beta-Lactamases have been identified as the major cause of antimicrobial resistance to beta-lactam antibiotics in Escherichia coli. The activities of ampicillin-sulbactam and amoxicillin-clavulanate as well as a range of beta-lactam antibiotics were studied with 87 clinical E. coli isolates from patients of the University Malaya Medical Center using the disc diffusion technique. Susceptible, intermediate and resistant categories were established based on the diameter of zones of inhibition set by the National Committee for Clinical Laboratory Standards (NCCLS). The isolates were then classified into 6 phenotypes according to the criteria stated in the methodology: S (susceptible to all beta-lactams); TL (resistant to aminopenicillins; amoxicillin-clavulanate susceptible and susceptible or intermediate to ampicillin-sulbactam); TI (resistant to aminopenicillins and ampicillin-sulbactam; susceptible to amoxicilin-clavulanate); TH-IRT (resistant to aminopenicillins; intermediate or resistant to amoxicillin-clavulanate; resistant to ampicillin-sulbactam); ESBL (resistant to aminopenicillins and oxyimino cephalosporins; positive results with the double-disc diffusion test); and CP (resistant to aminopenicillins, beta-lactam-beta-lactamase inhibitor combinations, oxyimino cephalosporins and cephamycins). Results showed that the TL phenotype was the commonest (40.2% of the isolates) followed by S (31%), TH-IRT (16.1%), ESBL and CP (3.4% each) and TI (2.3%). One isolate showed both ESBL and CP phenotypes while two isolates were classified as inconclusive. Representatives from each phenotype were further analysed for the presence of beta-lactamases which revealed a predominance of TEM and SHV enzyme producers. PCR-SSCP analysis of the SHV gene from all the ESBL and CP isolates revealed the predominance of SHV 5-type enzyme which was concurrent with our previous studies.
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.
The toxin-antitoxin (TA) systems are systems in which an unstable antitoxin inhibits a stable toxin. This review aims to introduce the TA system and its biological application in bacteria. For this purpose, first we introduce a new classification for the TA systems based on how the antitoxin can neutralize the toxin, we then describe the functions of TA systems and finally review the application of these systems in biotechnology.
Three transition metal derivatives (Zn, Cu, and Ni) of 2-[2-bromoethyliminomethyl]-4-[ethoxymethyl]phenol (L) were synthesized by the reaction of the metal salts with the Schiff base ligand in one pot. In the crystal structure of [Zn(L)Br], the Schiff base ligand binds to the metal center through its phenolate oxygen and imine nitrogen, and adopts a distorted tetrahedral geometry. These compounds were found to inhibit topoisomerase I (topo I) activity, induce DNA cleavage and show DNA binding activity. Moreover, these compounds were found to be cytotoxic towards several cancer cell lines (A2780, MCF-7, HT29, HepG2, A549, PC3, LNCaP) and prevent metastasis of PC3. Collectively, Cu(II) complex 2 shows superior activity relative to its Zn(II) and Ni(II) analogs.
This article comparatively reports the workability of Escherichia coli BL21(DE3) and Pseudomonas putida KT2440 cell factories for the expression of three model autodisplayed cellulases (i.e., endoglucanase, BsCel5A; exoglucanase, CelK; β-glucosidase, BglA). The differentiation of the recombinant cells was restricted to their cell growth and enzyme expression/activity attributes. Comparatively, the recombinant E. coli showed higher cell growth rates but lower enzyme activities than the recombinant P. putida. However, the endo-, exoglucanase, and β-glucosidase on the surfaces of both cell factories showed activity over a broad range of pH (4-10) and temperature (30-100 °C). The pH and temperature optima were pH 6, 60 °C (BsCel5A); pH 6, 60-70 °C (CelK); and pH 6, 50 °C (BglA). Overall, the P. putida cell factory with autodisplayed enzymes demonstrated higher bioactivity and remarkable biochemical characteristics and thus was chosen for the saccharification of filter paper. A volumetric blend of the three cellulases with P. putida as the host yielded a ratio of 1:1:1.5 of endoglucanase, exoglucanase, and β-glucosidase, respectively, as the optimum blend composition for filter paper degradation. At an optical density (578 nm) of 50, the blend generated a maximum sugar yield of about 0.7 mg/ml (~ 0.08 U/g) from Whatman filter paper (Ø 6 mm, ~ 2.5 mg) within 24 h.
Urinary tract infections (UTI) caused by uropathogenic Escherichia coli are one of the most common forms of human disease. In this study, the effect of the presence of newly acquired antibiotic resistance genes on biofilm formation of UTI-associated E. coli strains was examined. Two clinical UTI-associated E. coli strains (SMC18 and SMC20) carrying different combinations of virulence genes were transformed with pGEM-T, pGEM-T::KmΔAmp, or pGEM-T::Km to construct ampicillin-resistant (Km(S)Amp(R)), kanamycin-resistant (Km(R)Amp(S)), or ampicillin- and kanamycin-resistant (Km(R)Amp(R)) strains. Transformed and wild-type strains were characterized for biofilm formation, bacterial surface hydrophobicity, auto-aggregation, morphology, and attachment to abiotic surfaces. Transformation with a plasmid carrying an ampicillin resistance gene alone decreased (p < 0.05) biofilm formation by SMC18 (8 virulence marker genes) but increased (p < 0.05) biofilm formation by SMC20 (5 virulence marker genes). On the other hand, transformation with a plasmid carrying a kanamycin resistance gene alone or both ampicillin and kanamycin resistance genes resulted in a decrease (p < 0.05) in biofilm formation by SMC18 but did not affect (p > 0.05) the biofilm formation by SMC20. Our results suggest that transformation of UTI-associated E. coli with plasmids carrying different antibiotic resistance gene(s) had a significant impact on biofilm formation and that these effects were both strain dependent and varied between different antibiotics.
The ST131 multilocus sequence type (MLST) of Escherichia coli is a globally successful pathogen whose dissemination is increasing rates of antibiotic resistance. Numerous global surveys have demonstrated the pervasiveness of this clone; in some regions ST131 accounts for up to 30% of all E. coli isolates. However, many regions are underrepresented in these published surveys, including Africa, South America, and Asia. We collected consecutive bloodstream E. coli isolates from three countries in Southeast Asia; ST131 was the most common MLST type. As in other studies, the C2/H30Rx clade accounted for the majority of ST131 strains. Clinical risk factors were similar to other reported studies. However, we found that nearly all of the C2 strains in this study were closely related, forming what we denote the SEA-C2 clone. The SEA-C2 clone is enriched for strains from Asia, particularly Southeast Asia and Singapore. The SEA-C2 clone accounts for all of the excess resistance and virulence of ST131 relative to non-ST131 E. coli. The SEA-C2 strains appear to be locally circulating and dominant in Southeast Asia, despite the intuition that high international connectivity and travel would enable frequent opportunities for other strains to establish themselves.
Strains of Escherichia coli that are non-typeable by pulsed-field gel electrophoresis (PFGE) due to in-gel degradation can influence their molecular epidemiological data. The DNA degradation phenotype (Dnd(+)) is mediated by the dnd operon that encode enzymes catalyzing the phosphorothioation of DNA, rendering the modified DNA susceptible to oxidative cleavage during a PFGE run. In this study, a PCR assay was developed to detect the presence of the dnd operon in Dnd(+) E. coli strains and to improve their typeability. Investigations into the genetic environments of the dnd operon in various E. coli strains led to the discovery that the dnd operon is harboured in various diverse genomic islands.
Escherichia coli ST131 is now recognised as a leading contributor to urinary tract and bloodstream infections in both community and clinical settings. Here we present the complete, annotated genome of E. coli EC958, which was isolated from the urine of a patient presenting with a urinary tract infection in the Northwest region of England and represents the most well characterised ST131 strain. Sequencing was carried out using the Pacific Biosciences platform, which provided sufficient depth and read-length to produce a complete genome without the need for other technologies. The discovery of spurious contigs within the assembly that correspond to site-specific inversions in the tail fibre regions of prophages demonstrates the potential for this technology to reveal dynamic evolutionary mechanisms. E. coli EC958 belongs to the major subgroup of ST131 strains that produce the CTX-M-15 extended spectrum β-lactamase, are fluoroquinolone resistant and encode the fimH30 type 1 fimbrial adhesin. This subgroup includes the Indian strain NA114 and the North American strain JJ1886. A comparison of the genomes of EC958, JJ1886 and NA114 revealed that differences in the arrangement of genomic islands, prophages and other repetitive elements in the NA114 genome are not biologically relevant and are due to misassembly. The availability of a high quality uropathogenic E. coli ST131 genome provides a reference for understanding this multidrug resistant pathogen and will facilitate novel functional, comparative and clinical studies of the E. coli ST131 clonal lineage.
The endogenous production of long-chain polyunsaturated fatty acids (LC-PUFA) in carnivorous teleost species inhabiting freshwater environments is poorly understood. Although a predatory lifestyle could potentially supply sufficient LC-PUFA to satisfy the requirements of these species, the nutrient-poor characteristics of the freshwater food web could impede this advantage. In this study, we report the cloning and functional characterisation of an elongase enzyme in the LC-PUFA biosynthesis pathway from striped snakehead (Channa striata), which is a strict freshwater piscivore that shows high deposition of LC-PUFA in its flesh. We also functionally characterised a previously isolated fatty acyl desaturase cDNA from this species. Results showed that the striped snakehead desaturase is capable of Δ4 and Δ5 desaturation activities, while the elongase showed the characteristics of Elovl5 elongases. Collectively, these findings reveal that striped snakehead exhibits the genetic resources to synthesise docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Both genes are expressed at considerable levels in the brain and the liver. In liver, both genes were up-regulated by dietary C18 PUFA, although this increase did not correspond to a significant rise in the deposition of muscle LC-PUFA. Brain tissue of fish fed with plant oil diets showed higher expression of fads2 gene compared to fish fed with fish oil-based diet, which could ensure DHA levels remain constant under limited dietary DHA intake. This suggests the importance of DHA production from EPA via the ∆4 desaturation step in order to maintain an optimal reserve of DHA in the neuronal tissues of carnivores.
The alginate lyase AlyQ from Persicobacter sp. CCB-QB2 is a three-domained enzyme with a carbohydrate-binding module (CBM) from family 32. The CBM32 domain, AlyQB, binds enzymatically cleaved but not intact alginate. Co-crystallisation of AlyQB with the cleaved alginate reveals that it binds to the 4,5-unsaturated mannuronic acid of the non-reducing end. The binding pocket contains a conserved R248 that interacts with the sugar's carboxyl group, as well as an invariant W303 that stacks against the unsaturated pyranose ring. Targeting specifically the non-reducing end is more efficient than the reducing end since the latter consists of a mixture of mannuronic acid and guluronic acid. AlyQB also seems unable to bind these two saturated sugars as they contain OH groups that will clash with the pocket. Docking analysis of YeCBM32, which binds oligogalacturonic acid, shows that the stacking of the pyranose ring is shifted in order to accommodate the sugar's axial C1-OH, and its R69 is accordingly elevated to bind the sugar's carboxyl group. Unlike AlyQB, YeCBM32's binding pocket is able to accommodate both saturated and unsaturated galacturonic acid.
Over the past few decades, Escherichia coli (E. coli) remains the most favorable host among the microbial cell factories for the production of soluble recombinant proteins. Recombinant protein production (RPP) via E. coli is optimized at the level of gene expression (expression level) and the process condition of fermentation (process level). Presently, the reported studies do not give a clear view on the selection of methods employed in the optimization of RPP. Here, we have reviewed various optimization methods and their preferences with respect to the factors at expression and process levels to achieve the optimal levels of soluble RPP. With a greater understanding of these optimization methods, we proposed a stepwise methodology linking the factors from both levels for optimizing the production of soluble recombinant protein in E. coli. The proposed methodology is further explained through five sets of examples demonstrating the optimization of RPP at both expression and process levels.Key Points• Stepwise methodology of optimizing recombinant protein production is proposed.• In silico tools can facilitate the optimization of gene- and protein-based factors.• Optimization of gene- and protein-based factors aids host-vector selection.• Statistical optimization is preferred for achieving optimal levels of process factors.
The nucleocapsid (N) protein of Nipah virus (NiV) can be produced in three Escherichia coli strains [TOP10, BL21(DE3) and SG935] under the control of trc promoter. However, most of the product existed in the form of insoluble inclusion bodies. There was no improvement in the solubility of the product when this protein was placed under the control of T7 promoter. However, the solubility of the N protein was significantly improved by lowering the growth temperature of E. coli BL21(DE3) cell cultures. Solubility analysis of N- and C-terminally deleted mutants revealed that the full-length N protein has the highest solubility. The soluble N protein could be purified efficiently by sucrose gradient centrifugation and nickel affinity chromatography. Electron microscopic analysis of the purified product revealed that the N protein assembled into herringbone-like particles of different lengths. The C-terminal end of the N protein contains the major antigenic region when probed with antisera from humans and pigs infected naturally.
The prevalence of antibiotic-resistant bacteria in Southeast Asia is a significant concern, yet there is limited research on the gut resistome and its correlation with lifestyle and environmental factors in the region. This study aimed to profile the gut resistome of 200 individuals in Malaysia using shotgun metagenomic sequencing and investigate its association with questionnaire data comprising demographic and lifestyle variables. A total of 1038 antibiotic resistance genes from 26 classes were detected with a mean carriage rate of 1.74 ± 1.18 gene copies per cell per person. Correlation analysis identified 14 environmental factors, including hygiene habits, health parameters, and intestinal colonization, that were significantly associated with the resistome (adjusted multivariate PERMANOVA, p Escherichia coli as a major resistome network hub, with a positive correlation to 36 antibiotic-resistance genes. Our findings suggest that E. coli may play a pivotal role in shaping the resistome dynamics in Segamat, Malaysia, and its abundance is strongly associated with the community's health and lifestyle habits. Furthermore, the presence of yeast appears to be associated with the suppression of antibiotic-resistance genes.
The excretion of cyclodextrin glucanotransferase (CGTase) into the culture medium offers significant advantages over cytoplasmic expression. However, the limitation of Escherichia coli is its inability to excrete high amount of CGTase outside the cells. In this study, modification of the hydrophobic region of the N1R3 signal peptide using site-saturation mutagenesis improved the excretion of CGTase. Signal peptide mutants designated M9F, V10L and A15Y enhanced the excretion of CGTase three-fold and demonstrated two-fold higher secretion rate than the wild type. However, high secretion rate of these mutants was non-productive for recombinant protein production because it caused up to a seven-fold increase in cell death compared to the wild type. Our results indicated that the excretion of CGTase is highly dependent on hydrophobicity, secondary conformation and the type and position of amino acids at the region boundary and core segment of the h-region.
A broad substrate specificity enzyme that can act on a wide range of substrates would be an asset in industrial application. T1 lipase known to have broad substrate specificity in its native form apparently exhibits the same active sites as polyhydroxylalkanoate (PHA) depolymerase. PhaZ6Pl is one of the PHA depolymerases that can degrade semicrystalline P(3HB). The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. A structural study on PhaZ6Pl contains no lid in its structure and therefore T1 lipase was designed with removal of its lid region. BSLA lipase was chosen as the reference protein for T1 lipase modification since it contains no lid. Initially, structures of both enzymes were compared via protein-protein superimposition in 3D-space and the location of the lid region of T1 lipase was highlighted. A total of three variants of T1 lipase without lid were successfully designed by referring to BSLA lipase (a lipase without lid). The ability of T1 lipase without lid variants in degrading P(3HB) was investigated quantitatively. All the variants showed activity towards the substrate which confirmed that T1 lipase without lid is indeed able to degrade P(3HB). In addition, D2 was recorded to have the highest activity amongst other variants. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only record triglyceride degradation but also P(3HB) by simply removing the lid region.
Genetic polymorphism of the cytochrome P450 (CYP) genes particularly affects CYP2D6 and CYP2C19 to a functionally relevant extent, and it is therefore crucial to elucidate the enzyme kinetic and molecular basis for altered catalytic activity of these allelic variants. This study explored the expression and function of the reported alleles CYP2D6*2, CYP2D6*10, CYP2D6*17, CYP2C19*23, CYP2C19*24, and CYP2C19*25 with respect to gene polymorphisms. Site-directed mutagenesis (SDM) was carried out to generate these six alleles. After DNA sequencing, the CYP2D6 and CYP2C19 wild types alongside with their alleles were each independently co-expressed with NADPH-CYP oxidoreductase (OxR) in Escherichia coli. The expressed proteins were analyzed using Western blotting, reduced carbon monoxide (CO) difference spectral scanning, and cytochrome c reductase assay. Results from Western blot revealed the presence of all CYP wild-type and allelic proteins in E. coli membrane fractions. The reduced CO difference spectra scanning presented the distinct peak of absorbance at 450 nm, and the cytochrome c reductase assay has confirmed that spectrally active OxR was expressed in each protein preparation. As a conclusion, the results obtained from this study have proven the CYP variants to be immunoreactive and spectrally active and are suitable for use to examine biotransformation and interaction mechanism of the enzymes.
When recombineering bacterial artificial chromosomes (BACs), it is common practice to design the ends of the donor molecule with 50 bp of homology specifying its insertion site. We demonstrate that desired recombinants can be produced using intermolecular homologies as short as 15 bp. Although the use of shorter donor end regions decreases total recombinants by several fold, the frequency of recombinants with correctly inserted donor molecules was high enough for easy detection by simple polymerase chain reaction (PCR) screening. This observation may have important implications for the design of oligonucleotides for recombineering, including significant cost savings, especially for high-throughput projects that use large quantities of primers.