field, and recovering transformed cells-separately. We utilized a split-plot fractional design with five factors and a covariate to optimize the first step, response surface methodology (RSM) for the second step, and Plackett-Burman design for two categorical factors and one continuous factor for the final step. Following the experimental sequence with three bacterial models, we achieved efficiencies 10 to 100 times higher, reaching orders of 105 to 106 CFU/µg of circular plasmid DNA. These results highlight the significant potential for enhancing electroporation protocols for non-model bacteria.

From isotopically labeled DNA to fluorescently labeled dynamic pili: building a mechanistic model of DNA transport to the cytoplasmic membrane.

SUMMARYNatural competence, the physiological state wherein bacteria produce proteins that mediate extracellular DNA transport into the cytosol and the subsequent recombination of DNA into the genome, is conserved across the bacterial domain. DNA must successfully translocate across formidable permeability barriers during import, including the cell membrane(s) and the cell wall, that are normally impermeable to large DNA polymers. This review will examine the mechanisms underlying DNA transport from the extracellular space to the cytoplasmic membrane. First, the challenges inherent to DNA movement through the cell periphery will be discussed to provide context for DNA transport during natural competence. The following sections will trace the development of a comprehensive model for DNA translocation to the cytoplasmic membrane, highlighting the crucial studies performed over the last century that have contributed to building contemporary DNA import models. Finally, this review will conclude by reflecting on what is still unknown about the process and the possible solutions to overcome these limitations.

Optimization of plasmid electrotransformation into Bacillus subtilis using an antibacterial peptide.

Bacillus subtilis can potentially serve as an efficient expression host for biotechnology due to its ability to secrete extracellular proteins and enzymes directly into the culture medium. One of the important challenges in the biotechnology industry is to optimize the transformation conditions of B. subtilis bacteria. This study aims to provide a new method to optimize the transformation conditions and improve the transformation efficiency of B. subtilis WB600. To increase the transformation efficiency in B. subtilis, two methods of adding CM11 antibacterial peptides to the bacterial medium along with electroporation and optimizing the variables including the growth medium composition, time to adding CM11 peptide, electroporation voltage, recovery medium, and cell recovery time are used. The results of this study showed that the addition of antimicrobial peptides (AMPs) with a concentration of 2 µg/ml increases the transformation efficiency by 4 times compared to the absence of AMP in the bacterial medium. Additionally, the findings from our study indicated that the most optimal rate of transformation for B. subtilis was observed at a voltage of 7.5 kV/cm, with a recovery period of 12 h. With the optimized method, the transformation efficiency came up to 1.69 × 104 CFU/µg DNA. This improvement in transformation efficiency will be attributed to the research of expression of exogenous genes in B. subtilis, gene library construction for transformation of wild-type B. subtilis strains.

Dissemination of antibiotic resistance genes is regulated by iron oxides: Insight into the influence on bacterial transformation.

The transportation of antibiotic resistance genes (ARGs) in manure-soil-plant continuums poses risks to human health. Horizontal gene transfer, particularly for bacterial transformation, is an important way for ARG dissemination. As crucial components in soils, iron oxides impacted the fates of various abiotic and biotic contaminants due to their active properties. However, whether they can influence the transformation of ARGs is unknown, which waits to be figured out to boost the assessment and control of ARG spread risks. In this study, we have investigated the effects of goethite, hematite, and magnetite (0-250 mg/L, with sizes < 100 nm and > 100 nm) on the transfer of ampicillin resistance genes to Escherichia coli cells. At lower iron oxide concentrations, the transformation of ARGs was first facilitated (transformation frequency reached up to 3.38-fold higher), but the facilitating effects gradually weakened and eventually disappeared as concentrations further increased. Particle size and iron oxide type were not the universal determinants controlling the transformation. At lower concentrations, iron oxides interacted with proteins and phospholipids in E. coli envelope structures, and induced the overgeneration of intracellular reactive oxygen species. Consequently, they led to pore formation and permeability enhancement on the cell membrane, thus promoting the transformation. The facilitation was also associated with the carrier-like effect of iron oxides for antibiotic resistance plasmids. At higher concentrations, the weakened facilitations were attributed to the aggregation of iron oxides. In this study, we highlight the crucial roles of the concentrations (contents) of iron oxides on the dissemination of ARGs in soils; this study may serve as a reference for ARG pollution control in future agricultural production.

Genome editing in Acinetobacter baumannii through enhanced natural transformation.

Acinetobacter baumannii, a multidrug-resistant bacterium has become a significant cause of life-threatening infections acquired in hospitals worldwide. The existing drugs used to treat A. baumannii infections are rapidly losing efficacy, and the increasing antimicrobial resistance, which is expected to turn into a global health crisis, underscores the urgency to develop novel prevention and treatment strategies. We reasoned that the discovery of novel virulence targets for vaccine and therapy interventions requires a more enhanced method for the introduction of multiple elements of foreign DNA for genome editing than the current methods of natural transformation techniques. Herein, we employed a novel and a much-improved enhanced technique for the natural transformation of elements of the genome editing system CRISPR-Cas9 to suppress specific genomic regions linked to selectively suppress bacterial virulence. We modified the genome of the laboratory-adapted strain of A. baumannii BAA-747 by targeting the AmpC, as a marker gene, for disruption by three different genomic manipulation strategies, and created mutant strains of A. baumannii that are, at least, fourfold susceptible to ampicillin. This work has established an optimized enhanced natural transformation system that enables efficient genome editing of pathogenic bacteria in a laboratory setting, providing a valuable future tool for exploring the function of unidentified virulence genes in bacterial genomes.