Introduction of a stress-responsive gene, yggG, enhances the yield of l-phenylalanine with decreased acetic acid production in a recombinant Escherichia coli

A stress-responsive gene, yggG, was introduced into an l-phenylalanine producer, Escherichia coli AJ12741. In shake-flask culture, the yggG-containing recombinant strain (named AJ12741/pHYGG) produced 6.4 g l-phenylalanine l⁻¹ at the end of culture and its yield on glucose was 0.16 g l-phenylalanine g glucose⁻¹. These values are much higher than those of the original AJ12741 strain (3.7 g l-phenylalanine l⁻¹ and 0.09 g l-phenylalanine g glucose⁻¹, respectively). On the other hand, AJ12741/pHYGG strain produced only 4.5 g acetic acid l⁻¹ and its yield on glucose was about a half of that of the AJ12741 culture. Analysis of gene expression revealed that in late growth phase, the expression levels of genes involved in acetic acid production (pta, ackA, and poxB) were relatively low in AJ12741/pHYGG cells. In particular, the level of poxB expression in AJ12741/pHYGG strains was one-seventh of that of the original strain. These results suggest that the formation of a bottleneck for acetic acid production brings about a metabolic flow favorable to l-phenylalanine synthesis in the recombinant strain over-expressing the yggG gene. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular mass of poly[(R )-3-hydroxybutyric acid] produced in a recombinant Escherichia coli

 Poly[(R)-3-hydroxybutyric acid] (PHB) was produced at 37 °C by a recombinant Escherichia coli harboring the Alcaligenes eutrophus biosynthesis phbCAB genes in Luria-Bertani media containing glucose at 10–30 g/l at different pH values and the time-dependent changes in the molecular mass of PHB were studied. PHB polymers accumulated within cells while glucose was present in the medium. The number-average molecular mass of PHB decreased with time during the course of PHB accumulation, and the values for PHB were markedly dependent on the cultivation conditions of the E. coli, ranging from 0.5 MDa to 20 MDa. Under specific conditions (pH 6.0), E. coli produced PHB with an extremely high molecular mass (20 MDa). It has been suggested that a chain-transfer agent is generated in E. coli cells during the accumulation of PHB. Received: 18 July 1996 / Received revision: 4 November 1996 / Accepted: 4 November 1996     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

Molecular basis of the γ-aminobutyric acid A receptor α3 subunit interaction with the clustering protein gephyrin

The multifunctional scaffolding protein gephyrin is a key player in the formation of the postsynaptic scaffold at inhibitory synapses, clustering both inhibitory glycine receptors (GlyRs) and selected GABA(A) receptor (GABA(A)R) subtypes. We report a direct interaction between the GABA(A)R α3 subunit and gephyrin, mapping reciprocal binding sites using mutagenesis, overlay, and yeast two-hybrid assays. This analysis reveals that critical determinants of this interaction are located in the motif FNIVGTTYPI in the GABA(A)R α3 M3-M4 domain and the motif SMDKAFITVL at the N terminus of the gephyrin E domain. GABA(A)R α3 gephyrin binding-site mutants were unable to co-localize with endogenous gephyrin in transfected hippocampal neurons, despite being able to traffic to the cell membrane and form functional benzodiazepine-responsive GABA(A)Rs in recombinant systems. Interestingly, motifs responsible for interactions with GABA(A)R α2, GABA(A)R α3, and collybistin on gephyrin overlap. Curiously, two key residues (Asp-327 and Phe-330) in the GABA(A)R α2 and α3 binding sites on gephyrin also contribute to GlyR β subunit-E domain interactions. However, isothermal titration calorimetry reveals a 27-fold difference in the interaction strength between GABA(A)R α3 and GlyR β subunits with gephyrin with dissociation constants of 5.3 μm and 0.2 μm, respectively. Taken together, these observations suggest that clustering of GABA(A)R α2, α3, and GlyRs by gephyrin is mediated by distinct mechanisms at mixed glycinergic/GABAergic synapses.     

Identification of a cyclooxygenase gene from the red alga Gracilaria vermiculophylla and bioconversion of arachidonic acid to PGF(2α) in engineered Escherichia coli

Prostaglandins (PGs) are important local messenger molecules in many tissues and organs of animals including human. For applications in medicine and animal care, PGs are mostly purified from animal tissues or chemically synthesized. To generate a clean, reliable, and inexpensive source for PGs, we have now engineered expression of a suitable cyclooxygenase gene in Escherichia coli and achieved production levels of up to 2.7 mg l⁻¹ PGF_2α. The cyclooxygenase gene cloned from the red alga Gracilaria vermiculophylla appears to be fully functional without any eukaryotic modifications in E. coli. A crude extract of the recombinant E. coli cells is able to convert in vitro the substrate arachidonic acid (AA) to PGF_2α. Furthermore, these E. coli cells produced PGF_2α in a medium supplemented with AA and secreted the PGF_2α product. To our knowledge, this is the first report of the functional expression of a cyclooxygenase gene and concomitant production of PGF_2α in E. coli. The successful microbial synthesis of PGs with reliable yields promises a novel pharmaceutical tool to produce PGF_2α at significantly reduced prices and greater purity.     

Synthesis of 2-amino-2-deoxy- and 3-amino-3-deoxy-α-d-mannopyranosyl α-d-mannopyranoside by sequential osmylations of a dienic disaccharide

Partial oxyamination of 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranosyl 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranoside with chloramine-T and osmium tetraoxide gave 4,6-di-O-acetyl-2-deoxy-2-(p-toluene-sulfonamido)-α-d-mannopyranosyl 4,6-di-O-acetyl-2,3-dideoxy-α-d-erythro-hex-2-enopyranoside and its 3-deoxy-3-(p-toluenesulfonamido) regioisomer, each in 18–19% isolated yield. Osmium tetraoxide-catalyzed cis-hydroxylation of the remaining alkenic residue in these products led in high yields to the corresponding triols having the α-d-manno, α-d-manno configuration. These were N-desulfonylated (and simultaneously O-deacetylated) by the action of sodium in liquid ammonia to furnish 2-amino-2-deoxy-α-d-mannopyranosyl α-d-mannopyranoside and 3-amino-3-deoxy-α-d-mannopyranosyl α-d-mannopyranoside as new, trehalose-type amino sugars.     

Pathogen–pathogen interactions: a comparative study of Escherichia coli interactions with the clinical and environmental isolates of Acanthamoeba

In this study, we compared the interactions of invasive and non-invasive strains of E. coli with clinical and environmental isolates of Acanthamoeba. The environmental isolate of Acanthamoeba exhibited significantly higher association with E. coli compared with the clinical isolates of Acanthamoeba. The ratio of E. coli per amoebae was more than 8-fold higher in the environmental isolate compared with the clinical isolates of Acanthamoeba. Interestingly, non-pathogenic environmental Acanthamoeba showed uptake and/or survival of the non-invasive E. coli. In contrast, clinical isolates of Acanthamoeba did not support uptake and/or survival of non-invasive E. coli. Using several mutants derived from K1, we demonstrated that outer membrane protein A (OmpA) and lipopolysaccharide (LPS) are crucial bacterial determinants responsible for E. coli K1 interactions and in the intracellular survival of E. coli in Acanthamoeba. The use of Acanthamoeba as a model to study E. coli K1 pathogenesis and to understand bacterial immune evasion strategies is discussed further.     

Antibiotic-resistant Escherichia coli in karstic systems: a biological indicator of the origin of fecal contamination?

Occurrences of antibiotic-resistant Escherichia coli in two springs of a karstic system (NW France) providing drinking water were determined to study the role of aquifers in the dissemination of the resistance genes. Water samples were collected during wet and dry periods and after a heavy rainfall event to investigate E. coli density, antibiotic resistance patterns, and occurrences of class 1, 2, and 3 integrons. By observing patterns of the resistant isolates (i.e. number and type of resistances) and their occurrences, we were able to define two resistant subpopulations, introduced in the aquifer via surface water: (1) R1-2, characterized by one or two resistance(s), essentially to chloramphenicol and/or tetracycline (96.5%), was always found during the heavy rainfall event; (2) R3-10, characterized by three or more resistances, mostly resistant to tetracycline (94.1%) and beta-lactams (86%), was found transiently. Class 1 and 2 integrons were detected, mostly in the R3-10 subpopulation for class 1 integrons. The characteristics of these two subpopulations strongly suggest that the contamination originates from pasture runoff for the R1-2 subpopulation and from wastewater treatment plant effluents for the R3-10 subpopulation. These two subpopulations of E. coli could be used as biological indicators to determine the origin of groundwater contamination.     

Genetic stability and DNA rearrangements associated with a 2 × 1.1-Kb perfect palindrome in Escherichia coli

We show that circular plasmids containing perfect palindromic regions of 2?×?1.1 kb can be propagated in sbcC strains of Escherichia coli, a result that is at variance with the well known observation that λ DNA cannot tolerate palindromic regions larger than 2 × 265 bp. However, a significant fraction of these palindrome-containing plasmids can be recovered from E. coli strains either as linear molecules with hairpins at their ends or as head-to-head dimers, both in a RuvC-and RusA-independent manner. Our results suggests that large palindromes may form cruciforms in E. coli. However, palindrome-associated DNA rearrangements occur by a process that does not require any known cruciform resolvase activity. Our data support a replication-dependent model for the induction of DNA rearrangements by perfect palindromes.     

Molecular cloning of the β-cyclodextrin synthetase gene from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis

Summary The gene for -CGTase from an alkalophilic bacterium, Bacillus sp. #1011, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a B. subtilis plasmid pUB110. An E. coli recombinant plasmid pTUE202 and a B. subtilis plasmid pTUB703 were selected from ten plasmids, because the transformants by each of the two plasmids produced the highest amount of extracellular -CGTase in each strain. The plasmids were stably maintained and expressed in each bacterial strain. A common DNA region of approximately 2.5 kb was defined in the ten plasmids, and the enzymatic activity was lost when a part of the common region was deleted. The major product of hydrolysis from starch by the -CGTases of E. coli [pTUB202] and B. subtilis [pTUB703] was -CD as in the case of the enzyme of the parental Bacillus sp. #1011.Abbreviations -CGTase -cyclodextrin synthetase - -CD -cyclodextrin - -CD -cyclodextrin - -CD -cyclodextrin - [] designates plasmid-carrier state     

Multidrug-resistant Escherichia coli in Raw Milk: Molecular Characterization and the potential impact of camel’s Urine as an Antibacterial Agent

Raw milk is one of the most important vehicles for transmitting various pathogens, especially Escherichia coli (E. coli). Multidrug-resistant pathogens are highly prevalent among mastitic cows in various dairy farms worldwide. Therefore, our current study is based on the identification of E. coli from mastitic cow’s milk and their resistance to various antibacterial agents. As well, the impact of camel’s urine on multi-drug resistant E. coli were also evaluated. Thirty-three E. coli isolates were recovered from 254 milk samples. All strains were initially identified phenotypically by culturing on specific media and Vitek 2 Compact System. The protein fingerprinting technique was used as a confirmatory method. The Stx1, Stx2 and eae genes were also verified by polymerase chain reaction (PCR). The antimicrobial resistance of E. coli strains was tested by the Vitek 2 AST-GN69 cards. Thirty multi-drug resistant E. coli strains (20 from mastitic milk and 10 from clinical samples) were laboratory tested with different concentrations (100%, 75%, 50% and 25%) of virgin and breeding camel’s urine, using the paper disc diffusion method. Our findings showed that 93.94% of E. coli strains were recognized by the Vitek™ 2 system. The results of proteomic investigation illustrated that 100% of E. coli strains were identified at log values ≥2.00. The genotypic identification of the three virulence genes illustrated that 90.1%, 63.64%, and 30.55% of E. coli strains were able to carry the Stx1, eae, and Stx2 genes, respectively. Most strains of E. coli showed strong resistance against cefazolin (78.79%), ceftazidime (66.67%), cefotaxime (60.61%), ceftriaxone (54.55%), and cefepime (39.40%). The results of the antibacterial effect of camel’s urine revealed that the mean inhibitory zones of virgin camel’s urine were 28 mm, 17 mm, and 14 mm, for the concentrations of 100%, 75%, and 50%, respectively. Whereas; the inhibitory zones for the breeding camel’s urine were 18 mm, 0 mm, and 0 mm, for the concentrations of 100%, 75%, and 50%, respectively. We concluded that the majority of E. coli strains were able to harbor some virulence genes and resist many antibiotics. Our study also provided a robust evidence that the camel’s urine, particularly from the virgin camels has robust antimicrobial activity against multidrug-resistant E. coli strains.     

Opposite roles of domains 2 + 3 of Escherichia coli EF-Tu and Bacillus stearothermophilus EF-Tu in the regulation of EF-Tu GTPase activity

The effect of noncatalytic domains 2 + 3 on the intrinsic activity and thermostability of the EF-Tu GTPase center was evaluated in experiments with isolated domains 1 and six chimeric variants of mesophilic Escherichia coli (Ec) and thermophilic Bacillus stearothermophilus (Bst) EF-Tus. The isolated catalytic domains 1 of both EF-Tus displayed similar GTPase activities at their optimal temperatures. However, noncatalytic domains 2 + 3 of the EF-Tus influenced the GTPase activity of domains 1 differently, depending on the domain origin. Ecdomains 2 + 3 suppressed the GTPase activity of the Ecdomain 1, whereas those of BstEF-Tu stimulated the Bstdomain 1 GTPase. Domain 1 and domains 2 + 3 of both EF-Tus positively cooperated to heat-stabilize their GTPase centers to attain optimal activity at a temperature close to the optimal growth temperature of either organism. This can be explained by a stabilization effect of domains 2 + 3 on α-helical regions of the G-domain as revealed by CD spectroscopy.     

Expression in Escherichia coli and characterization of a bile acid-inducible 3α-hydroxysteroid dehydrogenase from Eubacterium sp. strain VPI 12708

We have previously cloned and sequenced three members of a bile acid-inducible gene family from Eubacterium sp. strain VPI 12708 that encode 27,000-M _r polypeptides. Two copies of these genes (baiA1 and baiA3) are identical, while the third copy (baiA2) encodes a polypeptide sharing 92% amino acid identity with the baiA1 and baiA3 gene products. We have overexpressed the baiA1 gene in Escherichia coli and analyzed the expressed activity. Thin-layer chromatography of ¹⁴C-labeled bile acid products from reactions using cell-free extracts revealed a 3-hydroxysteroid dehydrogenase activity for the BaiA1 protein. The BaiA1 protein could utilize both NAD⁺ and NADP⁺, and the preferred steroid substrate was the cholyl-coenzyme A conjugate rather than free cholic acid. These results show that the BaiA proteins are novel 3-hydroxysteroid dehydrogenases.     

Molecular cloning and expression in Escherichia coli of a thermophilic Bacillus sp. PDV endo-β-1,4-glucanase gene

The gene encoding endoglucanase in thermophilic Bacillus sp. PDV was cloned in Escherichia coli strain TB1 using pUC 8 as vector. The cloned 3.1 kb PstI DNA fragment was found to express the endoglucanase activity in either orientation. The deletion analysis of pSD 81 suggested that the Bacillus endoglucanase gene expressed in E. coli under the control of its own natural promoter, contained putatively in the 0.2 kb HindIII fragment at the 5′ end of the insert. The relative level of endoglucanase expression in E. coli was about three times higher than that in parent Bacillus sp. PDV. The cloned organism secreted about 84% of the total synthesized CMCase into the culture medium. The CMCase was stable up to 60°C and in the pH range of 4–10.     

Increase in the production of β-carotene in recombinant Escherichia coli cultured in a chemically defined medium supplemented with amino acids

Escherichia coli DH5α strain was selected as the recombinant host, and a chemically defined medium supplemented with amino acids was used instead of a complex medium for the efficient production of β-carotene. In a fed-batch culture using glycerol with a chemically defined medium supplemented with amino acids, the concentration, specific content, and productivity of β-carotene were 2,470 mg/l, 72 mg/g cells, and 77 mg/l h after 32 h, respectively. These values were, respectively, 43, 33, and 26 % higher than those obtained using the complex medium. This is the highest β-carotene production that has been reported among the recombinant cells to date.