Contribution of rpoS and bolA genes in biofilm formation in Escherichia coli K-12 MG1655

Flexibility of gene expression in bacteria permits its survival in varied environments. The genetic adaptation of bacteria through systematized gene expression is not only important, but also clinically relevant in their ability to grow biofilms in stress environments. Stress responses enable their survival under more severe conditions, enhanced resistance and/or virulence. In Escherichia coli (E. coli), two of the possible important genes for biofilm growth are rpoS and bolA gene. RpoS is also called as a master regulator of general stress response. Even though many studies have revealed the importance of rpoS in planktonic cells, little is known about the functions of rpoS in biofilms. In contrast, bolA which is a morphogene in E. coli is overexpressed under stressed environments resulting in round morphology. The hypothesis is that bolA could be implicated in biofilm development. This study reviewed the literature with the aim of understanding the stress tolerance response of E. coli in relation with rpoS and bolA genes in different environmental conditions including heat shock, cold shock, and stress in response to oxidation, acidic condition and in presence of cadmium. Knowledge of the genetic regulation of biofilm formation may lead to the understanding of the factors that drive the bacteria to switch to the biofilm mode of growth.     

Effect of Escherichia coli Morphogene bolA on Biofilms

Biofilm physiology is established under a low growth rate. The morphogene bolA is mostly expressed under stress conditions or in stationary phase, suggesting that bolA could be implicated in biofilm development. In order to verify this hypothesis, we tested the effect of bolA on biofilm formation. Overexpression of bolA induces biofilm development, while bolA deletion decreases biofilms.     

IscR Controls Iron-Dependent Biofilm Formation in Escherichia coli by Regulating Type I Fimbria Expression

Biofilm formation is a complex developmental process regulated by multiple environmental signals. In addition to other nutrients, the transition metal iron can also regulate biofilm formation. Iron-dependent regulation of biofilm formation varies by bacterial species, and the exact regulatory pathways that control iron-dependent biofilm formation are often unknown or only partially characterized. To address this gap in our knowledge, we examined the role of iron availability in regulating biofilm formation in Escherichia coli. The results indicate that biofilm formation is repressed under low-iron conditions in E. coli. Furthermore, a key iron regulator, IscR, controls biofilm formation in response to changes in cellular Fe-S homeostasis. IscR regulates the FimE recombinase to control expression of type I fimbriae in E. coli. We propose that iron-dependent regulation of FimE via IscR leads to decreased surface attachment and biofilm dispersal under iron-limiting conditions.     

The Role of the QseC Quorum-Sensing Sensor Kinase in Epinephrine-Enhanced Motility and Biofilm Formation by Escherichia coli

Biofilms play a pivotal role in infections related to devices. Biofilm formation in Escherichia coli is mediated by the quorum-sensing E. coli regulator C (QseC), the histidine sensor kinase that can sense epinephrine (EPI)/norepinephrine (NE). In this study, we evaluate the role of the QseC quorum-sensing sensor kinase in epinephrine-enhanced motility and biofilm formation by E. coli. An E. coli MC1000 qseC mutant was constructed. We investigated the role of the QseC in the formation of biofilms on the surface of medical-grade polyvinyl chloride using the E. coli K-12 MC1000 strain as well as a corresponding qseC mutant. Addition of EPI/NE increased biofilm formation by wild-type K-12 MC1000 but not by the isogenic qseC mutant. Scanning confocal laser microscopy corroborated these results by showing that EPI/NE addition significantly increased biofilm’s thickness. As expected, the addition of EPI/NE to the qseC mutant, which lacks the ability to sense the hormones, failed to stimulate biofilm formation. Since EPI/NE addition increased bacterial motility, we proposed that their stimulatory effects on biofilm formation occur by enhancing bacterial motility and altering biofilm architecture. We also found that EPI/NE regulate motility and the biofilm phenotype via QseC, as motility was diminished and biofilm formation was significantly decreased in a qseC deletion mutant. These results indicate that EPI/NE induce E. coli biofilm formation on the surface of polyvinyl chloride through QseC. Cross-talk between E. coli (quorum sensing) and host hormones may explain the pathogen-caused opportunistic infections that occur in patients with prosthetic devices used during hormone level fluctuations in the host.     

Biofilm Formation Protects Escherichia coli against Killing by Caenorhabditis elegans and Myxococcus xanthus

Enteric bacteria, such as Escherichia coli, are exposed to a variety of stresses in the nonhost environment. The development of biofilms provides E. coli with resistance to environmental insults, such as desiccation and bleach. We found that biofilm formation, specifically production of the matrix components curli and cellulose, protected E. coli against killing by the soil-dwelling nematode Caenorhabditis elegans and the predatory bacterium Myxococcus xanthus. Additionally, matrix-encased bacteria at the air-biofilm interface exhibited ∼40-fold-increased survival after C. elegans and M. xanthus killing compared to the non-matrix-encased cells that populate the interior of the biofilm. To determine if nonhost Enterobacteriaceae reservoirs supported biofilm formation, we grew E. coli on media composed of pig dung or commonly contaminated foods, such as beef, chicken, and spinach. Each of these medium types provided a nutritional environment that supported matrix production and biofilm formation. Altogether, we showed that common, nonhost reservoirs of E. coli supported the formation of biofilms that subsequently protected E. coli against predation.     

Biofilm Modifies Expression of Ribonucleotide Reductase Genes in Escherichia coli

Ribonucleotide reductase (RNR) is an essential enzyme for all living organisms since is the responsible for the last step in the synthesis of the four deoxyribonucleotides (dNTPs) necessary for DNA replication and repair. In this work, we have investigated the expression of the three-RNR classes (Ia, Ib and III) during Escherichia coli biofilm formation. We show the temporal and spatial importance of class Ib and III RNRs during this process in two different E. coli wild-type strains, the commensal MG1655 and the enteropathogenic and virulent E2348/69, the prototype for the enteropathogenic E. coli (EPEC). We have established that class Ib RNR, so far considered cryptic, play and important role during biofilm formation. The implication of this RNR class under the specific growth conditions of biofilm formation is discussed.     

High-Throughput Microfluidic Method To Study Biofilm Formation and Host-Pathogen Interactions in Pathogenic Escherichia coli

Biofilm formation and host-pathogen interactions are frequently studied using multiwell plates; however, these closed systems lack shear force, which is present at several sites in the host, such as the intestinal and urinary tracts. Recently, microfluidic systems that incorporate shear force and very small volumes have been developed to provide cell biology models that resemble in vivo conditions. Therefore, the objective of this study was to determine if the BioFlux 200 microfluidic system could be used to study host-pathogen interactions and biofilm formation by pathogenic Escherichia coli. Strains of various pathotypes were selected to establish the growth conditions for the formation of biofilms in the BioFlux 200 system on abiotic (glass) or biotic (eukaryotic-cell) surfaces. Biofilm formation on glass was observed for the majority of strains when they were grown in M9 medium at 30°C but not in RPMI medium at 37°C. In contrast, HRT-18 cell monolayers enhanced binding and, in most cases, biofilm formation by pathogenic E. coli in RPMI medium at 37°C. As a proof of principle, the biofilm-forming ability of a diffusely adherent E. coli mutant strain lacking AIDA-I, a known mediator of attachment, was assessed in our models. In contrast to the parental strain, which formed a strong biofilm, the mutant formed a thin biofilm on glass or isolated clusters on HRT-18 monolayers. In conclusion, we describe a microfluidic method for high-throughput screening that could be used to identify novel factors involved in E. coli biofilm formation and host-pathogen interactions under shear force.     

Escherichia coli harboring a natural IncF conjugative F plasmid develops complex mature biofilms by stimulating synthesis of colanic acid and Curli

It has been shown that Escherichia coli harboring the derepressed IncFI and IncFII conjugative F plasmids form complex mature biofilms by using their F-pilus connections, whereas a plasmid-free strain forms only patchy biofilms. Therefore, in this study we investigated the contribution of a natural IncF conjugative F plasmid to the formation of E. coli biofilms. Unlike the presence of a derepressed F plasmid, the presence of a natural IncF F plasmid promoted biofilm formation by generating the cell-to-cell mating F pili between pairs of F⁺ cells (approximately two to four pili per cell) and by stimulating the formation of colanic acid and curli meshwork. Formation of colanic acid and curli was required after the initial deposition of F-pilus connections to generate a three-dimensional mushroom-type biofilm. In addition, we demonstrated that the conjugative factor of F plasmid, rather than a pilus synthesis function, was involved in curli production during biofilm formation, which promoted cell-surface interactions. Curli played an important role in the maturation process. Microarray experiments were performed to identify the genes involved in curli biosynthesis and regulation. The results suggested that a natural F plasmid was more likely an external activator that indirectly promoted curli production via bacterial regulatory systems (the EnvZ/OmpR two-component regulators and the RpoS and HN-S global regulators). These data provided new insights into the role of a natural F plasmid during the development of E. coli biofilms.     

Nitrogen oxide is involved in the regulation of the Fe-S cluster assembly in proteins and the formation of biofilms by Escherichia coli cells

The functions of nitrogen oxide (NO) in the regulation of the reversible processes of Fe-S cluster assembly in proteins and the formation of Escherichia coli biofilms have been investigated. S-nitrosoglutathione (GSNO) and crystalline nitrosyl complexes of iron with sulfur-containing aliphatic ligands cisaconite (CisA) and penaconite have been used as NO donors for the first time. Wild-type E. coli cells of the strain MC4100, mutants ΔiscA and ΔsufA, and the double paralog mutant ΔiscA/sufA with deletions in the alternative pathways of Fe²⁺ supply for cluster assembly (all derived from the above-named strain) were used in this study. Plankton growth of bacterial cultures, the mass of mature biofilms, and the expression of the SoxRS[2Fe-2S] regulon have been investigated and shown to depend on strain genotype, the process of Fe-S cluster assembly in iron-sulfur proteins, NO donor structure, and the presence of Fe²⁺ chelator ferene in the incubation medium. The antibiotic ciprofloxacine (CF) was used as an inhibitor of E. coli biofilm formation in the positive control. NO donors regulating Fe-S cluster assembly in E. coli have been shown to control plankton growth of the cultures and the process of mature biofilm formation; toxic doses of NO caused a dramatic (3- to 4-fold) stimulation of cell entry into biofilms as a response to nitrosative stress; NO donors CisA and GSNO in physiological concentrations suppressed the formation of mature biofilms, and the activity of these compounds was comparable to that of CF. Regulation of both Fe-S cluster assembly in iron-sulfur proteins and biofilm formation by NO is indicative of the connection between these processes in E. coli.     

Crohn's Disease-Associated Adherent-Invasive Escherichia coli Adhesion Is Enhanced by Exposure to the Ubiquitous Dietary Polysaccharide Maltodextrin

Crohn''s disease (CD) is associated with intestinal dysbiosis evidenced by an altered microbiome forming thick biofilms on the epithelium. Additionally, adherent-invasive E. coli (AIEC) strains are frequently isolated from ileal lesions of CD patients indicating a potential role for these strains in disease pathogenesis. The composition and characteristics of the host microbiome are influenced by environmental factors, particularly diet. Polysaccharides added to food as emulsifiers, stabilizers or bulking agents have been linked to bacteria-associated intestinal disorders. The escalating consumption of polysaccharides in Western diets parallels an increased incidence of CD during the latter 20^th century. In this study, the effect of a polysaccharide panel on adhesiveness of the CD-associated AIEC strain LF82 was analyzed to determine if these food additives promote disease-associated bacterial phenotypes. Maltodextrin (MDX), a polysaccharide derived from starch hydrolysis, markedly enhanced LF82 specific biofilm formation. Biofilm formation of multiple other E. coli strains was also promoted by MDX. MDX-induced E. coli biofilm formation was independent of polysaccharide chain length indicating a requirement for MDX metabolism. MDX exposure induced type I pili expression, which was required for MDX-enhanced biofilm formation. MDX also increased bacterial adhesion to human intestinal epithelial cell monolayers in a mechanism dependent on type 1 pili and independent of the cellular receptor CEACAM6, suggesting a novel mechanism of epithelial cell adhesion. Analysis of mucosa-associated bacteria from individuals with and without CD showed increased prevalence of malX, a gene essential for MDX metabolism, uniquely in the ileum of CD patients. These findings demonstrate that the ubiquitous dietary component MDX enhances E. coli adhesion and suggests a mechanism by which Western diets rich in specific polysaccharides may promote dysbiosis of gut microbes and contribute to disease susceptibility.     

Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival

The protein RpoS is responsible for mediating cell survival during the stationary phase by conferring cell resistance to various stressors and has been linked to biofilm formation. In this study, the role of the rpoS gene in Escherichia coli O157:H7 biofilm formation and survival in water was investigated. Confocal scanning laser microscopy of biofilms established on coverslips revealed a nutrient-dependent role of rpoS in biofilm formation, where the biofilm biomass volume of the rpoS mutant was 2.4- to 7.5-fold the size of its rpoS⁺ wild-type counterpart in minimal growth medium. The enhanced biofilm formation of the rpoS mutant did not, however, translate to increased survival in sterile double-distilled water (ddH₂O), filter-sterilized lake water, or unfiltered lake water. The rpoS mutant had an overall reduction of 3.10 and 5.30 log₁₀ in sterile ddH₂O and filter-sterilized lake water, respectively, while only minor reductions of 0.53 and 0.61 log₁₀ in viable counts were observed for the wild-type form in the two media over a 13-day period, respectively. However, the survival rates of the detached biofilm-derived rpoS⁺ and rpoS mutant cells were comparable. Under the competitive stress conditions of unfiltered lake water, the advantage conferred by the presence of rpoS was lost, and both the wild-type and knockout forms displayed similar declines in viable counts. These results suggest that rpoS does have an influence on both biofilm formation and survival of E. coli O157:H7 and that the advantage conferred by rpoS is contingent on the environmental conditions.     

Novel Insights into E. coli’s Hexuronate Metabolism: KduI Facilitates the Conversion of Galacturonate and Glucuronate under Osmotic Stress Conditions

Using a gnotobiotic mouse model, we previously observed the upregulation of 2-deoxy-D-gluconate 3-dehydrogenase (KduD) in intestinal E. coli of mice fed a lactose-rich diet and the downregulation of this enzyme and of 5-keto 4-deoxyuronate isomerase (KduI) on a casein-rich diet. The present study aimed to define the role of the so far poorly characterized E. coli proteins KduD and KduI in vitro. Galacturonate and glucuronate induced kduD and kduI gene expression 3-fold and 7 to 11-fold, respectively, under aerobic conditions as well as 9 to 20-fold and 19 to 54-fold, respectively, under anaerobic conditions. KduI facilitated the breakdown of these hexuronates. In E. coli, galacturonate and glucuronate are normally degraded by UxaABC and UxuAB. However, osmotic stress represses the expression of the corresponding genes in an OxyR-dependent manner. When grown in the presence of galacturonate or glucuronate, kduID-deficient E. coli had a 30% to 80% lower maximal cell density and 1.5 to 2-fold longer doubling times under osmotic stress conditions than wild type E. coli. Growth on lactose promoted the intracellular formation of hexuronates, which possibly explain the induction of KduD on a lactose-rich diet. These results indicate a novel function of KduI and KduD in E. coli and demonstrate the crucial influence of osmotic stress on the gene expression of hexuronate degrading enzymes.     

c-di-GMP对大肠杆菌生物膜调控的研究进展

大肠杆菌生物膜是由聚集于特定介质上的大肠杆菌菌体细胞相互黏附并分泌胞外基质聚合物(extracellular polymeric substances,EPS)而产生的一种结构复杂的膜状聚集物。感染宿主后的致病性大肠杆菌在形成生物膜后会极大地逃避免疫系统以及环境中各种有害因素对其的影响,对宿主造成持续甚至致命的伤害。环二鸟苷酸(cyclic diguanosine monophosphate,c-di-GMP)是广泛存在于细菌中的第二信使,在调节生物膜形成过程中起到至关重要的作用。基于此,本文对近些年来有关c-di-GMP对大肠杆菌生物膜形成过程中菌体的运动、黏附以及EPS产生机制的研究进行了综述,以期为从c-di-GMP角度抑制大肠杆菌生物膜提供依据和思路。     

Cytosolic BolA Plays a Repressive Role in the Tolerance against Excess Iron and MV-Induced Oxidative Stress in Plants

The BolA-like protein is present in all eukaryotes, and it is able to form complex with monothiol glutaredoxin of the same subcellular compartments, suggesting that the BolA-like protein has essential function in eukaryotes, and that the function is associated with its partner glutaredoxin. Some studies have indicated a role for BolA proteins in Fe-S cluster synthesis or in redox homeostasis. However, the physiological function of BolA proteins remains to be elucidated. Here, we report the characterization of an insertion mutant of BolA3 in Arabidopsis. Among the four AtBolA proteins found in Arabidopsis, the AtBolA3 was the only BolA located in the cytosol of plant cells. It was highly expressed in roots. AtBolA3 was able to interact with the cytosolic monothiol glutaredoxin, AtGRXS17. The bola3 mutant did not show any notable phenotype under normal growth condition, but rather grew better than wild type under some stresses. The bola3 mutant was more tolerant to excess iron and the MV-induced oxidative stress than wild type. It displayed no necrosis in leaves, developed longer roots, accumulated more iron and higher Fe-S protein activities in roots. In addition, the mutant possessed a more potent antioxidant defense to scavenge ROS species. Taken together, our data indicated that the cytosolic AtBolA3 has a suppressive role in the tolerance to excess iron and the MV-induced oxidative stress in plants. AtBolA3 seems to be a repressor under some stress conditions.     

Characterization of Shiga Toxin-Producing Escherichia coli Isolates Associated with Two Multistate Food-Borne Outbreaks That Occurred in 2006

Shiga toxin-producing Escherichia coli isolates from two 2006 outbreaks were compared to other O157:H7 isolates for virulence genotype, biofilm formation, and stress responses. Spinach- and lettuce-related-outbreak strains had similar pulsed-field gel electrophoresis patterns, and all carried both stx₂ and stx_2c variant genes. Cooperative biofilm formation involving an E. coli O157:H7 strain and a non-O157:H7 strain was also demonstrated.     

Effects of Norspermidine and Spermidine on Biofilm Formation by Potentially Pathogenic Escherichia coli and Salmonella enterica Wild-Type Strains

Polyamines are present in all living cells. In bacteria, polyamines are involved in a variety of functions, including biofilm formation, thus indicating that polyamines may have potential in the control of unwanted biofilm. In the present study, the effects of the polyamines norspermidine and spermidine on biofilms of 10 potentially pathogenic wild-type strains of Escherichia coli serotype O103:H2, Salmonella enterica subsp. enterica serovar Typhimurium, and S. enterica serovar Agona were investigated. We found that exogenously supplied norspermidine and spermidine did not mediate disassembly of preformed biofilm of any of the E. coli and S. enterica strains. However, the polyamines did affect biofilm production. Interestingly, the two species reacted differently to the polyamines. Both polyamines reduced the amount of biofilm formed by E. coli but tended to increase biofilm formation by S. enterica. Whether the effects observed were due to the polyamines specifically targeting biofilm formation, being toxic for the cells, or maybe a combination of the two, is not known. However, there were no indications that the effect was mediated through binding to exopolysaccharides, as earlier suggested for E. coli. Our results indicate that norspermidine and spermidine do not have potential as inhibitors of S. enterica biofilm. Furthermore, we found that the commercial polyamines used contributed to the higher pH of the test medium. Failure to acknowledge and control this important phenomenon may lead to misinterpretation of the results.