• E. coli
• bacterial persistence
• biofilm formation
• phage infection
• type II toxin-antitoxin

• was funded by the Project of the key R & D program of the 2022 year of Ningbo Science and Technology Bureau (2022z2202022), Key Project of Ningbo Municipal Science and Technology Bureau (2023j020), China Population Welfare Foundation Medical Innovation Project-Comparative Study on the Comprehensive Effectiveness of Artificial Intelligence Blood Collection Robots and Manual Blood Collection in Clinical Blood Collection Practice (SLB-6-20230912-351), Ningbo Medical Science and Technology Program Project (2022Y05) and Faculty-level Project of the First Hospital Affiliated to Ningbo University

• Pseudomonas aeruginosa
• TCA cycle
• Virulence factor
• sRNA
• srbA

• Pseudomonas aeruginosa/genetics
• Pseudomonas aeruginosa/pathogenicity
• Pseudomonas aeruginosa/metabolism
• Citric Acid Cycle/genetics
• Gene Expression Regulation, Bacterial
• Virulence/genetics
• Virulence Factors/genetics
• Virulence Factors/metabolism
• RNA, Small Untranslated/genetics
• RNA, Small Untranslated/metabolism
• Bacterial Proteins/genetics
• Bacterial Proteins/metabolism
• RNA, Bacterial/genetics
• RNA, Bacterial/metabolism
• Biofilms/growth & development
• Microbial Viability

• Escherichia coli
• avian pathogenic Escherichia coli
• colibacillosis
• poultry
• public health

• cAMP
• cyaA
• mutation
• β-carotene

• Escherichia coli/genetics
• Escherichia coli/metabolism
• beta Carotene/biosynthesis
• beta Carotene/metabolism
• Cyclic AMP/metabolism
• Escherichia coli Proteins/genetics
• Escherichia coli Proteins/metabolism
• Adenylyl Cyclases/genetics
• Adenylyl Cyclases/metabolism
• Gene Deletion
• Genomics/methods
• Metabolic Engineering/methods
• Gene Expression Regulation, Bacterial
• Carotenoids/metabolism
• Genome, Bacterial
• Mutagenesis

• RS-2022-KS221581 Ministry of Oceans and Fisheries

• Biofilms/drug effects
• Biofilms/growth & development
• Escherichia coli K12/genetics
• Escherichia coli K12/metabolism
• Escherichia coli K12/drug effects
• Escherichia coli K12/physiology
• Escherichia coli Proteins/metabolism
• Escherichia coli Proteins/genetics
• Gene Expression Regulation, Bacterial
• Fimbriae, Bacterial/metabolism
• Fimbriae, Bacterial/genetics
• Oxidative Stress
• Promoter Regions, Genetic
• Erythritol/analogs & derivatives
• Erythritol/metabolism
• Erythritol/pharmacology

• R01 GM107311 NIGMS NIH HHS
• R01GM107311-8 U.S. Department of Health & Human Services | NIH | National Institute of General Medical Sciences (NIGMS)

• Escherichia coli
• biofilms
• glycopolymers
• phosphoethanolamine cellulose

• Biofilms/drug effects
• Cellulose/chemistry
• Cellulose/pharmacology
• Ethanolamines/chemistry
• Ethanolamines/pharmacology
• Uropathogenic Escherichia coli/drug effects
• Uropathogenic Escherichia coli/physiology
• Bacterial Adhesion/drug effects

• R01 HD090061 NICHD NIH HHS
• P30 DK058404 NIDDK NIH HHS
• P30 EY008126 NEI NIH HHS
• P30 CA068485 NCI NIH HHS
• R35 GM133602 NIGMS NIH HHS
• U24 DK059637 NIDDK NIH HHS
• S10 OD028704 NIH HHS
• R01 AI134036 NIAID NIH HHS
• I01 BX005352 BLRD VA
• P30 DK020593 NIDDK NIH HHS

• E. coli
• Salmonella
• Chaperone/Usher fimbriae
• Gene regulation
• UPEC

• Fimbriae, Bacterial/metabolism
• Fimbriae, Bacterial/genetics
• Molecular Chaperones/metabolism
• Molecular Chaperones/genetics
• Fimbriae Proteins/genetics
• Fimbriae Proteins/metabolism
• Gene Expression Regulation, Bacterial
• Bacterial Adhesion
• Operon

• 463840 Consejo Nacional de Ciencia y Tecnología
• 749442 Consejo Nacional de Ciencia y Tecnología
• FC-2015-2/950 Consejo Nacional de Ciencia y Tecnología
• IN209713, IN213516, IN215119 Dirección General de Asuntos del Personal Académico, Universidad Nacional Autónoma de México

• antimicrobial resistance genotype
• multidrug-resistant
• pulse field gel electrophoresis
• virulome expression

• Humans
• Anti-Bacterial Agents/pharmacology
• Anti-Bacterial Agents/therapeutic use
• Klebsiella pneumoniae
• Community-Acquired Infections/drug therapy
• Community-Acquired Infections/genetics
• Genotype
• Cross Infection/drug therapy
• Cross Infection/genetics
• Cross Infection/microbiology
• Hospitals
• Microbial Sensitivity Tests
• beta-Lactamases/genetics
• Drug Resistance, Multiple, Bacterial/genetics

• Enterobacterales
• multidrug-resistance
• virulence

• BT/PR40148/BTIS/137/20/2021 Department of Biotechnology

• Biofilm
• C-di-AMP
• C-di-GMP
• Efficacy of antibiotics
• Nucleotide
• Small molecule

• Ciprofloxacin/pharmacology
• Staphylococcus epidermidis
• Nucleotides
• Biofilms
• Anti-Bacterial Agents/pharmacology
• Cyclic GMP/metabolism
• Cyclic GMP/pharmacology
• Biocompatible Materials/pharmacology
• Adenosine Monophosphate

• R01 HL153231 NHLBI NIH HHS

• ESBL
• One Health
• blaCTX-M-15
• dog
• genomic surveillance

• Bacteremia
• Bloodstream infection
• Escherichia coli
• ExPEC
• Virulence

• Humans
• Escherichia coli/genetics
• Virulence/genetics
• HEK293 Cells
• Escherichia coli Infections/microbiology
• Caco-2 Cells
• Extraintestinal Pathogenic Escherichia coli/genetics
• Sepsis/microbiology
• Virulence Factors/genetics
• Urinary Tract Infections/microbiology
• Phylogeny

• 304760/2015-3 Conselho Nacional de Desenvolvimento Científico e Tecnológico
• 311283/2020-9 Conselho Nacional de Desenvolvimento Científico e Tecnológico
• 168193/2018-3 Conselho Nacional de Desenvolvimento Científico e Tecnológico
• 2018/17353-7 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 2019/21685-8 Fundação de Amparo à Pesquisa do Estado de São Paulo
• 88882.306532/2018-01 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior

• Escherichia coli
• PNPase
• RNA degradation
• cyclic AMP receptor protein
• knockout
• metabolism
• persister cells
• persister mechanisms

• Escherichia coli
• Escherichia coli Proteins/metabolism
• Polyribonucleotide Nucleotidyltransferase/genetics
• Polyribonucleotide Nucleotidyltransferase/metabolism
• Operon
• Anti-Bacterial Agents/pharmacology
• Anti-Bacterial Agents/metabolism
• Gene Expression Regulation, Bacterial

• LTA subunit
• LTB subunit
• bacterial adherence
• enterotoxigenic Escherichia coli
• heat-labile enterotoxin

• DNA gyrase
• DNA supercoiling
• persisters
• topoisomerases
• transcription bursting

The structural genes expressing type 1 fimbriae in Escherichia coli alternate between expressed (phase ON) and non-expressed (phase OFF) states due to inversion of the 314 bp fimS genetic switch. The FimB tyrosine integrase inverts fimS by site-specific recombination, alternately connecting and disconnecting the fim operon, encoding the fimbrial subunit protein and its associated secretion and adhesin factors, to and from its transcriptional promoter within fimS. Site-specific recombination by the FimB recombinase becomes biased towards phase ON as DNA supercoiling is relaxed, a condition that occurs when bacteria approach the stationary phase of the growth cycle. This effect can be mimicked in exponential phase cultures by inhibiting the negative DNA supercoiling activity of DNA gyrase. We report that this bias towards phase ON depends on the presence of the Fis nucleoid-associated protein. We mapped the Fis binding to a site within the invertible fimS switch by DNase I footprinting. Disruption of this binding site by base substitution mutagenesis abolishes both Fis binding and the ability of the mutated switch to sustain its phase ON bias when DNA is relaxed, even in bacteria that produce the Fis protein. In addition, the Fis binding site overlaps one of the sites used by the Lrp protein, a known directionality determinant of fimS inversion that also contributes to phase ON bias. The Fis–Lrp relationship at fimS is reminiscent of that between Fis and Xis when promoting DNA relaxation-dependent excision of bacteriophage λ from the E. coli chromosome. However, unlike the co-binding mechanism used by Fis and Xis at λ attR, the Fis–Lrp relationship at fimS involves competitive binding. We discuss these findings in the context of the link between fimS inversion biasing and the physiological state of the bacterium.

• DNA supercoiling
• Fis
• nucleoid-associated protein
• nucleoprotein complexes
• type 1 fimbriae

As one of the crucial enterotoxins secreted by enterotoxigenic Escherichia coli (ETEC), heat-labile enterotoxin (LT) enhances bacterial adherence both in vivo and in vitro; however, the underlying mechanism remains unclear. To address this, we evaluated the adherence of LT-producing and LT-deficient ETEC strains using the IPEC-J2 cell model. The expression levels of inflammatory cytokines and chemokines, and tight-junction proteins were evaluated in IPEC-J2 cells after infection with various ETEC strains. Further, the levels of adhesins and enterotoxins were also evaluated in F4ac-producing ETEC (F4 + ETEC) strains after treatment with cyclic AMP (cAMP). The adherence of the ΔeltAB mutant was decreased compared with the wild-type strain, whereas adherence of the 1836-2/pBR322-eltAB strain was markedly increased compared with the 1836-2 parental strain. Production of LT up-regulated the expression of TNF-α, IL-6, CXCL-8, and IL-10 genes. However, it did not appear to affect tight junction protein expression. Importantly, we found that cAMP leads to the upregulation of adhesin production and STb enterotoxin. Moreover, the F4 + ETEC strains treated with cAMP also had greater adhesion to IPEC-J2 cells, and the adherence of ΔfaeG, ΔfliC, and ΔestB mutants was decreased. These results indicate that LT enhances the adherence of F4 + ETEC due primarily to the upregulation of F4 fimbriae, flagellin, and STb enterotoxin expression and provide insights into the pathogenic mechanism of LT and ETEC.

• ETEC
• Heat-labile enterotoxin
• adherence
• fimbriae
• pathogenesis

Bacterial transcription factors (TFs) are widely studied in Escherichia coli. Yet it remains unclear how individual genes in the underlying pathways of TF machinery operate together during environmental challenge. Here, we address this by applying an unbiased, quantitative synthetic genetic interaction (GI) approach to measure pairwise GIs among all TF genes in E. coli under auxotrophic (rich medium) and prototrophic (minimal medium) static growth conditions. The resulting static and differential GI networks reveal condition-dependent GIs, widespread changes among TF genes in metabolism, and new roles for uncharacterized TFs (yjdC, yneJ, ydiP) as regulators of cell division, putrescine utilization pathway, and cold shock adaptation. Pan-bacterial conservation suggests TF genes with GIs are co-conserved in evolution. Together, our results illuminate the global organization of E. coli TFs, and remodeling of genetic backup systems for TFs under environmental change, which is essential for controlling the bacterial transcriptional regulatory circuits.

The bacterium E. coli has around 300 transcriptional factors, but the functions of many of them, and the interactions between their respective regulatory networks, are unclear. Here, the authors study genetic interactions among all transcription factor genes in E. coli, revealing condition-dependent interactions and roles for uncharacterized transcription factors.

Functionally related genes in bacteria are often organized and transcribed as polycistronic transcriptional units. Examples are the fim operon, which codes for biogenesis of type 1 fimbriae in Escherichia coli, and the atp operon, which codes for the FoF1 ATP synthase. We tested the hypothesis that markerless polar mutations could be efficiently engineered using CRISPR/Cas12a in these loci.

Cas12a-mediated engineering of a terminator sequence inside the fimA gene occurred with efficiencies between 10 and 80% and depended on the terminator’s sequence, whilst other types of mutations, such as a 97 bp deletion, occurred with 100% efficiency. Polar mutations using a terminator sequence were also engineered in the atp locus, which induced its transcriptional shutdown and produced identical phenotypes as a deletion of the whole atp locus (ΔatpIBEFHAGDC). Measuring the expression levels in the fim and atp loci showed that many supposedly non-polar mutants induced a significant polar effect on downstream genes. Finally, we also showed that transcriptional shutdown or deletion of the atp locus induces elevated levels of intracellular ATP during the exponential growth phase.

We conclude that Cas12a-mediated mutagenesis is an efficient simple system to generate polar mutants in E. coli. Different mutations were induced with varying degrees of efficiency, and we confirmed that all these mutations abolished the functions encoded in the fim and atp loci. We also conclude that it is difficult to predict which mutagenesis strategy will induce a polar effect in genes downstream of the mutation site. Furthermore the strategies described here can be used to manipulate the metabolism of E. coli as showcased by the increase in intracellular ATP in the markerless ΔatpIBEFHAGDC mutant.

The online version contains supplementary material available at 10.1186/s12934-022-01844-y.

• CRISPR mutagenesis
• Cas12a
• Intracellular ATP
• Polycistronic operons
•  Markerless genome editing

Enterotoxigenic Escherichia coli (ETEC) strains are one of the most common etiological agents of diarrhea in both human and farm animals. In addition to encoding toxins that cause diarrhea, ETEC have evolved numerous strategies to interfere with host defenses. These strategies most likely depend on the sensing of host factors, such as molecules secreted by gut epithelial cells. The present study tested whether the exposure of ETEC to factors secreted by polarized IPEC-J2 cells resulted in transcriptional changes of ETEC-derived virulence factors. Following the addition of host-derived epithelial factors, genes encoding enterotoxins, secretion-system-associated proteins, and the key regulatory molecule cyclic AMP (cAMP) receptor protein (CRP) were substantially modulated, suggesting that ETEC recognize and respond to factors produced by gut epithelial cells. To determine whether these factors were heat sensitive, the IEC-conditioned medium was incubated at 56 °C for 30 min. In most ETEC strains, heat treatment of the IEC-conditioned medium resulted in a loss of transcriptional modulation. Taken together, these data suggest that secreted epithelial factors play a role in bacterial pathogenesis by modulating the transcription of genes encoding key ETEC virulence factors. Further research is warranted to identify these secreted epithelial factors and how ETEC sense these molecules to gain a competitive advantage in the early engagement of the gut epithelium.

• enterotoxigenic Escherichia coli
• enterotoxins
• gut epithelium

While primarily Gram-positive bacteria cause bacterial eye infections, several Gram-negative species also pose eye health risks. Currently, few studies have tried to understand the pathogenic mechanisms involved in E. coli eye infections. Therefore, this study aimed to establish the pathogenic potential of E. coli strains isolated from eye infections. Twenty-two strains isolated between 2005 and 2019 from patients with keratitis or conjunctivitis were included and submitted to traditional polymerase chain reactions (PCR) to define their virulence profile, phylogeny, clonal relationship, and sequence type (ST). Phenotypic assays were employed to determine hemolytic activity, antimicrobial susceptibility, and adhesion to human primary corneal epithelial cells (PCS-700-010). The phylogenetic results indicated that groups B2 and ST131 were the most frequent. Twenty-five virulence genes were found among our strains, with ecp, sitA, fimA, and fyuA being the most prevalent. Two strains presented a hemolytic phenotype, and resistance to ciprofloxacin and ertapenem was found in six strains and one strain, respectively. Regarding adherence, all but one strains adhered in vitro to corneal cells. Our results indicate significant genetic and virulence variation among ocular strains and point to an ocular pathogenic potential related to multiple virulence mechanisms.

In Escherichia coli the expression of type 1 pili (T1P) is determined by the site-specific inversion of the fimS ON–OFF switch located immediately upstream of major fimbrial subunit gene fimA. Here we investigated the role of virulence (Ler, GrlR, and GrlA) and global regulators (H-NS, IHF, and Fis) in the regulation of the fimS switch in the human enteropathogenic E. coli (EPEC) O127:H6 strain E2348/69. This strain does not produce detectable T1P and PCR analysis of the fimS switch confirmed that it is locked in the OFF orientation. Among the regulator mutants analyzed, only the ∆fis mutant produced significantly high levels of T1P on its surface and yielded high titers of agglutination of guinea pig erythrocytes. Expression analysis of the fimA, fimB, and fimE promoters using lacZ transcriptional fusions indicated that only PfimA activity is enhanced in the absence of Fis. Collectively, these data demonstrate that Fis is a negative regulator of T1P expression in EPEC and suggest that it is required for the FimE-dependent inversion of the fimS switch from the ON-to-OFF direction. It is possible that a similar mechanism of T1P regulation exists in other intestinal and extra-intestinal pathogenic classes of E. coli.

• EPEC
• Fis
• fimS switch
• phase variation
• type 1 pilus

Uropathogenic Escherichia coli (UPEC) is the principal etiology of more than half of urinary tract infections (UTI) in humans with diabetes mellitus. Epidemiological data and studies in mouse model of ascending UTI have elucidated various host factors responsible for increasing the susceptibility of diabetic hosts to UPEC-UTI. In contrast, diabetic urinary microenvironment-mediated alterations in UPEC physiology and its contributions to shaping UPEC-UTI pathogenesis in diabetes have not been examined. To address our central hypothesis that glycosuria directly induces urinary virulence of UPEC, we compared virulence characteristics and gene expression in human UPEC strains UTI89 (cystitis) and CFT073 (pyelonephritis), exposed for 2 h in vitro to urine from either male or female donors that was either plain or supplemented with glucose to mimic glycosuria. Compared to control UPEC exposed to nutrient-rich culture medium, lysogeny broth, glycosuria-exposed UPEC exhibited significant increase in biofilm formation and reduction in the hemagglutination of Guinea pig erythrocytes (a measure of type 1 piliation). In addition, the analysis of UTI89 transcriptome by RNA sequencing revealed that 2-h-long, in vitro exposure to glycosuria also significantly alters expression of virulence and metabolic genes central to urinary virulence of UPEC. Addition of galactose as an alternative carbon source affected biofilm formation and gene expression profile of UPEC to an extent similar to that observed with glucose exposure. In summary, our results provide novel insights into how glycosuria-mediated rapid changes in UPEC fitness may facilitate UTI pathogenesis in the diabetic urinary microenvironment.

IMPORTANCE Uropathogenic Escherichia coli (UPEC) is an important causative agent of urinary tract infections in diabetic humans. We examined the effects of in vitro exposure to glycosuria (presence of glucose in urine) on the virulence and gene expression by UPEC. Our results show that glycosuria rapidly (in 2 h) alters UPEC gene expression, induces biofilm formation, and suppresses type 1 piliation. These results offer novel insights into the pathogenesis of UPEC in the urinary tract.

• RNA-seq
• UPEC
• carbon metabolism
• diabetes
• glycosuria
• iron utilization
• urinary tract infection
• uropathogenic Escherichia coli
• virulence factors

• Adherent bacteria
• Gene expression profiles
• Strain CFT073
• UPEC
• Uropathogenic Escherichia coli

Urinary tract infections (UTIs) are one of the most common bacterial infections with uropathogenic Escherichia coli (UPEC) being the most prevalent causative agent in both complicated and uncomplicated UTIs. Antibiotic resistance among UPEC has been already demonstrated against a wide variety of antibiotics and the situation is continuing to deteriorate increasing the rate of recurrence and the difficulty of treatment and prophylaxis. Recently, a big attention has been paid to non-antibiotic approaches as an alternative to conventional antibiotics. Among many strategies, phytotherapy has gained a special attention worldwide. Herbal remedies have been used in traditional medicine since ancient times and they are well known for their effectiveness in treating many health conditions including UTIs. Researches are conducted continuously to validate the use of many medicinal plants against UPEC, investigate their mechanisms of action, and determine their active constituents. Our extensive review of the recent literature revealed that many phytochemicals are shown to target and inhibit a wide variety of bioprocesses in UPEC, such as adhesion, motility, biofilm formation, and quorum sensing. Such natural approaches are very promising in confronting the antibiotic resistance of UPEC and can be further used to develop plant-based strategies and pharmaceutical products to treat and prevent UTIs caused by UPEC.

• Escherichia coli
• adhesion
• stress response
• type 1 fimbriae

• NSERC Discovery program, project 2019-06642 Natural Sciences and Engineering Research Council

• Escherichia coli K-12
• H-NS silencer
• anti-silencer LeuO
• gSELEX
• genome regulation
• network hierarchy
• transcription factor

• Adaptation, Physiological
• Adhesins, Bacterial/genetics
• Adhesins, Bacterial/metabolism
• Animals
• Biofilms/growth & development
• Escherichia coli Infections/microbiology
• Fimbriae, Bacterial/metabolism
• Gene Expression Profiling
• Humans
• Macrophages/microbiology
• Mice
• Osmoregulation
• Oxidative Stress
• RNA, Bacterial/genetics
• RNA, Small Untranslated/genetics
• Sequence Deletion
• Urinary Tract Infections/microbiology
• Uropathogenic Escherichia coli/genetics
• Uropathogenic Escherichia coli/growth & development
• Uropathogenic Escherichia coli/physiology
• Virulence

• CIHR
• Natural Sciences and Engineering Research Council of Canada
• Canadian Institutes of Health Research
• Fonds de recherche du Québec – Santé
• Fonds de Recherche du Québec - Nature et Technologies

• Escherichia coli
• FimA
• Flagellin
• FliC
• OMV

• Crp
• Std
• fimbriae
• promoter
• regulation

• dapagliflozin, diabetic complication, diabetic drug, hyperglucosuria, urinary tract infection, uropathogenic Escherichia coli

• Animals
• Benzhydryl Compounds
• Escherichia coli Infections/complications
• Female
• Glucosides
• Mice
• Mice, Inbred Strains
• Urinary Tract
• Urinary Tract Infections/chemically induced
• Uropathogenic Escherichia coli

• R01 DK114224 NIDDK NIH HHS
• R21 AI135645 NIAID NIH HHS
• UL1 TR001420 NCATS NIH HHS

Biofilm formation is a complex phenomenon of bacterial cells, involved in several human infections. Its formation is regulated and controlled by several protein factors. The BolA-like proteins (bolA gene) are conserved in both prokaryotes and eukaryotes. The BolA protein is a transcription factor involved in bacterial cell motility and biofilm formation. This study was initiated to elucidate the role of the bolA gene in the curli biogenesis and amyloid production as well as to observe changes in the expression of fimH, a fimbriae gene.

Knockdown mutants of Escherichia coli MG1655 bolA gene (bolA-KD) were generated using CRISPR interference. The results obtained, were validated through gene expression using RT-PCR, microscopic analysis and different biofilm and amyloid assays.

The bolA knockdown mutants showed a decrement in curli amyloid fibers, in fimbriae production and biofilm formation. We have also observed a reduction in EPS formation, eDNA production and extracellular protein content. Gene expression data showed that bolA downregulation caused the suppression of csgA and csgD of curli that led to the reduction in curli fiber and the amyloid formation and also the suppression of fimH, leading to the loss of fimbriae.

Curli fibers and fimbriae are found to be involved in biofilm formation leading to the pathogenicity of the bacterial cell. BolA is a conserved protein and is found to play a significant role in curli and fimbriae formation in E. coli. This study further proved that CRISPRi mediated suppression of the bolA gene leads to inhibition of biofilm formation through curli and fimbriae inhibition. Hence, it may be proposed as a possible target for intervention of biofilm mediated infections.

• Biofilm
• CRISPRi
• Escherichia coli
• Extra polymeric substance (EPS)
• bolA
• bolA gene knockdown strain (bol-KD)
• dCas9

Biofilms are communities of microorganisms that live in a self-produced extracellular matrix in order to survive in hostile environments. Second messengers, such as c-di-GMP and cAMP, participate in the regulation of biofilm formation. c-di-GMP is a major molecule that is involved in modulating the bacterial transition between a planktonic lifestyle and biofilm formation. Aside from regulating carbon catabolism repression in most bacteria, cAMP has also been found to mediate biofilm formation in many bacteria. Although the underlying mechanisms of biofilm formation mediated by cAMP-CRP have been well-investigated in several bacteria, the regulatory pathways of cAMP-CRP are still poorly understood compared to those of c-di-GMP. Moreover, some bacteria appear to form biofilm in response to changes in carbon source type or concentration. However, the relationship between the carbon metabolisms and biofilm formation remains unclear. This mini-review provides an overview of the cAMP-CRP-regulated pathways involved in biofilm formation in some bacteria. This information will benefit future investigations of the underlying mechanisms that connect between biofilm formation with nutrient metabolism, as well as the cross-regulation between multiple second messengers.

• biofilm
• c-di-GMP
• cAMP-CRP
• carbon catabolite repression
• second messengers

• Biofilm
• Cell motility
• Lysobacter
• Mass spectrometric imaging
• Rhizosphere
• Secondary metabolites

Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

The Shigella species are Gram-negative, facultative intracellular pathogens that invade the colonic epithelium and cause significant diarrheal disease. Despite extensive research on the pathogen, a comprehensive understanding of how Shigella initiates contact with epithelial cells remains unknown. Shigella maintains many of the same Escherichia coli adherence gene operons; however, at least one critical gene component in each operon is currently annotated as a pseudogene in reference genomes. These annotations, coupled with a lack of structures upon microscopic analysis following growth in laboratory media, have led the field to hypothesize that Shigella is unable to produce fimbriae or other traditional adherence factors. Nevertheless, our previous analyses have demonstrated that a combination of bile salts and glucose induces both biofilm formation and adherence to colonic epithelial cells. The goal of this study was to perform transcriptomic and genetic analyses to demonstrate that adherence gene operons in Shigella flexneri strain 2457T are functional, despite the gene annotations. Our results demonstrate that at least three structural genes facilitate S. flexneri 2457T adherence for epithelial cell contact and biofilm formation. Furthermore, our results demonstrate that host factors, namely, glucose and bile salts at their physiological concentrations in the small intestine, offer key environmental stimuli required for adherence factor expression in S. flexneri. This research may have a significant impact on Shigella vaccine development and further highlights the importance of utilizing in vivo-like conditions to study bacterial pathogenesis.

IMPORTANCE Bacterial pathogens have evolved to regulate virulence gene expression at critical points in the colonization and infection processes to successfully cause disease. The Shigella species infect the epithelial cells lining the colon to result in millions of cases of diarrhea and a significant global health burden. As antibiotic resistance rates increase, understanding the mechanisms of infection is vital to ensure successful vaccine development. Despite significant gains in our understanding of Shigella infection, it remains unknown how the bacteria initiate contact with the colonic epithelium. Most pathogens harbor multiple adherence factors to facilitate this process, but Shigella was thought to have lost the ability to produce these factors. Interestingly, we have identified conditions that mimic some features of gastrointestinal transit and that enable Shigella to express adherence structural genes. This work highlights aspects of genetic regulation for Shigella adherence factors and may have a significant impact on future vaccine development.

• Shigella flexneri
• adherence factors
• bile salts
• biofilm
• curli
• epithelial cells
• glucose
• in vivo-like conditions
• intestinal
• long polar fimbriae
• type 1 fimbriae

• Escherichia coli
• carbon metabolism
• iron metabolism
• nitrogen metabolism
• nucleotide metabolism
• urinary tract infection

• Escherichia coli
• luciferase
• stress
• type 1 fimbriae
• urinary tract

• Adhesins, Bacterial/metabolism
• Adult
• Animals
• Disease Models, Animal
• Escherichia coli Infections/microbiology
• Escherichia coli Proteins/genetics
• Escherichia coli Proteins/metabolism
• Extraintestinal Pathogenic Escherichia coli
• Female
• Fimbriae, Bacterial/metabolism
• Gene Expression Regulation, Bacterial
• Humans
• Intracellular Signaling Peptides and Proteins/genetics
• Intracellular Signaling Peptides and Proteins/metabolism
• Mice
• Oxidative Stress/physiology
• Urinary Tract Infections/microbiology
• Uropathogenic Escherichia coli/genetics
• Uropathogenic Escherichia coli/metabolism
• Virulence
• Virulence Factors/genetics
• Young Adult

• ExPEC
• UPEC
• UTI
• central metabolism
• glycolysis
• urinary tract infection
• uropathogenic

• DNA supercoiling
• RNA polymerase
• conditional knockdown
• relaxation-stimulated transcription
• supercoiling-sensitive promoters

• CRP
• Escherichia coli (E. coli)
• GTPase
• chaperone
• cold adaptation
• cyclic AMP (cAMP)
• ribosome assembly

• autotransporter proteins
• phase switch
• tyrosine recombinases
• urinary tract infection
• uropathogenic E. coli

• Animals
• Attachment Sites, Microbiological
• Cell Line
• Disease Models, Animal
• Epithelial Cells/microbiology
• Escherichia coli Infections/microbiology
• Escherichia coli Proteins/genetics
• Female
• Fimbriae, Bacterial/genetics
• Gene Expression Regulation, Bacterial
• Humans
• Kidney/microbiology
• Mice
• Mice, Inbred CBA
• Recombinases/genetics
• Recombinases/metabolism
• Regulatory Sequences, Nucleic Acid
• Type V Secretion Systems/genetics
• Urinary Tract Infections/microbiology
• Uropathogenic Escherichia coli/genetics
• Virulence Factors/genetics
• Virulence Factors/metabolism

• R01 DK063250 NIDDK NIH HHS
• DH | National Institute for Health Research (NIHR)
• Department of Health | National Health and Medical Research Council (NHMRC)

Bacterial persistence is a transient, nonheritable physiological state that provides tolerance to bactericidal antibiotics. The stringent response, toxin-antitoxin modules, and stochastic processes, among other mechanisms, play roles in this phenomenon. How persistence is regulated is relatively ill defined. Here we show that cyclic AMP, a global regulator of carbon catabolism and other core processes, is a negative regulator of bacterial persistence in uropathogenic Escherichia coli, as measured by survival after exposure to a β-lactam antibiotic. This phenotype is regulated by a set of genes leading to an oxidative stress response and SOS-dependent DNA repair. Thus, persister cells tolerant to cell wall-acting antibiotics must cope with oxidative stress and DNA damage and these processes are regulated by cyclic AMP in uropathogenic E. coli.

Bacterial persister cells are important in relapsing infections in patients treated with antibiotics and also in the emergence of antibiotic resistance. Our results show that in uropathogenic E. coli, the second messenger cyclic AMP negatively regulates persister cell formation, since in its absence much more persister cells form that are tolerant to β-lactams antibiotics. We reveal the mechanism to be decreased levels of reactive oxygen species, specifically hydroxyl radicals, and SOS-dependent DNA repair. Our findings suggest that the oxidative stress response and DNA repair are relevant pathways to target in the design of persister-specific antibiotic compounds.

• CRP
• DNA damage
• SOS response
• Tn-Seq
• antibiotics
• cAMP
• dormancy
• oxidative stress
• persister cells

Salmonella is one of the most frequently reported causes of foodborne illness worldwide. Non-typhoidal serovars cause gastroenteritis in humans. Salmonella can grow on surfaces forming biofilms, contributing to its persistence since biofilms are difficult to eradicate due to the high resistance to antimicrobials and disinfectants. It has been described that there are two crucial biofilm promoting factors in Salmonella: curli and cellulose. The expression of both factors is coordinately regulated by the transcriptional regulator CsgD. Most biofilm studies of Salmonella have been performed by growing bacteria in low osmolarity rich medium and low temperature (25°C). In such conditions, the biofilm is formed at the air–liquid interface (pellicle biofilm). Remarkably, when Salmonella grow in minimal medium, biofilm formation switches from the air–liquid interface to the solid–liquid interface (bottom biofilm). In this report, the switching between pellicle and bottom biofilm has been characterized. Our data indicate that curli, but not cellulose, is crucial for the formation of both kinds of biofilms. In minimal medium, conditions promoting formation of bottom biofilm, a high transcriptional expression of csgD and consequently of the genes involved in the synthesis of curli and cellulose was detected. The nutritional status of the cells seems to be pivotal for the spatial distribution of the biofilms formed. When bacteria is growing in minimal medium the addition of amino acids downregulates the expression of csgB and causes the switch between bottom and pellicle biofilm. The crosstalk between general metabolism and biofilm formation is also highlighted by the fact that the metabolic sensor cAMP modulates the type of biofilm generated by Salmonella. Moreover, cAMP regulates transcriptional expression of csgD and stimulates pellicle biofilm formation, suggesting that the physiological conditions define the type of biofilm formed by Salmonella. The consequences of the switching between pellicle and bottom biofilm during either infection or survival in natural environments remain undercover.

• Salmonella
• adenylate cyclase
• amino acids
• biofilm
• cellulose
• curli
• nutrient composition studies

Cyclic AMP receptor protein (Crp) is a major transcriptional regulator in bacteria. This study demonstrated that Crp affects numerous virulence-related phenotypes, including colonization of mice, motility, fimbria-mediated adhesion, and glucose stress tolerance in uropathogenic Proteus mirabilis. Diabetic mice were more susceptible to kidney colonization by wild-type strain than nondiabetic mice, in which the crp mutant exhibited increased kidney colonization. Loss of crp or addition of 10% glucose increased the P. mirabilis adhesion to kidney cells. Direct negative regulation of pmpA (which encodes the major subunit of P-like fimbriae) expression by Crp was demonstrated using a reporter assay and DNase I footprinting. Moreover, the pmpA/crp double mutant exhibited reduced kidney adhesion comparable to that of the pmpA mutant, and mouse kidney colonization by the pmpA mutant was significantly attenuated. Hence, the upregulation of P-like fimbriae in the crp mutant substantially enhanced kidney colonization. Moreover, increased survival in macrophages, increased stress tolerance, RpoS upregulation, and flagellum deficiency leading to immune evasion may promote kidney colonization by the crp mutant. This is the first study to elucidate the role of Crp in the virulence of uropathogenic P. mirabilis, underlying mechanisms, and related therapeutic potential.