Escherichia coli is an important human pathogen that is able to cause a variety of infections, which can result in diarrhoea, urinary tract infections, sepsis, and even meningitis, depending on the pathotype of the infecting strain. Like many Gram-negative bacteria, E. coli is becoming increasingly resistant to many frontline antibiotics, including third-generation cephalosporins and carbapenems, which are often considered the antibiotics of last resort for these infections. This is particularly the case in Egypt, where multidrug-resistant (MDR) E. coli is highly prevalent. However, in spite of this, few Egyptian MDR E. coli strains have been fully characterised by genome sequencing. Here, we present the genome sequences of ten highly MDR E. coli strains, which were isolated from children who presented with diarrhoea at the Outpatients Clinic of Assiut University Children’s Hospital in Assiut, Egypt. We report that they carry multiple antimicrobial resistance genes, which includes extended spectrum β-lactamase genes, as well as blaNDM and blaOXA carbapenemase genes, likely encoded on IncX3 and IncF plasmids. Many of these strains were also found to be high-risk extra-intestinal pathogenic E. coli (ExPEC) clones belonging to sequence types ST167, ST410, and ST617. Thus, their presence in the Egyptian paediatric population is particularly worrying, and this highlights the need for increased surveillance of high-priority pathogens in this part of the world.

Escherichia coli is increasingly recognised as an important cause of ventilator-associated pneumonia (VAP), particularly in intensive care units (ICUs) with high antimicrobial selective pressure. Unlike classical respiratory pathogens, ICU-associated E. coli often originates from the patient’s intestinal microbiota and harbours a complex mobilome enriched with antimicrobial resistance determinants. In this study, a total of 200 nosocomial endotracheal aspirate samples were aseptically collected from patients admitted to the Respiratory ICU at Assiut University hospital. Antimicrobial susceptibility testing, serotyping and screening for various virulence and antimicrobial resistance genes (e.g., extended-spectrum β-lactamase (ESBL) and carbapenems genes) were carried out. In total, E. coli isolates were recovered from 54/200 (27%) endotracheal aspirates, with a high prevalence of multidrug resistance (MDR) observed (74.1%). Resistance to β-lactams was common with phenotypic evidence suggestive of ESBL production detected in 64.8% of isolates. Genome sequencing of three MDR E. coli isolates confirmed that they carried multiple antimicrobial resistance genes, which included ESBL genes (e.g., blaCTX-M-15 and blaTEM-1B). Each strain was also found to be high-risk extraintestinal pathogenic E. coli (ExPEC) clones, belonging to either sequence type ST131 or ST405. These findings support an endogenous infection model for VAP, whereby ICU selective pressure favours highly mobile, multidrug-resistant E. coli lineages adapted for extraintestinal survival. The high production of ESBLs and the prevalence of carbapenemase genes highlight the urgent need for molecular surveillance and antimicrobial stewardship strategies for the control of such high-priority pathogens in this part of the world.

Bacterial pathogens use a wide array of virulence factors to colonise and subsequently elicit disease in their host. These factors are often subject to extensive regulation at the transcriptional level, to ensure that their expression is timely. Although many pathogens use bespoke transcription factors that primarily target virulence genes, global transcription factors also sometimes play a role in controlling these genes. Enteroaggregative Escherichia coli (EAEC) is a significant cause of watery and mucoid diarrhoea globally. The organism colonises the small intestine before producing toxins that elicit disease, using a multitude of virulence factors that are encoded both chromosomally and on virulence plasmids. In this work, we have studied the cAMP Receptor Protein (CRP), a well-characterised bacterial global transcription factor, focusing on its role in pathogenicity of the prototype EAEC strain 042. We show that, although most functional CRP binding sites on the chromosome are conserved between E. coli K-12 and 042, CRP has been co-opted to couple the expression of some virulence genes to the nutritional state of the cell. We report novel mechanisms for CRP-dependent regulation of genes, whose products contribute to adhesion, production of a bacterial antibiotic, and export of a polysaccharide capsule. ### Competing Interest Statement The authors have declared no competing interest. Biotechnology and Biological Sciences Research Council, BB/R017689/1, BB/W00285X/1 Wellcome Trust, Investigator Award 212193/Z/18/Z Princess Nourah bint Abdulrahman University, PNURSP2025R898

Bacterial pathogens employ a diverse array of virulence factors to colonize and subsequently elicit disease in their host. These factors are often subject to extensive regulation at the transcriptional level to ensure that their expression is timely. Although many pathogens use bespoke transcription factors that primarily target virulence genes, global transcription factors also sometimes play a role in controlling these genes. Enteroaggregative Escherichia coli (EAEC) is a significant cause of watery and mucoid diarrhoea globally. The organism colonizes the small intestine before producing toxins that elicit disease, using a multitude of virulence factors that are encoded both chromosomally and on virulence plasmids. In this work, we have studied the cAMP receptor protein (CRP), a well-characterized bacterial global transcription factor, focusing on its role in the pathogenicity of the prototype EAEC strain 042. We show that, although most functional CRP binding sites on the chromosome are conserved between E. coli K-12 and 042, CRP has been co-opted to couple the expression of some virulence genes to the nutritional state of the cell. We report novel mechanisms for CRP-dependent regulation of genes whose products contribute to the maturation of a bacterial antibiotic, export of a polysaccharide capsule and production of a putative adhesin.

Despite the increased reporting of Carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKp) in Egypt, there is a paucity of information regarding the molecular characteristics of such strains. Herein, we present the genome sequence of two CR-hvKp strains, K22 and K45, which were isolated from VAP (ventilator-associated-pneumonia) patients admitted to pediatric ICU at Assiut University Children’s Hospital, Egypt. K22 and K45 isolates were subjected to antimicrobial susceptibility testing and whole-genome sequencing. Genomic analysis was performed to characterize each strain, determining their plasmids, antimicrobial resistance (AMR) genes, and virulence determinants. K22 possessed an extensive drug resistance phenotype (XDR), whilst K45 exhibited a multidrug resistance phenotype (MDR), with genome sequencing revealing the presence of a diverse array of AMR genes. Both strains were resistant to the carbapenem antibiotic imipenem, carrying the OXA-48 carbapenemase, with K22 additionally possessing an NDM-1 carbapenemase. Each strain was considered high-risk, with K22 and K45 respectively belonging to sequence types ST383 and ST14 and possessing virulence genes implicated in hypervirulence (e.g., iucABCD-iutA and rmpA). Importantly, both strains carried multiple plasmid replicons, including an AMR/virulence IncHI1B/FIB hybrid plasmid and MDR IncL/M plasmids. This report highlights the critical role of plasmids in the evolution of virulent K. pneumoniae strains and suggests the circulation of an IncHI1B/FIB hybrid plasmid, simultaneously disseminating AMR and hypervirulence, amongst K. pneumoniae strains within Assiut University Children’s Hospital.

Promoter-specific activation of transcript initiation provides an important regulatory device in Escherichia coli and Salmonella. Here, we describe the different mechanisms that operate, focusing on how they have evolved to manage the "housekeeping" bacterial transcription machinery. Some mechanisms involve assisting the bacterial DNA-dependent RNA polymerase or replacing or remodeling one of its subunits. Others are directed to chromosomal DNA, improving promoter function, or relieving repression. We discuss how different activators work together at promoters and how the present complex network of transcription factors evolved.

Holliday 4-way junctions are key to important biologicalDNA processes(insertion, recombination, and repair) and are dynamic structuresthat adopt either open or closed conformations, the open conformationbeing the biologically active form. Tetracationic metallo-supramolecularpillarplexes display aryl faces about a cylindrical core, an idealstructure to interact with open DNA junction cavities. Combining experimentalstudies and MD simulations, we show that an Au pillarplex can bindDNA 4-way (Holliday) junctions in their open form, a binding modenot accessed by synthetic agents before. Pillarplexes can bind 3-wayjunctions too, but their large size leads them to open up and expandthat junction, disrupting the base pairing, which manifests in anincreased hydrodynamic size and lower junction thermal stability.At high loading, they rearrange both 4-way and 3-way junctions intoY-shaped forks to increase the available junction-like binding sites.Isostructural Ag pillarplexes show similar DNA junction binding behaviorbut lower solution stability. This pillarplex binding contrasts with(but complements) that of metallo-supramolecular cylinders, whichprefer 3-way junctions and can rearrange 4-way junctions into 3-wayjunction structures. The pillarplexes' ability to bind open4-way junctions creates exciting possibilities to modulate and switchsuch structures in biology, as well as in synthetic nucleic acid nanostructures.In human cells, the pillarplexes do reach the nucleus, with antiproliferativeactivity at levels similar to those of cisplatin. The findings providea new roadmap for targeting higher-order junction structures usinga metallo-supramolecular approach, as well as expanding the toolboxavailable to design bioactive junction binders into organometallicchemistry.

Escherichia coli K-12 was originally isolated 100 years ago and since then it has become an invaluable model organism and a cornerstone of molecular biology research. However, despite its pedigree, since its initial isolation E. coli K-12 has been repeatedly cultured, passaged and mutagenized, resulting in an organism that carries many genetic changes. To understand more about this important model organism, we have sequenced the genomes of two ancestral K-12 strains, WG1 and EMG2, considered to be the progenitors of many key laboratory strains. Our analysis confirms that these strains still carry genetic elements such as bacteriophage lambda (λ) and the F plasmid, but also indicates that they have undergone extensive laboratory-based evolution. Thus, scrutinizing the genomes of ancestral E. coli K-12 strains leads us to examine whether E. coli K-12 is a sufficiently robust model organism for 21st century microbiology.

Over the decades, the bacterium Escherichia coli (E. coli) has become the cornerstone of recombinant protein production, used for heterologous synthesis of a variety of membrane proteins. Due to its rapid growth to high densities in cheap media, and its ease of manipulation and handling, E. coli is an excellent host cell for a range of membrane protein targets. Furthermore, its genetic tractability allows for a variety of gene constructs to be screened for optimal expression conditions, resulting in relatively high yields of membrane protein in a short amount of time. Here, we describe the general workflow for the production of membrane proteins in E. coli. The protocols we provide show how the gene of interest is modified, transferred to an expression vector and host, and how membrane protein yields can be optimized and analyzed. The examples we illustrate are well suited for scientists who are starting their journey into the world of membrane protein production.

Most Escherichia coli overexpression vectors used for recombinant protein production (RPP) depend on organic inducers, for example, sugars or simple conjugates. However, these can be expensive and, sometimes, chemically unstable. To simplify this and to cut the cost of RPP, we have developed vectors controlled by the Escherichia coli nitrate‐responsive NarL transcription activator protein, which use nitrate, a cheap, stable, and abundant inorganic ion, to induce high‐level controlled RPP. We show that target proteins, such as green fluorescent protein, human growth hormone, and single‐chain variable region antibody fragments can be expressed to high levels using our promoter systems. As nitrate levels are high in many commercial fertilizers, we demonstrate that controlled RPP can be achieved using readily available and inexpensive garden products.

We previously identified a 50-residue bacteriophage protein, gp7, which interacts with and modulates the function of the LexA transcription factor from Bacillus thuringiensis . Here, we present data that indicates that the small DdrR protein from A. baumannii likely coordinates the SOS response and prophage processes by also interacting with LexA superfamily members.

The serine protease autotransporters of the Enterobacteriaceae (SPATEs) are a large family of virulence factors commonly found in enteric bacteria. These secreted virulence factors have diverse functions during bacterial infection, including adhesion, aggregation and cell toxicity. One such SPATE, the Pic mucinase (protein involved in colonisation) cleaves mucin, allowing enteric bacterial cells to utilise mucin as a carbon source and to penetrate the gut mucus lining, thereby increasing mucosal colonisation. The pic gene is widely distributed within the Enterobacteriaceae, being found in human pathogens, such as enteroaggregative Escherichia coli (EAEC), uropathogenic E. coli (UPEC) and Shigella flexneri 2a. As the pic promoter regions from EAEC strain 042 and UPEC strain CFT073 differ, we have investigated the regulation of each promoter. Here, using in vivo and in vitro techniques, we show that both promoters are activated by the global transcription factor, CRP (cyclic AMP receptor protein), but the architectures of the EAEC and the UPEC pic promoter differ. Expression from both pic promoters is repressed by the nucleoid-associated factor, Fis, and maximal promoter activity occurs when cells are grown in minimal medium. As CRP activates transcription in conditions of nutrient depletion, whilst Fis levels are maximal in nutrient-rich environments, the regulation of the EAEC and UPEC pic promoters is consistent with Pic's nutritional role in scavenging mucin as a suitable carbon source during colonisation and infection.

We have developed a novel urea-inducible recombinant protein production system by exploiting the Proteus mirabilis urease ureR-ureD promoter region and the ureR AraC-family transcriptional regulator. Experiments using the expression of β-galactosidase and green fluorescent protein (GFP) showed that promoter activity is tightly regulated and that varying the concentration of urea can give up to 100-fold induction. Production of proteins of biopharmaceutical interest has been demonstrated, including human growth hormone (hGH), a single chain antibody fragment (scFv) against interleukin-1β and a potential Neisserial vaccine candidate (BamAENm). Expression levels can be fine-tuned by temperature and different urea concentrations, and can be induced with readily available garden fertilisers and even urine. As urea is an inexpensive, stable inducer, a urea-induced expression system has the potential to considerably reduce the costs of large-scale recombinant protein production.

Escherichia coli K-12 was originally isolated 100 years ago and since then, it has become an invaluable model organism and a cornerstone of molecular biology research. However, despite its apparent pedigree, since its initial isolation, E. coli K-12 has been repeatedly cultured, passaged, and mutagenized, resulting in an organism that carries extensive genetic changes. To understand more about the evolution of this important model organism, we have sequenced the genomes of two ancestral K-12 strains, WG1 and EMG2, considered to be the progenitors of many key laboratory strains. Our analysis confirms that these strains still carry genetic elements such as bacteriophage lambda (λ) and the F plasmid, but also indicates that they have undergone extensive lab-based evolution. Thus, scrutinizing the genomes of ancestral E. coli K-12 strains, leads us to question whether E. coli K-12 is a sufficiently robust model organism for 21st century microbiology. DATA SUMMARY All supporting data are provided within the article or through supplementary data files. Supplementary Figs. S1 to S14 and Supplementary File S1 are available with the online version of this article. All genome sequence data has been deposited in NCBI GenBank under Bioproject ID PRJNA848777. The assembled and annotated genomes of WG1 and EMG2 have been deposited with the accession numbers, CP099590 and CP099591 (WG1) and CP099588 and CP099589 (EMG2). Impact Statement Since its isolation in 1922, Escherichia coli K-12, has become arguably the premier model organism for contemporary science. The adoption of E. coli K-12 by many microbiologists across the globe, means that it has a complex pedigree, and, although many E. coli K-12 strains have been sequenced, little is known about the early versions of K-12, which still carry the F plasmid and bacteriophage λ. To understand more about the lab-based evolution that has shaped this important model organism, we have sequenced two ancestral K-12 strains, WG1 and EMG2, that are considered to be the progenitors of many of the laboratory strains used today.

Ecologically beneficial traits in bacteria are encoded by intrinsic and horizontally acquired genes. However, such traits are not universal, and the highly mosaic nature of bacterial genomes requires control at the transcriptional level to drive these processes. It has emerged that regulatory flexibility is widespread in the Escherichia coli species, whereby preexisting transcription factors can acquire new and unrelated roles in regulating beneficial traits. DsdC is the regulator of D-serine tolerance in E. coli, is essential for D-serine catabolism, and is often encoded by two copies in neonatal meningitis-associated E. coli (NMEC). Here, we reveal that DsdC is a global regulator of transcription in NMEC and does not require D-serine for the control of novel beneficial traits. We show that DsdC binds the chromosome in an unusual manner, with many binding sites arranged in clusters spanning entire operons and within gene coding sequences, such as neuO. Importantly, we identify neuO as the most significantly down-regulated gene in a strain deleted for both dsdC copies, in both the presence and absence of D-serine. NeuO is prophage encoded in several NMEC K1 isolates and mediates capsule O-acetylation but has no effect on attachment to or invasion of human brain endothelial cells. Instead, we demonstrate that NeuO provides resistance against K1 bacteriophage attack and that this critical function is regulated by DsdC. This work highlights how a horizontally acquired enzyme that functions in cell-surface modulation can be controlled by an intrinsic regulator to provide a key ecological benefit to an E. coli pathotype.

Many commonly used bacterial promoters employed for recombinant protein production (RPP) in Escherichia coli are capable of high-level protein expression. However, such promoter systems are often too strong, being ill suited for expressing proteins that are difficult to fold, targeted to the membrane or secreted out of the cytoplasm. To circumvent this problem, a suite of bacterial promoters has been constructed with a range of different promoter strengths, assigning them specific "promoter activity ratings" (PARs). Selecting three of these PAR promoters, with low, intermediate and high strengths, it is demonstrated that the expression of target proteins, such as green fluorescent protein (GFP), human growth hormone (hGH) and single chain variable region antibody fragments (scFvs), can be set to three levels when expressed in E. coli. It is shown that the PAR promoter system is extremely flexible, operating in a variety of E. coli strains and under various different culture regimes. Furthermore, due to its tight regulation, it is shown that this system can also express a toxic outer membrane protein, at levels which do not affect bacterial growth. Thus, the PAR promoter system can be used to tailor the expression levels of target proteins in E. coli and maximize RPP.

The BAM complex in Escherichia coli is composed of five proteins, BamA-E. BamA and BamD are essential for cell viability and are required for the assembly of β-barrel outer membrane proteins. Consequently, BamA and BamD are indispensable for secretion via the classical autotransporter pathway (Type 5a secretion). In contrast, BamB, BamC and BamE are not required for the biogenesis of classical autotransporters. Recently, we demonstrated that TamA, a homologue of BamA, and its partner protein TamB, were required for efficient secretion of proteins via the classical autotransporter pathway. The trimeric autotransporters are a subset of the Type 5-secreted proteins. Unlike the classical autotransporters, they are composed of three identical polypeptide chains which must be assembled together to allow secretion of their cognate passenger domains. In contrast to the classical autotransporters, the role of the Bam and Tam complex components in the biogenesis of the trimeric autotransporters has not been investigated fully. Here, using the Salmonella enterica trimeric autotransporter SadA and the structurally similar YadA protein of Yersinia spp., we identify the importance of BamA and BamD in the biogenesis of the trimeric autotransporters and reveal that BamB, BamC, BamE, TamA and TamB are not required for secretion of functional passenger domain on the cell surface. Importance The secretion of trimeric autotransporters (TAA’s) has yet to be fully understood. Here we show that efficient secretion of TAAs requires the BamA and D proteins, but does not require BamB, C or E. In contrast to classical autotransporter secretion, neither trimeric autotransporter tested required TamA or B proteins to be functionally secreted.

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis , a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5’ region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.

Klebsiella pneumoniae is an important human pathogen in both developing and industrialised countries that can causes a variety of human infections, such as pneumonia, urinary tract infections and bacteremia. Like many Gram-negative bacteria, it is becoming resistant to many frontline antibiotics, such as carbapenem and cephalosporin antibiotics. In Egypt, K. pneumoniae is increasingly recognised as an emerging pathogen, with high levels of antibiotic resistance. However, few Egyptian K. pneumoniae strains have been sequenced and characterised. Hence, here, we present the genome sequence of a multidrug resistant K. pneumoniae strain, KPE16, which was isolated from a child in Assiut, Egypt. We report that it carries multiple antimicrobial resistance genes, including a blaNDM-1 carbapenemase and extended spectrum β-lactamase genes (i.e., blaSHV-40, blaTEM-1B, blaOXA-9 and blaCTX-M-15). By comparing this strain with other Egyptian isolates, we identified common plasmids, resistance genes and virulence determinants. Our analysis suggests that some of the resistance plasmids that we have identified are circulating in K. pneumoniae strains in Egypt, and are likely a source of antibiotic resistance throughout the world.

Rising levels of antibiotic resistance dictate that new antibiotics with novel modes of action must be found. Here, we investigated the mode of action of a novel antibiotic that is a member of a family of synthetic DNA minor groove binding (MGB) molecules. MGB-BP-3 has successfully completed a Phase II clinical trial in humans as an orally administered drug for the treatment of chronic Clostridioides (Clostridium) difficile infections, where it outperformed the existing benchmark (vancomycin). MGB-BP-3 is active against a variety of Gram-positive pathogens including Staphylococcus aureus, which was used as the model for this study. The transcriptomic response of S. aureus to MGB-BP-3 identified downregulated promoters. DNase I and permanganate footprinting demonstrated binding to essential SigA promoters and the inhibition of promoter isomerisation by RNA polymerase holoenzyme. Promoters controlling DNA replication and peptidoglycan biosynthesis are amongst those affected by MGB-BP-3. Thus, MGB-BP-3 binds to and inhibits multiple essential promoters on the S. aureus chromosome, suggesting that evolution of resistance by drug target mutation should be unlikely. In confirmation, laboratory-directed evolution against sub-inhibitory concentrations of MGB-BP-3 resulted in no resistance whereas resistance to the single target RNA-polymerase inhibitor rifampicin arose rapidly.