Sequence and origin of the Streptomyces intergenetic-conjugation helper plasmid pUZ8002.

Conjugation of plasmids from Escherichia coli is essential for the genetic manipulation of Streptomyces spp. To facilitate intergeneric conjugation from E. coli to Streptomyces the conjugative machinery required for genetic transfer is usually provided by the non-transferable helper plasmid, pUZ8002. Here we present the complete nucleotide sequence of pUZ8002, describe the previously undocumented creation process, and provide details of the sequence relative to the parental pUZ8 plasmid and another previously published pUZ8002 sequence.

Enhancement of growth media for extreme iron limitation in Escherichia coli.

Iron is an essential nutrient for microbial growth and bacteria have evolved numerous routes to solubilize and scavenge this biometal, which is often present at very low concentrations in host tissue. We recently used a MOPS-based medium to induce iron limitation in Escherichia coli K-12 during the characterization of novel siderophore-conjugated antibiotics. In this study we confirm that growth media derived from commercially available M9 salts are unsuitable for studies of iron-limited growth, probably through the contamination of the sodium phosphate buffer components with over 100 µM iron. In contrast, MOPS-based media that are treated with metal-binding Chelex resin allow the free iron concentration to be reduced to growth-limiting levels. Despite these measures a small amount of E. coli growth is still observed in these iron-depleted media. By growing E. coli in conditions that theoretically increase the demand for iron-dependent enzymes, namely by replacing the glucose carbon source for acetate and by switching to a microaerobic atmosphere, we can reduce background growth even further. Finally, we demonstrate that by adding an exogeneous siderophore to the growth media which is poorly used by E. coli, we can completely prevent growth, perhaps mimicking the situation in host tissue. In conclusion, this short study provides practical experimental insight into low iron media and how to augment the growth conditions of E. coli for extreme iron-limited growth.

AutoBioTech─A Versatile Biofoundry for Automated Strain Engineering.

The inevitable transition from petrochemical production processes to renewable alternatives has sparked the emergence of biofoundries in recent years. Manual engineering of microbes will not be sufficient to meet the ever-increasing demand for novel producer strains. Here we describe the AutoBioTech platform, a fully automated laboratory system with 14 devices to perform operations for strain construction without human interaction. Using modular workflows, this platform enables automated transformations of Escherichia coli with plasmids assembled via modular cloning. A CRISPR/Cas9 toolbox compatible with existing modular cloning frameworks allows automated and flexible genome editing of E. coli. In addition, novel workflows have been established for the fully automated transformation of the Gram-positive model organism Corynebacterium glutamicum by conjugation and electroporation, with the latter proving to be the more robust technique. Overall, the AutoBioTech platform excels at versatility due to the modularity of workflows and seamless transitions between modules. This will accelerate strain engineering of Gram-negative and Gram-positive bacteria.

Characterization of antimicrobial resistance profiles in Escherichia coli isolated from captive mammals in Ecuador.

BACKGROUND: This study focuses on the AMR profiles in E. coli isolated from captive mammals at EcoZoo San Martín, Baños de Agua Santa, Ecuador, highlighting the role of wildlife as reservoirs of resistant bacteria. AIMS: The aim of this research is to investigate the antimicrobial resistance profiles of E. coli strains isolated from various species of captive mammals, emphasizing the potential zoonotic risks and the necessity for integrated AMR management strategies. MATERIALS & METHODS: A total of 189 fecal samples were collected from 70 mammals across 27 species. These samples were screened for E. coli, resulting in 90 identified strains. The resistance profiles of these strains to 16 antibiotics, including 10 ß-lactams and 6 non-ß-lactams, were determined using the disk diffusion method. Additionally, the presence of Extended-Spectrum Beta-Lactamase (ESBL) genes and other resistance genes was analyzed using PCR. RESULTS: Significant resistance was observed, with 52.22% of isolates resistant to ampicillin, 42.22% to ceftriaxone and cefuroxime, and 27.78% identified as ESBL-producing E. coli. Multiresistance (resistance to more than three antibiotic groups) was found in 35.56% of isolates. Carnivorous and omnivorous animals, particularly those with prior antibiotic treatments, were more likely to harbor resistant strains. DISCUSSION: These findings underscore the role of captive mammals as indicators of environmental AMR. The high prevalence of resistant E. coli in these animals suggests that zoos could be significant reservoirs for the spread of antibiotic-resistant bacteria. The results align with other studies showing that diet and antibiotic treatment history influence resistance profiles. CONCLUSION: The study highlights the need for an integrated approach involving veterinary care, habitat management, and public awareness to prevent captive wildlife from becoming reservoirs of antibiotic-resistant bacteria. Improved waste management practices and responsible antibiotic use are crucial to mitigate the risks of AMR in zoo environments and reduce zoonotic threats.

The Scr and Csc pathways for sucrose utilization co-exist in E. coli, but only the Scr pathway is widespread in other Enterobacteriaceae.

Most Escherichia coli isolates from humans do not utilize D-sucrose as a substrate for fermentation or growth. Previous work has shown that the Csc pathway allows some E. coli to utilize sucrose for slow growth, and this pathway has been engineered in E. coli W strains to enhance use of sucrose as a feedstock for industrial applications. An alternative sucrose utilization pathway, Scr, was first identified in Klebsiella pneumoniae and has been reported in some E. coli and Salmonella enterica isolates. We show here that the Scr pathway is native to an important subset of E. coli phylogroup B2 lineages that lack the Csc pathway but grow rapidly on sucrose. Laboratory E. coli strains derived from MG1655 (phylogroup A, ST10) are unable to utilize sucrose and lack the scr and csc genes, but a recombinant plasmid-borne scr locus enables rapid growth on and fermentation of sucrose. Genome analyses of Enterobacteriaceae indicate that the scr locus is widespread in other Enterobacteriaceae; including Enterobacter and Klebsiella species, and some Citrobacter and Proteus species. In contrast, the Csc pathway is limited mostly to E. coli, some Shigella species (in which csc loci are rendered non-functional by various mutations), and Citrobacter freundii. The more efficient Scr pathway likely has greater potential than the Csc pathway for bioindustrial applications of E. coli and other Enterobacteriaceae using sucrose as a feedstock.

A natural approach to combating antibiotic-resistant pathogens in livestock: Hibiscus sabdariffa-derived hibiscus acid as a promising solution.

We examined the antibacterial efficacy of streptomycin, hibiscus acid, and their combination against multidrug-resistant Shiga-toxin-producing Escherichia coli (STEC) and Salmonella Typhimurium in mice. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for streptomycin, hibiscus acid, and their combination against STEC and Salmonella. Fifteen sets of six mice in each set were utilised: six groups were orally exposed to 4 log10 colony forming units (CFUs) of S. Typhimurium and another six to STEC, and three acted as the controls. Six hours post-inoculation, specific groups of mice received either oral solutions containing hibiscus acid at 5 and 7 mg/ml; streptomycin at 50 and 450 µg/ml; hibiscus acid/streptomycin (5 mg/ml hibiscus acid and 50 µg/ml streptomycin); or isotonic saline. The study determined the MIC and MBC of 7 mg/ml of hibiscus acid; 300 and 450 µg/ml of streptomycin; and two concentrations of hibiscus/streptomycin (3 mg/ml / 20 µg/ml and 5 mg/ml / 50 µg/ml). Interestingly, the mice that were infected and subsequently treated with hibiscus acid at 7 mg/ml alone or in conjunction with streptomycin did not have either STEC or Salmonella in their faecal samples, and none of the mice died. In contrast, the untreated mice and those exclusively treated with streptomycin had the pathogens present in their stool, leading to the mortality of all the subjects.

Impact of a Switch From Ciprofloxacin to Ceftriaxone Prophylaxis on Infectious Complications After Transrectal Ultrasound-Guided Biopsy of the Prostate.

In a 12-year single-center quasi-experimental study, a switch from ciprofloxacin to ceftriaxone prophylaxis for transrectal ultrasound-guided prostate biopsy procedures was associated with a significant reduction in 30-day postprocedure urinary tract infection, urinary tract infection-related hospitalizations, antibiotic prescriptions, and isolation of fluoroquinolone-resistant organisms from urine or blood cultures.

Perforation of the Terminal Ileum Secondary to Mucosal Damage of Enteroaggregative Escherichia coli and a Toothpick.

Enteroaggregative Escherichia coli (EAEC) is a common form of E. coli that causes gastroenteritis and diarrhea worldwide. Biofilm formation on the intestinal mucosa initiates an inflammatory cascade in the gastrointestinal tissue, which has significant destructive effects on the mucosa of the small and large intestines. Small bowel obstruction and perforation due to a foreign body are uncommon, but the risk increases with pre-existing conditions such as the presence of intestinal strictures, inflammation, and mucosal ulceration. We present a unique case of acute enteritis from EAEC with mucosal ulceration and perforation because of co-ingestion of foreign body and impaction with the presence of stricture in the terminal ileum. This was treated with small bowel resection and primary anastomosis. The patient was successfully discharged from the hospital. The clinical features and pathological findings of enteric EAEC infection are described. To our knowledge, intestinal perforation and secondary peritonitis related to EAEC enteric infection, with mucosal ulceration and perforation secondary to co-ingestion of a foreign body with intestinal stricture, have not been documented. In this case, EAEC was associated with terminal ileum mucosal ulceration and complicated by perforation secondary to foreign body impaction along with ileal stricture. These compounding effects likely explain gastrointestinal tract perforation and secondary peritonitis.

Metabolic Engineering of Escherichia coli for Bioproduction of (R)-3-Hydroxybutyric Acid through a Three-Pronged Approach.

(R)-3-Hydroxybutyric acid (R-3HB) is an important chiral chemical with extensive applications in the agricultural, food, and chemical industries. The synthesis of R-3HB by microbial fermentation is of interest due to its remarkable stereoselectivity and economy. However, the low production of R-3HB failed to meet the needs of large-scale industrial production. In this study, an engineered strain for the efficient biosynthesis of R-3HB was constructed through a three-pronged approach encompassing biosynthetic pathway optimization, engineering of NADPH regenerators, and central metabolism regulation. The engineered strain Q5081 produced 75.7 g/L R-3HB, with a productivity of 1.26 g/L/h and a yield of 0.34 g/g glucose in fed-batch fermentation, showing the highest reported titer and productivity of R-3HB to date. We also performed transcriptome sequencing and annotation to illustrate the mechanism underlying the enhanced R-3HB production. The systematic metabolic engineering by a three-pronged approach demonstrated the feasibility of improving the biosynthesis, and the engineered strain Q5081 has the potential for widespread applications in the industrial production of R-3HB.

Characterization of the molecular role that ST3GAL1 plays in porcine susceptibility to E. coli F18 infection.

Escherichia coli F18 (E. coli F18) is the main cause of bacterial diarrhea in piglets. Previous transcriptome reported that ST3GAL1 was associated to E. coli F18 infection. However, its role in mediating the resistance to E. coli F18 remains elusive. Here, we revealed that the downregulation of ST3GAL1 expression contributed to the enhancement of E. coli F18 resistance in IPEC-J2 cells. Bisulfite sequencing identified 26 methylated CpG sites in the ST3GAL1 core promoter. Among these, the ST3GAL1 mRNA levels significantly correlated with methylation levels of the mC-8 site in the specificity protein 1 (SP1) transcription factor (P < 0.01). Interestingly, ST3GAL1 expression may enhances the immune response by activating TLRs signaling, meanwhile decreases the production of the E. coli F18 receptor by inhibiting glycosphingolipid biosynthesis signaling, thereby leading to enhance the resistance to E. coli F18 infection. Besides, low ST3GAL1 expression may increase E. coli resistance by reducing sialylation. Together, these results support the status of ST3GAL1 as a viable target for efforts to modulate E. coli F18 susceptibility, offering a theoretical foundation for the use of this gene as a key biomarker for molecular breeding to improve porcine disease resistance.

The microbiological quality of flour products in the UK with respect to Salmonella and Shiga-toxin-producing E. coli (STEC).

AIM: To investigate the possible contamination of raw flour and raw flour-based products, such as pancake/batter mixes, with Salmonella, generic E. coli and STEC. Samples included flours available for sale in the UK over a period of four months (January to April 2020). The Bread and Flour regulations, 1998 state the permitted ingredients in flour and bread but it does not specify the regular monitoring of the microbiological quality of flour and flour-based products. METHODS AND RESULTS: Samples of raw flour were collected by local authority sampling officers in accordance with current guidance on microbiological food sampling then transported to the laboratory for examination. Microbiological testing was performed to detect Salmonella spp., generic E. coli and Shiga-toxin-producing Escherichia coli (STEC) characterised for the presence of STEC virulence genes: stx1, stx2 and subtypes, eae, ipah, aggR, lt, sth and stp, using molecular methods (PCR). Of the 882 flours sampled, the incidence of Salmonella was 0.1% (a single positive sample that contained multiple ingredients such as flour, dried egg and dried milk, milled in the UK), and 68 samples (7.7%) contained generic E. coli at a level of >20 CFU/g. Molecular characterisation of flour samples revealed the presence of the Shiga-toxin (stx) gene in 10 samples (5 imported and 5 from the UK) (1.1%), from which STEC was isolated from 7 samples (0.8%). Salmonella and STEC isolates were sequenced to provide further characterisation of genotypes and to compare to sequences of human clinical isolates held in the UKHSA archive. Using our interpretive criteria based on genetic similarity, none of the STEC flour isolates correlated with previously observed human cases, while the singular Salmonella serotype Newport isolate from the mixed ingredient product was similar to a human case in 2019, from the UK, of S. Newport. Although there have been no reported human cases of STEC matching the isolates from these flour samples, some of the same serotypes and stx subtypes detected are known to have caused illness in other contexts. CONCLUSION: Results indicate that while the incidence was low, there is a potential for the presence of Salmonella and STEC in flour, and a genetic link was demonstrated between a Salmonella isolate from a flour-based product and a human case of salmonellosis.

Genomic SELEX Screening of Regulatory Targets of Transcription Factors.

The genome of Escherichia coli K-12 is transcribed by a single species of RNA polymerase. The selectivity of transcriptional targets is determined via interaction with one of seven species of the sigma subunit and a total of approximately 300 species of transcription factor (TFs). For comprehensive identification of the regulatory targets of these two groups of regulatory proteins on the genome, we developed an in vitro approach, "Genomic SELEX" (gSELEX) screening. Here we describe a detailed protocol of the gSELEX screening system, which uses purified regulatory proteins and fragments of genomic DNA from E. coli. Moreover, we describe methods and examples of results using cell-free synthetic proteins.

ChIP-qPCR of FLAG-Tagged Proteins in Bacteria.

DNA-protein interactions occur in biological processes such as genome replication, gene transcription, DNA repair, and chromatin compaction and organization. Mapping the distribution of the DNA-bound proteins on the chromosome is essential for understanding their associated biological process. Chromatin immunoprecipitation (ChIP) involves the antibody-mediated enrichment of DNA fragments bound by a target protein and has become one of the most powerful techniques for exploring the distribution of proteins on the chromosome. By incorporating quantitative polymerase chain reaction (qPCR) downstream of the ChIP assay, ChIP-qPCR was developed to describe binding profiles of DNA-associated proteins at a candidate locus. In this chapter, we describe ChIP-qPCR. We provide a step-by-step protocol for the preparation of a ChIP library of a 3× FLAG-tagged protein in bacteria, describe how downstream qPCR experiments can be performed with the appropriate controls, and explain how the data is analyzed. This chapter provides reliable technical guidance for ChIP-qPCR studies in bacteria.

Modular Assembly of Synthetic Secondary Chromosomes.

The development of novel DNA assembly methods in recent years has paved the way for the construction of synthetic replicons to be used for basic research and biotechnological applications. A learning-by-building approach can now answer questions about how chromosomes must be constructed to maintain genetic information. Here we describe an efficient pipeline for the design and assembly of synthetic, secondary chromosomes in Escherichia coli based on the popular modular cloning (MoClo) system.

Exploring the De Novo NMN Biosynthesis as an Alternative Pathway to Enhance NMN Production.

Nicotinamide mononucleotide (NMN) serves as a precursor for NAD+ synthesis and has been shown to have positive effects on the human body. Previous research has predominantly focused on the nicotinamide phosphoribosyltransferase-mediated route (NadV-mediated route) for NMN biosynthesis. In this study, we have explored the de novo NMN biosynthesis route as an alternative pathway to enhance NMN production. Initially, we systematically engineered Escherichia coli to enhance its capacity for NMN synthesis and accumulation, resulting in a remarkable over 100-fold increase in NMN yield. Subsequently, we progressively enhanced the de novo NMN biosynthesis route to further augment NMN production. We screened and identified the crucial role of MazG in catalyzing the enzymatic cleavage of NAD+ to NMN. And the de novo NMN biosynthesis route was optimized and integrated with the NadV-mediated NMN biosynthetic pathways, leading to an intracellular concentration of 844.10 ± 17.40 µM NMN. Furthermore, the introduction of two transporters enhanced the uptake of NAM and the excretion of NMN, resulting in NMN production of 1293.73 ± 61.38 µM. Finally, by engineering an E. coli strain with optimized PRPP synthetase, we achieved the highest NMN production, reaching 3067.98 ± 27.25 µM after 24 h of fermentation at the shake flask level. In addition to constructing an efficient E. coli cell factory for NMN production, our findings provide new insights into understanding the NAD+ salvage pathway and its role in energy metabolism within E. coli.

Whole-genome sequencing of multidrug-resistant Escherichia coli causing urinary tract infection in an immunocompromised patient: a case report.

BACKGROUND: Escherichia coli is a major human pathogen responsible for a broad range of clinical illnesses. It has been linked to endemic and epidemic nosocomial diseases caused by multidrug-resistant pathogens in Sudan as well as throughout the globe. CASE PRESENTATION: A 76-year-old African woman arrived at Saad Rashwan Medical Centre complaining of backaches and discomfort during urination. Throughout the preceding 5 years, the patient had recurrent urinary tract infections. Following overnight incubation at 37 °C, Escherichia coli was found in her midstream urine specimen on cysteine lactose electrolyte deficient agar media. Minimum inhibitory concentration (colorimetric/turbidimetric method) was employed to test a wide range of antimicrobial drugs against this bacterial strain, and the results revealed significant multidrug resistance. QIAamp® DNA Mini Kit was used to obtain DNA Template from the purified Escherichia coli (Qiagen, Hilden, Germany). The bacterial whole-genome sequence was done by Novogene company (Hong Kong) using Illumina HiSeq 2500 (Illumina, San Diego, CA, USA), followed by whole genome reconstructions, and identification of antibiotic-resistant genes. Phylogenetic analysis revealed that our strain was related to the Escherichia coli DSM30083 ( genome sequence ID: CP033092.2) from the USA. Our strain possessed the following antimicrobial-resistant genes: aminoglycoside (kdpE, baeR, cpxA, aadA5), nitroimidazole (msbA), phosphonic acid (mdtG), tetracycline (emrY), macrolide, penam, tetracycline, (evgA, TolC, H-NS), fluoroquinolone, cephalosporin, glycylcycline, penam, tetracycline, rifamycin, phenicol antibiotic, disinfecting agents and antiseptics (acrB; marA), sulfonamide (sul1), macrolide (Mrx), cephalosporin, penam (CTX-M-15), carbapenem, cephalosporin, and penam (OXA-1). CONCLUSION: This study found that the isolated Escherichia coli strain had varied antimicrobial resistance genes on the basis of whole-genome sequencing and phenotypic resistance analyses. Whole-genome sequencing is critical for control and preventative methods to battle the growing threat of antimicrobial resistance. A larger investigation is recommended for improved generalization of results.

A novel SERS method for detecting E. coli based on an aptamer-functionalized Au-Ag@Si triangular pyramid substrate.

Escherichia coli contamination in food and pharmaceutical preparations needs to be strictly controlled according to the regulations in many countries. However, rapid and sensitive E. coli detection is still a challenge. In this study, an aptamer-based surface-enhanced Raman spectroscopy (SERS) sandwich method was developed for the rapid and sensitive detection of E. coli using an aptamer-functionalized Au-Ag@Si triangular pyramid (TP) substrate. The Au-Ag@Si TP substrate was functionalized with E. coli aptamer to work as both the capture probe and SERS tag by integrating with Raman reporter (6-carboxy-X-rhodamine). The fabrication of the capture probe, SH-apt@Au-Ag@Si TP, was simple and rapid (20.5 h). This method could selectively and rapidly detect E. coli with a limit of detection of 2.8 CFU/mL within approximately 3 h. It was successfully applied to a traditional Chinese medicine preparation, Xinhuang tablets, with recoveries ranging from 90.19 % to 104.17 %. The results indicated that the developed method was simple and rapid, and it could be a promising alternative for the on-site detection of E. coli in pharmaceutical and food samples.

Molecularly imprinted Photoelectrochemical sensor for Escherichia coli based on Cu:ZIF-8/KZ3TTz heterojunction.

Herein, a signal stable molecularly imprinted photoelectrochemical (MIP-PEC) sensing platform was designed to sensitively detect Escherichia coli by incorporating polythiophene film with Cu: ZIF-8/KZ3TTz heterojunction. Attributed to the formation of a staggered type II heterostructure between KZ3TTz and Cu: ZIF-8 semiconductors, the Cu: ZIF-8/KZ3TTz heterojunction exhibited stable and significant cathode PEC response. Impressively, selective MIP film was grown on the surface of Cu: ZIF-8/KZ3TTz/GCE by electro-polymerization of 2,2-Dimethyl-5-(3-thienyl)-1,3-dioxane-4,6-dione (DTDD) in the presence of E. coli. After removing E. coli, more electrons were transferred to the electrolyte solution through the imprinting cavity on the MIP film, which was eliminated by O2 in the electrolyte, causing further enhancement of the cathode PEC response. On the contrary, when the imprinted cavity was filled with E. coli, the cathodic PEC response gradually decreased due to steric hindrance effect. The sensor showed excellent linearity in the range of 101 to 108 CFU/mL with a detection limit of 4.09 CFU/mL (S/N = 3). This strategy offered a novel approach for pathogenic bacteria detection in food safety and environmental monitoring.

The Antibacterial Properties of a Reinforced Zinc Oxide Eugenol Combined with Cloisite 5A Nanoclay: An In-Vitro Study.

Pulpotomies and pulpectomies are the most common clinical approach for dental caries in the primary dentition. Reinforced zinc oxide eugenol (ZOE) is an ideal material for filling in the pulp chamber after pulp therapies. The aim of this study was to assess the addition of Cloisite 5A nanoclay material to ZOE and evaluate its antibacterial properties. In this case-control study, the nanoclay nanoparticles were dissolved using a solvent (Eugenol) in different concentrations and their antibacterial properties were assessed using the agar diffusion test and biofilm analysis of Streptococcus mutans (S. mutans), Enterococcus faecalis (E. faecalis), and Escherichia coli (E. coli) in in vitro conditions using the AATCC 100 standards. The diameter of the inhibition zone was measured and assessed statistically using the SPSS software (Version 28, IBM, Chicago, IL, USA) with a significance level of 0.05. The antibacterial properties of the ZOE with nanoclay particles were significantly greater in comparison to the plain ZOE against E. faecalis, S. mutans, and E. coli. The inhibition zone against E. coli under the effect of the ZOE and nanoclay particles combined was significantly higher than that against E. faecalis and S. mutans. The current study showed that the addition of Cloisite 5A nanoclay particles can improve the antibacterial properties of ZOE significantly at certain concentrations.

Role of the antiparallel double-stranded filament form of FtsA in activating the Escherichia coli divisome.

The actin-like FtsA protein is essential for function of the cell division machinery, or divisome, in many bacteria including Escherichia coli. Previous in vitro studies demonstrated that purified wild-type FtsA assembles into closed mini-rings on lipid membranes, but oligomeric variants of FtsA such as FtsAR286W and FtsAG50E can bypass certain divisome defects and form arc and double-stranded (DS) oligomeric states, respectively, which may reflect conversion of an inactive to an active form of FtsA. However, it remains unproven which oligomeric forms of FtsA are responsible for assembling and activating the divisome. Here, we used an in vivo crosslinking assay for FtsA DS filaments to show that they largely depend on proper divisome assembly and are prevalent at later stages of cell division. We also used a previously reported variant that fails to assemble DS filaments, FtsAM96E R153D, to investigate the roles of FtsA oligomeric states in divisome assembly and activation. We show that FtsAM96E R153D cannot form DS filaments in vivo, fails to replace native FtsA, and confers a dominant negative phenotype, underscoring the importance of the DS filament stage for FtsA function. Surprisingly, however, activation of the divisome through the ftsL* or ftsW* superfission alleles suppressed the dominant negative phenotype and rescued the functionality of FtsAM96E R153D. Our results suggest that FtsA DS filaments are needed for divisome activation once it is assembled, but they are not essential for divisome assembly or guiding septum synthesis.IMPORTANCECell division is fundamental for cellular duplication. In simple cells like Escherichia coli bacteria, the actin homolog FtsA is essential for cell division and assembles into a variety of protein filaments at the cytoplasmic membrane. These filaments not only help tether polymers of the tubulin-like FtsZ to the membrane at early stages of cell division but also play crucial roles in recruiting other cell division proteins to a complex called the divisome. Once assembled, the E. coli divisome subsequently activates synthesis of the division septum that splits the cell in two. One recently discovered oligomeric conformation of FtsA is an antiparallel double-stranded filament. Using a combination of in vivo crosslinking and genetics, we provide evidence suggesting that these FtsA double filaments have a crucial role in activating the septum synthesis enzymes.