Proteomic analysis of uropathogenic Escherichia coli

Urinary tract infections (UTIs) are among the most common of bacterial infections in humans. Although a number of Gram-negative bacteria can cause UTIs, most cases are due to infection by uropathogenic E. coli (UPEC). Genomic studies have shown that UPEC encode a number of specialized activities that allow the bacteria to initiate and maintain infections in the environment of the urinary tract. Proteomic analyses have complemented the genomic data and have documented differential patterns of protein synthesis for bacteria growing ex vivo in human urine or recovered directly from the urinary tracts of infected mice. These studies provide valuable insights into the molecular basis of UPEC pathogenesis and have aided the identification of putative vaccine targets. Despite the substantial progress that has been achieved, many future challenges remain in the application of proteomics to provide a comprehensive view of bacterial pathogenesis in both acute and chronic UTIs.     

Defining genomic islands and uropathogen-specific genes in uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) strains are responsible for the majority of uncomplicated urinary tract infections, which can present clinically as cystitis or pyelonephritis. UPEC strain CFT073, isolated from the blood of a patient with acute pyelonephritis, was most cytotoxic and most virulent in mice among our strain collection. Based on the genome sequence of CFT073, microarrays were utilized in comparative genomic hybridization (CGH) analysis of a panel of uropathogenic and fecal/commensal E. coli isolates. Genomic DNA from seven UPEC (three pyelonephritis and four cystitis) isolates and three fecal/commensal strains, including K-12 MG1655, was hybridized to the CFT073 microarray. The CFT073 genome contains 5,379 genes; CGH analysis revealed that 2,820 (52.4%) of these genes were common to all 11 E. coli strains, yet only 173 UPEC-specific genes were found by CGH to be present in all UPEC strains but in none of the fecal/commensal strains. When the sequences of three additional sequenced UPEC strains (UTI89, 536, and F11) and a commensal strain (HS) were added to the analysis, 131 genes present in all UPEC strains but in no fecal/commensal strains were identified. Seven previously unrecognized genomic islands (>30 kb) were delineated by CGH in addition to the three known pathogenicity islands. These genomic islands comprise 672 kb of the 5,231-kb (12.8%) genome, demonstrating the importance of horizontal transfer for UPEC and the mosaic structure of the genome. UPEC strains contain a greater number of iron acquisition systems than do fecal/commensal strains, which is reflective of the adaptation to the iron-limiting urinary tract environment. Each strain displayed distinct differences in the number and type of known virulence factors. The large number of hypothetical genes in the CFT073 genome, especially those shown to be UPEC specific, strongly suggests that many urovirulence factors remain uncharacterized.     

Comparative analysis of essential genes and nonessential genes in Escherichia coli K12

Genes can be classified as essential or nonessential based on their indispensability for a living organism. Previous researches have suggested that essential genes evolve more slowly than nonessential genes and the impact of gene dispensability on a gene’s evolutionary rate is not as strong as expected. However, findings have not been consistent and evidence is controversial regarding the relationship between the gene indispensability and the rate of gene evolution. Understanding how different classes of genes evolve is essential for a full understanding of evolutionary biology, and may have medical relevance in the design of new antibacterial agents. We therefore performed an investigation into the properties of essential and nonessential genes. Analysis of evolutionary conservation, protein length distribution and amino acid usage between essential and nonessential genes in Escherichia coli K12 demonstrated that essential genes are relatively preserved throughout the bacterial kingdom when compared to nonessential genes. Furthermore, results show that essential genes, compared to nonessential genes, have a significantly higher proportion of large (>534 amino acids) and small proteins (<139 amino acids) relative to medium-sized proteins. The pattern of amino acids usage shows a similar trend for essential and nonessential genes, although some notable exceptions are observed. These findings help to clarify our understanding of the evolutionary mechanisms of essential and nonessential genes, relevant to the study of mutagenesis and possibly allowing prediction of gene properties in other poorly understood organisms.     

A genome-wide inducible phenotypic screen identifies antisense RNA constructs silencing Escherichia coli essential genes

Regulated antisense RNA (asRNA) expression has been employed successfully in Gram-positive bacteria for genome-wide essential gene identification and drug target determination. However, there have been no published reports describing the application of asRNA gene silencing for comprehensive analyses of essential genes in Gram-negative bacteria. In this study, we report the first genome-wide identification of asRNA constructs for essential genes in Escherichia coli. We screened 250?000 library transformants for conditional growth inhibitory recombinant clones from two shotgun genomic libraries of E.?coli using a paired-termini expression vector (pHN678). After sequencing plasmid inserts of 675 confirmed inducer sensitive cell clones, we identified 152 separate asRNA constructs of which 134 inserts came from essential genes, while 18 originated from nonessential genes (but share operons with essential genes). Among the 79 individual essential genes silenced by these asRNA constructs, 61 genes (77%) engage in processes related to protein synthesis. The cell-based assays of an asRNA clone targeting fusA (encoding elongation factor G) showed that the induced cells were sensitized 12-fold to fusidic acid, a known specific inhibitor. Our results demonstrate the utility of the paired-termini expression vector and feasibility of large-scale gene silencing in E.?coli using regulated asRNA expression.     

Conditional lethal amber mutations in essential Escherichia coli genes

The essential genes of microorganisms encode biological functions important for survival and thus tend to be of high scientific interest. Drugs that interfere with essential functions are likely to be interesting candidates for antimicrobials. However, these genes are hard to study genetically because knockout mutations in them are by definition inviable. We recently described a conditional mutation system in Escherichia coli that uses a plasmid to produce an amber suppressor tRNA regulated by the arabinose promoter. This suppressor was used here in the construction of amber mutations in seven essential E. coli genes. Amber stop codons were introduced as "tagalong" mutations in the flanking DNA of a downstream antibiotic resistance marker by lambda red recombination. The drug marker was removed by expression of I-SceI meganuclease, leaving a markerless mutation. We demonstrate the method with the genes frr, gcpE, lpxC, map, murA, ppa, and rpsA. We were unable to isolate an amber mutation in ftsZ. Kinetics of cell death and morphological changes were measured following removal of arabinose. As expected given the wide range of cellular mechanisms represented, different mutants showed widely different death curves. All of the mutations were bactericidal except the mutation in gcpE, which was bacteriostatic. The strain carrying an amber mutation in murA was by far the most sensitive, showing rapid killing in nonpermissive medium. The MurA protein is critical for peptidoglycan synthesis and is the target for the antibiotic fosfomycin. Such experiments may inexpensively provide valuable information for the identification and prioritization of targets for antibiotic development.     

Experimental determination and system level analysis of essential genes in Escherichia coli MG1655

Defining the gene products that play an essential role in an organism's functional repertoire is vital to understanding the system level organization of living cells. We used a genetic footprinting technique for a genome-wide assessment of genes required for robust aerobic growth of Escherichia coli in rich media. We identified 620 genes as essential and 3,126 genes as dispensable for growth under these conditions. Functional context analysis of these data allows individual functional assignments to be refined. Evolutionary context analysis demonstrates a significant tendency of essential E. coli genes to be preserved throughout the bacterial kingdom. Projection of these data over metabolic subsystems reveals topologic modules with essential and evolutionarily preserved enzymes with reduced capacity for error tolerance.     

Adhesion and entry of uropathogenic Escherichia coli

To effectively colonize a host animal and cause disease, many bacterial pathogens have evolved the mechanisms needed to invade and persist within host cells and tissues. Recently it was discovered that uropathogenic Escherichia coli, the primary causative agent of urinary tract infections, can invade and replicate within uroepithelial cells. This can provide E. coli with a survival advantage, allowing the microbes to better resist detection and clearance by both innate and adaptive immune defence mechanisms. Adhesive organelles, including type 1, P, and S pili along with Dr adhesins, promote both bacterial attachment to and invasion of host tissues within the urinary tract. Interactions mediated by these adhesins can also stimulate a number of host responses that can directly influence the outcome of a urinary tract infection.     

In-frame deletion of Escherichia coli essential genes in complex regulon

A conditional knockout-rescue system was developed to construct an in-frame deletion strain ofEscherichia coli essential genes. The target was flanked with marker genes and FRT (FLP recognition target) sites, and a plasmid containing arabinose-induced FLP recombinase was transformed. After arabinose induction, cells could survive only when target protein activity was provided in trans. We selected three essential genes as targets, yaeT, fabZ, and dnaE, which are components of the complex eight-gene regulon yaeT-hlpA-lpxD-fabZ-lpxA-1pxB-rnhB-dnaE. Deletion of these three genes exhibit no polar effects on their adjacent genes in terms of cell viability, meaning that this system not only allows for the simplified study of protein interactions and homolog screening in other organisms, but also facilitates the null mutant construction of essential genes.     

产异丁醇关键基因在大肠杆菌中表达的研究

[目的]改造大肠杆菌缬氨酸合成途径,使其能够代谢合成异丁醇.[方法]将乳酸乳球菌(Lactococcus lactis) 1.2829的2-酮异戊酸脱羧酶基因(kivD)和醇脱氢酶基因(adhA)串联克隆到大肠杆菌DH5α宿主中表达.[结果]经过改造的宿主菌发酵24 h后异丁醇产量为0.12 g/L.酶活测定实验发现,kivD和adhA基因在宿主菌中均得到表达,但由于KivD的低表达量导致宿主菌最终的异丁醇合成能力偏低.通过研究温度和pH对KivD和AdhA酶活的影响,最终选定二者的最适温度为30℃,最适pH为6.5. [结论]通过向宿主菌导入外源异丁醇合成基因能够改造其自身代谢途径,从而合成异丁醇.     

Genetic method to analyze essential genes of Escherichia coli

The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-beta-d-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.     

The contribution of tellurite resistance genes to the fitness of Escherichia coli uropathogenic strains

The presence of tellurite resistance gene operons has been reported in several human pathogens despite the fact that tellurium, as well as its soluble salts, are both rare in nature and are no longer in use as antimicrobial agents. We have introduced the cloned terWZA-F genes from an uropathogenic Escherichia coli isolate into another clinical E. coli isolate that was shown to be ter-gene free. The presence of the introduced genes increased the level of potassium tellurite resistance, as well as the level of resistance to oxidative stress mediated by hydrogen peroxide; and prolonged the ability of particular strains to survive in macrophages. We therefore propose that the contribution of tellurite resistance genes to oxidative stress resistance in bacteria is at least one reason for their presence in the genomes of a broad range of pathogenic microorganisms.     

sucAB and sucCD are mutually essential genes in Escherichia coli

sucAB and sucCD of Escherichia coli encode enzymes that generate succinyl-CoA from 2-oxoglutarate and succinate, respectively. Their mutual essentiality was studied. sucAB and sucCD could be deleted individually, but not simultaneously. The mutual essentiality of sucAB and sucCD was further confirmed by the conditional expression of sucABCD, sucAB, and sucCD under the control of a P(BAD) in E. coli MG1655, E. coli MG1655 (DeltasucCD), and E. coli MG1655 (DeltasucAB), respectively. These strains grew well in Luria-Bertani medium containing 0.1% arabinose, but not in the absence of arabinose unless the medium was supplemented with succinyl-CoA. Our results indicate that either sucAB or sucCD is enough to produce succinyl-CoA that is essential for cell viability.     

Cytotoxic and hemolytic activities in uropathogenic Escherichia coli

Fifty nine Escherichia coli strains obtained from patients with upper or lower urinary tract infections (UTI) and 30 E. coli strains isolated from stools of healthy individuals were tested for hemolytic and cytotoxic activities. Forty four percent of uropathogenic E. coli (UPEC) and 3.3% of fecal E. coli were hemolytic. Among the hemolytic UPEC, 92% produced alpha-hemolysin. A cytotoxic activity was detected in culture filtrates of 71% of UPEC strains and 30% of fecal E. coli. No relationship was found between cytotoxic and hemolytic activities or between cytotoxic titers and UPEC origin (upper or lower UTI). E. coli cytotoxin has a cytocidal activity against some epithelioid cultured cell lines (Vero, HeLa and Hep-2) but was almost inactive for avian-fibroblast cells. Cytotoxin-affected cells appeared rounded, refractile and detached from the surface of the vessel. Some characteristics exhibited by the cytotoxin as the morphological response induced on cells, the increasing of cytopathic effect with time, its irreversible cytocidal activity and its heat-lability resemble the properties described for E. coli Verotoxin (VT). Adherence to uroepithelial cells is recognized as a virulence factor for UPEC. It is suggested that cell damage by cytotoxic and adhering UPEC might contribute to E. coli virulence to urinary tract.     

Instability of pathogenicity islands in uropathogenic Escherichia coli 536

The uropathogenic Escherichia coli strain 536 carries at least five genetic elements on its chromosome that meet all criteria characteristic of pathogenicity islands (PAIs). One main feature of these distinct DNA regions is their instability. We applied the so-called island-probing approach and individually labeled all five PAIs of E. coli 536 with the counterselectable marker sacB to evaluate the frequency of PAI-negative colonies under the influence of different environmental conditions. Furthermore, we investigated the boundaries of these PAIs. According to our experiments, PAI II536 and PAI III536 were the most unstable islands followed by PAI I536 and PAI V536, whereas PAI IV536 was stable. In addition, we found that deletion of PAI II536 and PAI III536 was induced by several environmental stimuli. Whereas excision of PAI I536, PAI II536, and PAI V536 was based on site-specific recombination between short direct repeat sequences at their boundaries, PAI III536 was deleted either by site-specific recombination or by homologous recombination between two IS100-specific sequences. In all cases, deletion is thought to lead to the formation of nonreplicative circular intermediates. Such extrachromosomal derivatives of PAI II536 and PAI III536 were detected by a specific PCR assay. Our data indicate that the genome content of uropathogenic E. coli can be modulated by deletion of PAIs.