Aug 2026· Access Microbiology· Vol 8· 0 citations· 28 references
Medicine
TL;DR
Growth conditions that limit the effects of ϕ80-dependent lysis when using GR536 to identify metal-uptake genes by complementation are clarified, specifically, removal of the antibiotic resistance markers and the avoidance of using intact pBAD30 as a negative control.
Abstract
Abstract We report the genome sequence of Escherichia coli GR536, a previously constructed metal-uptake-deficient strain derived from E. coli W3110. Growth of GR536 in an iron-restricted liquid medium resulted in apparent lysis during the early exponential growth phase. This effect was exacerbated in cells transformed with pBAD30, a commonly used arabinose-inducible expression vector. Whole-genome sequencing confirmed the expected gene disruptions (entC, feoABC, mntH, zupT::cat and fecABCDE::kan). However, comparison to E. coli W3110 identified the presence of the ϕ80 prophage (46.16 kbp) and cryptic prophage CPZ-55 (6.763 kbp), as well as the absence of cryptic prophage e14 (15.193 kbp). We also identified 9 IS-element deletions, 3 IS-element insertions, 7 other deletions or insertions and 74 candidate individual nucleotide changes. The growth defect in GR536 correlated with lysis due to the production of ϕ80 virions as determined by the inability of isolated phage to infect an E. coli strain lacking the phage receptor (∆fhuA) and the BamHI digestion pattern of the purified phage DNA. We further determined that the ϕ80-dependent lysis in GR536 is exacerbated by the presence of the chloramphenicol- and kanamycin-resistance markers introduced during construction of GR536 and the pBAD30 plasmid multiple cloning site. Removal of the markers (E. coli GR536*) and disruption of the pBAD30 multiple cloning site generated a strain that showed a 104-fold reduction in ϕ80 production. Furthermore, construction of a W3110 lysogen containing the ϕ80 prophage and comparison with GR536* grown under the same conditions showed a ~104-fold higher level of phage production by the parent strain, indicating that phage-dependent lysis was not increased by the deletion of the metal-uptake genes and thus independent of iron availability. These observations clarify growth conditions that limit the effects of ϕ80-dependent lysis when using GR536 to identify metal-uptake genes by complementation, specifically, removal of the antibiotic resistance markers and the avoidance of using intact pBAD30 as a negative control.
Lipopolysaccharide (LPS) is a major component of the outer membrane of Gram-negative bacteria, contributing to membrane integrity and environmental interactions. Genome-wide studies defining bacterial gene functions have been extensively performed in the model strain Escherichia coli K-12 which lacks O-antigen (OAg), and therefore does not produce smooth LPS (S-LPS). Consequently, the genetic requirements for S-LPS production in this model system remain incompletely defined. Here, a functional wbbL gene was introduced into the E. coli K-12 KEIO single-gene deletion mutant library to restore OAg synthesis, enabling genome-wide analysis of S-LPS production by screening with colicin E2 (ColE2) and validated with LPS silver staining. This identified 319 mutants with increased sensitivity to ColE2 in the presence of OAg, suggesting broader envelope-associated effects during screening. In addition, 27 mutants showed defects in S-LPS production, corresponding to genes involved in OAg biosynthesis, LPS core and sugar precursor synthesis, OAg ligation and regulation, and enterobacterial common antigen biosynthesis. A further 18 mutants initially appeared defective in S-LPS production but could not be validated upon reconstruction, and whole-genome sequencing revealed secondary mutations responsible for the observed phenotypes. This study provides a validated genetic framework for S-LPS production in E. coli K-12 and highlights the importance of rigorous validation in genome-wide screening approaches.
Jilong Qin, E. Tran, Vincenzo Leo et al.· bioRxiv· 0 citations
ABSTRACT Cefiderocol (FDC) is a new siderophore-conjugated cephalosporin that enters the periplasm via iron transport systems. However, the specific contribution of individual iron uptake pathways to FDC activity remains unclear. We investigated the role of 13 iron acquisition systems using several Escherichia coli strain collections. FDC MICs were determined in iron-depleted and iron-supplemented media for E. coli mutants (Keio and pathogenic island [PAI]-deleted collections) and for no-acquired β-lactamase or TEM (NoBL/TEM-Ec) and NDM-producing E. coli (NDM-Ec) clinical isolates. Prevalence of iron uptake genes was assessed in these isolates, and fec operon prevalence and genomic location were investigated in E. coli genomes from EnteroBase and RefSeq databases. Compared to K-12 and 536 reference E. coli, only ΔcirA and Δfiu (enterobactin system) showed increased FDC MICs (8- and 3-fold, respectively), while ΔfecA and ΔfecB had lower MICs (3-fold decrease). The fec operon, a known extraintestinal virulence factor, was significantly more prevalent in isolates with FDC MICs above median than below (96% vs. 33% in NoBL/TEM-Ec; 100% vs. 0% in NDM-Ec). According to EUCAST breakpoints, 63.6% of fec-positive NDM-Ec were resistant to FDC, whereas none of the fec-negative were. The fec operon was found in 46.5% of E. coli genomes, including virulent clones (ST131, 77%) and was mostly chromosome-borne (99%). Plasmid-borne fec closely resembled that of Enterobacter hormaechei or Klebsiella pneumoniae, suggesting interspecies transfer. Our findings highlight the role of Fec in reducing FDC susceptibility and promoting resistance in NDM-producing E. coli. They challenge the virulence-resistance trade-off demonstrating the “liaisons dangereuses” between iron and antibiotics.
Emilie Mallecot, Valentine Berti, Marie Petitjean et al.· Antimicrobial Agents and Che...· 0 citations
Escherichia coli cells can grow in a DnaA- and oriC-independent manner in rnhA-deficient cells that lack the major RNaseH activity. This mode of replication is named cSDR (constitutive stable DNA replication) and is believed to initiate from the sites of RNA-DNA hybrids, although the detailed mechanisms of cSDR are unclear. In this study, we discovered that dif sequence and XerC/D are essential for cSDR-dependent growth. cSDR was observed in either dif or xerC/D mutant cells as efficiently as in the control cells, as measured by the incorporation of [3H]-thymine. However, we found that these mutants accumulate cells with extra DNA contents in the presence of rifampicin and cefalexin, suggesting accumulation of unresolved dimeric or multimeric chromosomes or that of catenated chromosomes due to potential problems in resolution of the replicated chromosomes or in decatenation process. These results indicate that site-specific recombination mediated by dif-XerC/D is essential for cSDR-dependent growth.
Taku Tanaka, Rino Fukatsu, Y. Seki et al.· Molecular and Cellular Biolo...· 0 citations
Results identify P SD13 as a phage-derived promoter exhibiting strong activity in both Streptomyces and E. coli, suggesting its potential as a useful genetic element for Streptomyces engineering and heterologous gene expression.
Nana Lu· Applied Microbiology and Bio...· 0 citations
Avian pathogenic Escherichia coli (APEC) is a major cause of colibacillosis in poultry, yet the role of the ecpA gene, which encodes the major structural subunit of the Escherichia coli common pilus (ECP), remains incompletely defined in APEC pathogenesis. To investigate the role of ecpA in the biological characteristics and pathogenicity of Avian Pathogenic Escherichia coli (APEC) strain FJLY68, an ecpA deletion mutant (ΔecpA) and its corresponding complemented strain (CΔecpA) were constructed using the CRISPR/Cas9 system and verified by PCR and Sanger sequencing. Phenotypic analyses revealed that the ΔecpA mutation significantly impaired bacterial motility, biofilm formation, adherence to chicken embryonic fibroblast (DF-1) cells, and fimbriae assembly. Transcriptomic analysis identified 1720 differentially expressed genes in the ΔecpA mutant, significantly enriched in pathways associated with flagellar assembly, chemotaxis, and metabolism, consistent with the observed phenotypic changes. Although in vitro growth was unaffected, the ΔecpA mutant exhibited markedly attenuated virulence in a chick infection model, as indicated by an increased LD50, attenuated clinical signs and pathological lesions, and reduced bacterial colonisation in tissues. Full genetic complementation restored all observed defects to wild-type levels. This study identifies ecpA as a critical determinant of APEC pathogenesis, directly linking its function to bacterial motility, biofilm formation, adhesion, and in vivo virulence, and provides a theoretical basis for developing novel control strategies targeting this virulence factor.
The spread of antimicrobial-resistant Enterobacterales is a major One Health issue, with aquatic environments increasingly recognized as potential reservoirs. However, data on bacterial adaptation to water is limited. We examined the survival and transcriptomic adaptation of Escherichia coli and Klebsiella pneumoniae, including plasmid-cured variants (PCVs), during 14 days in sterilized tap and river water. The strains belonged to sequence types (ST)648 and ST307, representing international high-risk clonal lineages. Viable cell counts of wild-type strains and PCVs remained stable in both water types. We used RNA sequencing, followed by functional analysis of the differentially expressed genes. Considerable transcriptomic changes occurred, especially in K. pneumoniae, with extensive regulation of genes related to inorganic ion transport, and coenzyme and nutrient transport and metabolism. Adaptational differences between wild-type strains and PCVs highlighted plasmid-associated effects. These findings demonstrate strain-specific adaptive responses to aquatic environments and underline the context-dependent influence of plasmids in shaping adaptation.
Phillip Lübcke, S. Knauf, Elias Eger et al.· npj Emerging Contaminants· 0 citations