Pseudogenes are considered to be nonfunctional genes that lack a physiological role. By screening 3985 Escherichia coli mutants using chemochromic membranes, we found four pseudogenes involved in hydrogen metabolism. Knockouts of pseudogenes ydfW and ypdJ had a defective hydrogen phenotype on glucose and formate, respectively. Also, the knockout of pseudogene yqiG formed hydrogen from formate but not from glucose. For the yqiG mutant, 100% hydrogen recovery was obtained by the complementation of YqiG via a plasmid. The knockout of pseudogene ylcE showed hydrogen deficiency in minimal media which suggested that the role of YlcE is associated with cell growth. Hence, the products of these four pseudogenes play an important physiological role in hydrogen production in E. coli.
Succinic acid is an important platform chemical with a variety of applications. Model-guided metabolic engineering strategies in Escherichia coli for strain improvement to increase succinic acid production using glucose and glycerol remain largely unexplored. Herein, we report what are, to our knowledge, the first metabolic knockout of the atpE gene to have increased succinic acid production using both glucose and alternative glycerol carbon sources in E. coli. Guided by a genome-scale metabolic model, we engineered the E. coli host to enhance anaerobic production of succinic acid by deleting the atpE gene, thereby generating additional reducing equivalents by blocking H(+) conduction across the mutant cell membrane. This strategy produced 1.58 and .49 g l(-1) of succinic acid from glycerol and glucose substrate, respectively. This work further elucidates a model-guided and/or system-based metabolic engineering, involving only a single-gene deletion strategy for enhanced succinic acid production in E. coli.
Protein-ligand binding free energy values of wild-type and mutant C-terminal domain of Escherichia coli arginine repressor (ArgRc) protein systems bound to L-arginine or L-citrulline molecules were calculated using the linear interaction energy (LIE) method by molecular dynamics (MD) simulation. The binding behaviour predicted by the dissociation constant (K(d)) calculations from the binding free energy values showed preferences for binding of L-arginine to the wild-type ArgRc but not to the mutant ArgRc(D128N). On the other hand, L-citrulline do not favour binding to wild-type ArgRc but prefer binding to mutant ArgRc(D128N). The dissociation constant for the wild-type ArgRc-L-arginine complex obtained in this study is in agreement with reported experimental results. Our results also support the experimental data for the binding of L-citrulline to the mutant ArgRc(D128N). These showed that LIE method for protein-ligand binding free energy calculation could be applied to the wild-type and the mutant E. coli ArgRc-L-arginine and ArgRc-L-citrulline protein-ligand complexes and possibly to other transcriptional repressor-co-repressor systems as well.
The hemolysin transport system was found to mediate the release of cyclodextrin glucanotransferase (CGTase) into the extracellular medium when it was fused to the C-terminal 61 amino acids of HlyA (HlyAs(61)). To produce an improved-secretion variant, the hly components (hlyAs, hlyB and hlyD) were engineered by directed evolution using error-prone PCR. Hly mutants were screened on solid LB-starch plate for halo zone larger than the parent strain. Through screening of about 1 × 10(4) Escherichia coli BL21(DE3) transformants, we succeeded in isolating five mutants that showed a 35-217% increase in the secretion level of CGTase-HlyAs(61) relative to the wild-type strain. The mutation sites of each mutant were located at HlyB, primarily along the transmembrane domain, implying that the corresponding region was important for the improved secretion of the target protein. In this study we describe the finding of novel site(s) of HlyB responsible for enhancing secretion of CGTase in E. coli.
The role of membrane permeabilisation and disruption in the mechanism of action of some polymyxin analogues against Gram-negative organisms is contentious. The effects of polymyxin B (PMB) and its analogue polymyxin B nonapeptide (PMBN) on Escherichia coli envelopes should correlate, but previous work by other workers suggests that PMBN has a different mode of action. This study has reassessed the biochemical techniques used previously and has shown that, in contrast to previous studies, PMBN (a well-characterised antibacterial synergist) readily releases periplasmic proteins and lipopolysaccharide from treated E. coli at subinhibitory concentrations in normal physiological buffer conditions. We conclude that, when tested with appropriate methodology, PMBN closely correlates with the early effects of PMB on the cell envelope of E. coli and this study shows that it is now consistent with the accepted interactions of membrane-active agents against Gram-negative cells.
Maintenance of recombinant plasmid vectors in host bacteria relies on the presence of selection antibiotics in the growth media to suppress plasmid -free segregants. However, presence of antibiotic resistance genes and antibiotics themselves is not acceptable in several applications of biotechnology. Previously, we have shown that FabV-Triclosan selection system can be used to select high and medium copy number plasmid vectors in E. coli. Here, we have extended our previous work and demonstrated that expression vectors containing FabV can be used efficiently to express heterologous recombinant proteins in similar or better amounts in E. coli host when compared with expression vectors containing β-lactamase. Use of small amount of non-antibiotic Triclosan as selection agent in growth medium, enhanced plasmid stability, applicability in various culture media, and compatibility with other selection systems for multiple plasmid maintenance are noteworthy features of FabV-Triclosan selection system.
Among their numerous physiological effects, heat shock proteins (Hsps) are potent immunomodulators, a characteristic reflecting their potential as therapeutic agents and which led to their application in combating infection. As an example, the up-regulation of endogenous Hsp70 in the branchiopod crustacean Artemia franciscana (Kellogg) is concurrent with shielding against bacterial infection. To better understand this protective mechanism, gnotobiotic Artemia were fed with Escherichia coli treated to over-produce different prokaryotic Hsps. This was shown to increase larval resistance to experimental Vibrio campbellii exposure. Immunoprobing of Western blots showed that the enhanced resistance to V. campbellii correlated with DnaK production in E coli. A definitive role for DnaK was then demonstrated by feeding Artemia larvae with transformed bacteria over-producing only this protein, although other Hsps such as DnaJ and grpE also provided tolerance against Vibrio infection. Feeding of bacteria synthesizing selected Hsps is therefore suggested as an alternative to antibiotic use as a means of enhancing resistance of Artemia larvae to bacterial infection, which may have potential applications in aquaculture.
Here in this study regarding the over expression of TP, which causes some physical, mental and socio problems like psoriasis, chronic inflammatory disease, tumor angiogenesis and rheumatoid arthritis etc. By this consideration, the inhibition of this enzyme is vital to secure life from serious threats. In connection with this, we have synthesized twenty derivatives of isoquinoline bearing oxadiazole (1-20), characterized through different spectroscopic techniques such as HREI-MS, 1H- NMR and 13C-NMR and evaluated for thymidine phosphorylase inhibition. All analogues showed outstanding inhibitory potential ranging in between 1.10 ± 0.05 to 54.60 ± 1.50 µM. 7-Deazaxanthine (IC50 = 38.68 ± 1.12 µM) was used as a positive control. Through limited structure activity relationships study, it has been observed that the difference in inhibitory activities of screened analogs are mainly affected by different substitutions on phenyl ring. The effective binding interactions of the most active analogs were confirmed through docking study.
Curli are bacterial surface-associated amyloid fibers that bind to the dye Congo red (CR) and facilitate uropathogenic Escherichia coli (UPEC) biofilm formation and protection against host innate defenses. Here we sequenced the genome of the curli-producing UPEC pyelonephritis strain MS7163 and showed it belongs to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. MS7163 produced curli at human physiological temperature, and this correlated with biofilm growth, resistance of sessile cells to the human cationic peptide cathelicidin, and enhanced colonization of the mouse bladder. We devised a forward genetic screen using CR staining as a proxy for curli production and identified 41 genes that were required for optimal CR binding, of which 19 genes were essential for curli synthesis. Ten of these genes were novel or poorly characterized with respect to curli synthesis and included genes involved in purine de novo biosynthesis, a regulator that controls the Rcs phosphorelay system, and a novel repressor of curli production (referred to as rcpA). The involvement of these genes in curli production was confirmed by the construction of defined mutants and their complementation. The mutants did not express the curli major subunit CsgA and failed to produce curli based on CR binding. Mutation of purF (the first gene in the purine biosynthesis pathway) and rcpA also led to attenuated colonization of the mouse bladder. Overall, this work has provided new insight into the regulation of curli and the role of these amyloid fibers in UPEC biofilm formation and pathogenesis.IMPORTANCE Uropathogenic Escherichia coli (UPEC) strains are the most common cause of urinary tract infection, a disease increasingly associated with escalating antibiotic resistance. UPEC strains possess multiple surface-associated factors that enable their colonization of the urinary tract, including fimbriae, curli, and autotransporters. Curli are extracellular amyloid fibers that enhance UPEC virulence and promote biofilm formation. Here we examined the function and regulation of curli in a UPEC pyelonephritis strain belonging to the highly virulent O45:K1:H7 neonatal meningitis-associated clone. Curli expression at human physiological temperature led to increased biofilm formation, resistance of sessile cells to the human cationic peptide LL-37, and enhanced bladder colonization. Using a comprehensive genetic screen, we identified multiple genes involved in curli production, including several that were novel or poorly characterized with respect to curli synthesis. In total, this study demonstrates an important role for curli as a UPEC virulence factor that promotes biofilm formation, resistance, and pathogenesis.
Chemical modification of medicines from natural product-based molecules has become of interest in recent years. In this study, a series of halogenated azo derivatives 1a-d were synthesised via coupling reaction, followed by Steglich esterification with aspirin (a natural product derivative) to form azo derivatives 2a-d. While, halogenated azo-aspirin 3a-d were synthesised via direct coupling reaction of aspirin and diazonium salt. Bacteriostatic activity was demonstrated against E. coli and S. aureus via turbidimetric kinetic method. Compound 3a-d showed excellent antibacterial activities against E. coli (MIC 75-94 ppm) and S. aureus (MIC 64-89 ppm) compared to ampicillin (MIC 93 and 124 ppm respectively), followed by 1a-d and 2a-d. The presence of reactive groups of -OH, N=N, C=O and halogens significantly contribute excellent interaction towards E. coli and S. aureus. Molecular dockings analysis of 3a against MIaC protein showed binding free energy of -7.2 kcal/mol (E. coli) and -6.6 kcal/mol (S. aureus).
Uropathogenic Escherichia coli (UPEC) is a major cause of urinary tract and bloodstream infections and possesses an array of virulence factors for colonization, survival, and persistence. One such factor is the polysaccharide K capsule. Among the different K capsule types, the K1 serotype is strongly associated with UPEC infection. In this study, we completely sequenced the K1 UPEC urosepsis strain PA45B and employed a novel combination of a lytic K1 capsule-specific phage, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing (TraDIS) to identify the complement of genes required for capsule production. Our analysis identified known genes involved in capsule biosynthesis, as well as two additional regulatory genes (mprA and lrhA) that we characterized at the molecular level. Mutation of mprA resulted in protection against K1 phage-mediated killing, a phenotype restored by complementation. We also identified a significantly increased unidirectional Tn5 insertion frequency upstream of the lrhA gene and showed that strong expression of LrhA induced by a constitutive Pcl promoter led to loss of capsule production. Further analysis revealed loss of MprA or overexpression of LrhA affected the transcription of capsule biosynthesis genes in PA45B and increased sensitivity to killing in whole blood. Similar phenotypes were also observed in UPEC strains UTI89 (K1) and CFT073 (K2), demonstrating that the effects were neither strain nor capsule type specific. Overall, this study defined the genome of a UPEC urosepsis isolate and identified and characterized two new regulatory factors that affect UPEC capsule production.IMPORTANCE Urinary tract infections (UTIs) are among the most common bacterial infections in humans and are primarily caused by uropathogenic Escherichia coli (UPEC). Many UPEC strains express a polysaccharide K capsule that provides protection against host innate immune factors and contributes to survival and persistence during infection. The K1 serotype is one example of a polysaccharide capsule type and is strongly associated with UPEC strains that cause UTIs, bloodstream infections, and meningitis. The number of UTIs caused by antibiotic-resistant UPEC is steadily increasing, highlighting the need to better understand factors (e.g., the capsule) that contribute to UPEC pathogenesis. This study describes the original and novel application of lytic capsule-specific phage killing, saturated Tn5 transposon mutagenesis, and high-throughput transposon-directed insertion site sequencing to define the entire complement of genes required for capsule production in UPEC. Our comprehensive approach uncovered new genes involved in the regulation of this key virulence determinant.
The role of Escherichia coli H antigens in hydrophobicity and attachment to glass, Teflon and stainless steel (SS) surfaces was investigated through construction of fliC knockout mutants in E. coli O157:H7, O1:H7 and O157:H12. Loss of FliC(H12) in E. coli O157:H12 decreased attachment to glass, Teflon and stainless steel surfaces (p<0.05). Complementing E. coli O157:H12 ΔfliC(H12) with cloned wildtype (wt) fliC(H12) restored attachment to wt levels. The loss of FliCH7 in E. coli O157:H7 and O1:H7 did not always alter attachment (p>0.05), but complementation with cloned fliC(H12), as opposed to cloned fliCH7, significantly increased attachment for both strains compared with wt counterparts (p<0.05). Hydrophobicity determined using bacterial adherence to hydrocarbons and contact angle measurements differed with fliC expression but was not correlated to the attachment to materials included in this study. Purified FliC was used to functionalise silicone nitride atomic force microscopy probes, which were used to measure adhesion forces between FliC and substrates. Although no significant difference in adhesion force was observed between FliC(H12) and FliCH7 probes, differences in force curves suggest different mechanism of attachment for FliC(H12) compared with FliCH7. These results indicate that E. coli strains expressing flagellar H12 antigens have an increased ability to attach to certain abiotic surfaces compared with E. coli strains expressing H7 antigens.
The emergence of Escherichia coli that produce extended spectrum beta-lactamases (ESBLs) and are multidrug resistant (MDR) poses antibiotic management problems. Forty-seven E. coli isolates from various public hospitals in Malaysia were studied. All isolates were sensitive to imipenem whereas 36 were MDR (resistant to 2 or more classes of antibiotics). PCR detection using gene-specific primers showed that 87.5% of the ESBL-producing E. coli harbored the blaTEM gene. Other ESBL-encoding genes detected were blaOXA, blaSHV, and blaCTX-M. Integron-encoded integrases were detected in 55.3% of isolates, with class 1 integron-encoded intI1 integrase being the majority. Amplification and sequence analysis of the 5'CS region of the integrons showed known antibiotic resistance-encoding gene cassettes of various sizes that were inserted within the respective integrons. Conjugation and transformation experiments indicated that some of the antibiotic resistance genes were likely plasmid-encoded and transmissible. All 47 isolates were subtyped by PFGE and PCR-based fingerprinting using random amplified polymorphic DNA (RAPD), repetitive extragenic palindromes (REPs), and enterobacterial repetitive intergenic consensus (ERIC). These isolates were very diverse and heterogeneous. PFGE, ERIC, and REP-PCR methods were more discriminative than RAPD in subtyping the E. coli isolates.