Bacterial Hemoglobins and Flavohemoglobins for Alleviation of Nitrosative Stress in Escherichia coli

Escherichia coli MG1655 cells expressing novel bacterial hemoglobin and flavohemoglobin genes from a medium-copy-number plasmid were grown in shake flask cultures under nitrosative and oxidative stress. E. coli cells expressing these proteins display enhanced resistance against the NO^· releaser sodium nitroprusside (SNP) relative to that of the control strain bearing the parental plasmid. Expression of bacterial hemoglobins originating from Campylobacter jejuni (CHb) and Vitreoscilla sp. (VHb) conferred resistance on SNP-challenged cells. In addition, it has been shown that NO^· detoxification is also a common feature of flavohemoglobins originating from different taxonomic groups and can be transferred to a heterologous host. These observations have been confirmed in a specific in vitro NO^· consumption assay. Protein extracts isolated from E. coli strains overexpressing flavohemoglobins consumed authentic NO^· more readily than protein extracts from the wild-type strain. Oxidative challenge to the cells evoked nonuniform responses from the various cell cultures. Improved oxidative-stress-sustaining properties had also been observed when the flavohemoglobins from E. coli, Klebsiella pneumoniae, Deinococcus radiodurans, and Pseudomonas aeruginosa were expressed in E. coli.     

Novel hemoglobins to enhance microaerobic growth and substrate utilization in Escherichia coli

Limited oxygen availability is a prevalent problem in microbial biotechnology. Recombinant Escherichia coli expressing the hemoglobin from Vitreoscilla (VHb) or the flavohemoglobin from Ralstonia eutropha (formerly Alcaligenes eutrophus) (FHP) demonstrate significantly increased cell growth and productivity under microaerobic conditions. We identify novel bacterial hemoglobin-like proteins and examine if these novel bacterial hemoglobins can elicit positive effects similar to VHb and FHP and if these hemoglobins alleviate oxygen limitation under microaerobic conditions when expressed in E. coli. Several finished and unfinished bacterial genomes were screened using R. eutropha FHP as a query sequence for genes (hmp) encoding hemoglobin-like proteins. Novel hmp genes were identified in Pseudomonas aeruginosa, Salmonella typhi, Klebsiella pneumoniae, Deinococcus radiodurans, and Campylobacter jejuni. Previously characterized hmp genes from E. coli and Bacillus subtilis and the novel hmp genes from P. aeruginosa, S. typhi, C. jejuni, K. pneumoniae, and D. radiodurans were PCR amplified and introduced into a plasmid for expression in E. coli. Biochemically active hemoproteins were expressed in all constructs, as judged by the ability to abduct carbon monoxide. Growth behavior and byproduct formation of E. coli K-12 MG1655 cells expressing various hemoglobins were analyzed in microaerobic fed-batch cultivations and compared to plasmid-bearing control and to E. coli cells expressing VHb. The clones expressing hemoglobins from E. coli, D. radiodurans, P.aeruginosa, and S. typhi reached approximately 10%, 27%, 23%, and 36% higher final optical density values, respectively, relative to the plasmid bearing E. coli control (A(600) 5.5). E. coli cells expressing hemoproteins from P. aeruginosa, S. typhi, and D. radiodurans grew to similar final cell densities as did the strain expressing VHb (A(600) 7.5), although none of the novel constructs was able to outgrow the VHb-expressing E. coli strain. Additionally, increased yield of biomass on glucose was measured for all recombinant strains, and an approximately 2-fold yield enhancement was obtained with D. radiodurans hemoprotein-expressing E. coli relative to the E. coli control carrying the parental plasmid without any hemoglobin gene.     

Recombinational repair is critical for survival of Escherichia coli exposed to nitric oxide

Nitric oxide (NO(.)) is critical to numerous biological processes, including signal transduction and macrophage-mediated immunity. In this study, we have explored the biological effects of NO(.)-induced DNA damage on Escherichia coli. The relative importance of base excision repair, nucleotide excision repair (NER), and recombinational repair in preventing NO(.)-induced toxicity was determined. E. coli strains lacking either NER or DNA glycosylases (including those that repair alkylation damage [alkA tag strain], oxidative damage [fpg nei nth strain], and deaminated cytosine [ung strain]) showed essentially wild-type levels of NO(.) resistance. However, apyrimidinic/apurinic (AP) endonuclease-deficient cells (xth nfo strain) were very sensitive to killing by NO(.), which indicates that normal processing of abasic sites is critical for defense against NO(.). In addition, recA mutant cells were exquisitely sensitive to NO(.)-induced killing. Both SOS-deficient (lexA3) and Holliday junction resolvase-deficient (ruvC) cells were very sensitive to NO(.), indicating that both SOS and recombinational repair play important roles in defense against NO(.). Furthermore, strains specifically lacking double-strand end repair (recBCD strains) were very sensitive to NO(.), which suggests that NO(.) exposure leads to the formation of double-strand ends. One consequence of these double-strand ends is that NO(.) induces homologous recombination at a genetically engineered substrate. Taken together, it is now clear that, in addition to the known point mutagenic effects of NO(.), it is also important to consider recombination events among the spectrum of genetic changes that NO(. ) can induce. Furthermore, the importance of recombinational repair for cellular survival of NO(.) exposure reveals a potential susceptibility factor for invading microbes.     

The function of cytoplasmic flavin reductases in the reduction of azo dyes by bacteria

A flavin reductase, which is naturally part of the ribonucleotide reductase complex of Escherichia coli, acted in cell extracts of recombinant E. coli strains under aerobic and anaerobic conditions as an "azo reductase." The transfer of the recombinant plasmid, which resulted in the constitutive expression of high levels of activity of the flavin reductase, increased the reduction rate for different industrially relevant sulfonated azo dyes in vitro almost 100-fold. The flavin reductase gene (fre) was transferred to Sphingomonas sp. strain BN6, a bacterial strain able to degrade naphthalenesulfonates under aerobic conditions. The flavin reductase was also synthesized in significant amounts in the Sphingomonas strain. The reduction rates for the sulfonated azo compound amaranth were compared for whole cells and cell extracts from both recombinant strains, E. coli, and wild-type Sphingomonas sp. strain BN6. The whole cells showed less than 2% of the specific activities found with cell extracts. These results suggested that the cytoplasmic anaerobic "azo reductases," which have been described repeatedly in in vitro systems, are presumably flavin reductases and that in vivo they have insignificant importance in the reduction of sulfonated azo compounds.     

Stimulation of bacterial decolorization of an azo dye by extracellular metabolites from Escherichia coli strain NO3

This study provides an attempt to use the extracellular metabolites of a dye-decolorizing strain, Escherichia coli strain NO3, as a biostimulator to entice E. coli strain NO3 into a beneficial mode of metabolism for an economically feasible decolorization. Addition of supernatants containing metabolites from growth and decolorization cultures triggered an enhancement of decolorization rates. Although extracellular metabolites play a crucial role for stimulating decolorization, they still cannot enable decolorization alone without involvement of biodecolorizers (E. coli strain NO3). Substantial decreases in oxidation-reduction potential to negative levels were observed during anaerobic decolorization in the presence of the metabolites. Results of repeated batch cultures also showed that serial acclimation of E. coli strain NO3 significantly increased decolorization capability. Supplements of metabolites seem to derepress glucose inhibition on decolorization by E. coli strain NO3. The metabolites produced from higher dye-concentration decolorization led to more enhancement of the bacterial decolorization.     

Identification and cloning of gusA, encoding a new beta-glucuronidase from Lactobacillus gasseri ADH

The gusA gene, encoding a new beta-glucuronidase enzyme, has been cloned from Lactobacillus gasseri ADH. This is the first report of a beta-glucuronidase gene cloned from a bacterial source other than Escherichia coli. A plasmid library of L. gasseri chromosomal DNA was screened for complementation of an E. coli gus mutant. Two overlapping clones that restored beta-glucuronidase activity in the mutant strain were sequenced and revealed three complete and two partial open reading frames. The largest open reading frame, spanning 1,797 bp, encodes a 597-amino-acid protein that shows 39% identity to beta-glucuronidase (GusA) of E. coli K-12 (EC 3.2.1.31). The other two complete open reading frames, which are arranged to be separately transcribed, encode a putative bile salt hydrolase and a putative protein of unknown function with similarities to MerR-type regulatory proteins. Overexpression of GusA was achieved in a beta-glucuronidase-negative L. gasseri strain by expressing the gusA gene, subcloned onto a low-copy-number shuttle vector, from the strong Lactobacillus P6 promoter. GusA was also expressed in E. coli from a pET expression system. Preliminary characterization of the GusA protein from crude cell extracts revealed that the enzyme was active across an acidic pH range and a broad temperature range. An analysis of other lactobacilli identified beta-glucuronidase activity and gusA homologs in other L. gasseri isolates but not in other Lactobacillus species tested.     

The effect of small concentrations of antibiotics blocking the synthesis of bacterial cell wall on the permeability of Escherichia coli cell wall for plasmid DNA

Short treatment of Escherichia coli cells with antibiotics disturbing synthesis of bacterial cell wall in small concentrations renders the cells capable of absorbing foreign plasmid DNA. A novel express-method for transformation of E. coli cells by plasmid DNA has been developed on the basis of the results obtained. The whole procedure can be performed at room temperature. Depending on cell strain and the plasmid size, the efficiency of transformation can vary from 1.10(4) to 5.10(5) transformants per 1 mkg of DNA. The method suggested improves significantly the every-day work aimed at constructing plasmids.     

Expression of the yeast galactokinase gene in Escherichia coli.

In Saccharomyces cerevisiae the genes for three of the enzymes involved in galactose metabolism are tightly linked near the centromere of chromosome II (Douglas and Hawthorne, 1964). However, the molecular mechanisms which control the expression of these genes are not well understood. A DNA fragment containing at least one of these yeast genes, the galactokinase gene (gal1), has been joined to the bacterial plasmid pBR322 and maintained in an Escherichia coli strain that carries a deletion in its own galactokinase gene, galK. The presence of the yeast gene was demonstrated by (i) complementation of the E. coli galactokinase deletion, (ii) by hybridization of the cloned DNA fragment to restriction enzyme digests of total yeast DNA and (iii) by assaying for yeast galactokinase activity in bacterial cell extracts. The yeast DNA fragment is 4700 base pairs long, and enables the host E. coli K-12 strain to grow in minimal medium containing galactose as the sole carbon source with a generation time of 14.3 h. The yeast galactokinase activity in the bacterial extracts is 0.7% of the bacterial galactokinase activity found in wild-type E. coli fully induced with fucose.     

Nitric oxide formation by Escherichia coli. Dependence on nitrite reductase,the NO-sensing regulator Fnr,and flavohemoglobin Hmp

Nitric oxide (NO) is a key signaling and defense molecule in biological systems. The bactericidal effects of NO produced, for example, by macrophages are resisted by various bacterial NO-detoxifying enzymes, the best understood being the flavohemoglobins exemplified by Escherichia coli Hmp. However, many bacteria, including E. coli, are reported to produce NO by processes that are independent of denitrification in which NO is an obligatory intermediate. We demonstrate using an NO-specific electrode that E. coli cells, grown anaerobically with nitrate as terminal electron acceptor, generate significant NO on adding nitrite. The periplasmic cytochrome c nitrite reductase (Nrf) is shown, by comparing Nrf+ and Nrf- mutants, to be largely responsible for NO generation. Surprisingly, an hmp mutant did not accumulate more NO but, rather, failed to produce detectable NO. Anaerobic growth of the hmp mutant was not stimulated by nitrate, and the mutant failed to produce periplasmic cytochrome(s) c, leading to the hypothesis that accumulating NO in the absence of Hmp inactivates the global anaerobic regulator Fnr by reaction with the [4Fe-4S]2+ cluster (Cruz-Ramos, H., Crack, J., Wu, G., Hughes, M. N., Scott, C., Thomson, A. J., Green, J., and Poole, R. K. (2002) EMBO J. 21, 3235-3244). Fnr thus failed to up-regulate nitrite reductase. The model is supported by the inability of an fnr mutant to generate NO and by the restoration of NO accumulation to hmp mutants upon introducing a plasmid encoding Fnr* (D154A) known to confer activity in the presence of oxygen. A cytochrome bd-deficient mutant retained NO-generating activity. The present study reveals a critical balance between NO-generating and -detoxifying activities during anaerobic growth.     

Silver accumulation and resistance in Escherichia coli R1

E. coli R1 contains at least 2 large plasmids (83 and 77 kb) while E. coli S1 was previously cured of the 83 kb plasmid. Transformation using artificial competence, high-voltage electroporation, and plasmid mobilization experiments with the mobilizing plasmid RP4, failed to ascertain the 83 kb plasmid was responsible for Ag(+)-resistance. Silver accumulation by an Ag(+)-sensitive E. coli S1 strain was 5-fold higher than an Ag(+)-resistant E. coli R1 strain. The Ag(+)-resistant E. coli R1 strain produced 33% more H2S and 32% more intracellular acid labile SH than the Ag(+)-sensitive E. coli R1 strain when grown in the absence of AgNO3. Hydrophobic interaction chromatography revealed E. coli R1 displayed higher cell surface hydrophobicity than E. coli S1. HPLC protein analysis of cell-free extracts also revealed differences between protein fractions in E. coli R1 and S1 strains.     

Mycobacterial mammalian cell entry protein 1A (Mce1A)-mediated adherence enhances the chemokine production by A549 alveolar epithelial cells

Mycobacterial mammalian cell entry protein 1A (Mce1A) is involved in the uptake of bacteria in non-phagocytic cells and also possibly in granuloma formation. However, it has not been clarified whether the interaction between mycobacterial Mce1A and epithelial cell induces chemokine and cytokine production which is required for granuloma formation. To this end, we infected A549 alveolar epithelial cells in vitro with E. coli expressing Mce1A on the cell surface and examined the resultant chemokine/cytokine production. Mce1A promoted bacterial adherence and internalization of E. coli into A549 cells, and these recombinant bacteria induced high levels of MCP-1 and IL-8 production, compared to E. coli harboring the plasmid vector alone. Chemokine production was enhanced by the internalization of recombinant E. coli expressing Mce1A because cytochalasin D treatment partially inhibited MCP-1 and IL-8 production. However, Mce1A-coated latex beads did not induce the chemokine production. These results suggest that although Mce1A does not induce production of chemokines, it may promote chemokine induction by augmenting the interaction between bacteria and epithelial cells.     

Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli

An alternative approach to the use of antibiotic selection markers for maintenance of recombinant plasmid vectors in Escherichia coli based on an aminoacid auxotrophy complementation has been developed. An E. coli M15-derivated glycine-auxotrophic strain of has been constructed by means of a PCR-based approach. This mutant strain contains a deletion in the glyA gene, which encodes for serine hydroxymethyl transferase, an enzyme involved in the main glycine biosynthesis pathway in E. coli. Also, we have constructed the complementation plasmid pQEalphabetarham derived from the commercially available expression vector pQE40 (QIAGEN) containing the glyA homologous gene under the control of the constitutive weak promoter P3. By using the E. coli M15DeltaglyA strain combined with the pQEalphabetarham plasmid, a successful complementation system was achieved, allowing transformants to grow on minimal media without glycine supplementation. The capability of the new system E. coli M15DeltaglyA/pQEalphabetarham for recombinant overproduction of rhamnulose 1-phosphate aldolase was evaluated in antibiotic free fed-batch cultures at controlled specific growth rate, obtaining high cell density cultures and high RhuA production and productivity levels comparable to those obtained with the conventional system. The new selection marker based on glycine-auxotrophy is a promising genetic tool, not only for recombinant protein production, but also for plasmid DNA production processes, where antibiotics can not be present in the medium formulation.     

Human cDNA expressing a functional DNA glycosylase excising 3-methyladenine and 7-methylguanine.

A cDNA expression library from a human cell line was introduced into an E. coli strain deficient in the repair of 3-meAde bases in DNA. E. coli strains deficient in the repair of 3-meAde are unusually sensitive to DNA methylating agents. A plasmid pANPG10 (Alkyl-N-Purine-DNA Glycosylase) was rescued from the library based on its ability to reduce the sensitivity of the mutant strain to methylmethane sulfonate. Crude extracts of the E. coli mutant strain hosting the plasmid pANPG10 release both 3-meAde and 7-meGua from DNA. The longest open reading frame in the sequence codes for a polypeptide of 230 amino acids of molecular weight 25.5 kD, with a pI of 9.1. The derived amino acid sequence of the human 3-meAde-DNA glycosylase has 85% sequence identity with the 3-meAde-DNA glycosylase from rat hepatoma cells.     

Plant transformation by coinoculation with a disarmed Agrobacterium tumefaciens strain and an Escherichia coli strain carrying mobilizable transgenes

Transformation of Nicotiana tabacum leaf explants was attempted with Escherichia coli as a DNA donor either alone or in combination with Agrobacterium tumefaciens. We constructed E. coli donor strains harboring either the promiscuous IncP-type or IncN-type conjugal transfer system and second plasmids containing the respective origins of transfer and plant-selectable markers. Neither of these conjugation systems was able to stably transform plant cells at detectable levels, even when VirE2 was expressed in the donor cells. However, when an E. coli strain expressing the IncN-type conjugation system was coinoculated with a disarmed A. tumefaciens strain, plant tumors arose at high frequencies. This was caused by a two-step process in which the IncN transfer system mobilized the entire shuttle plasmid from E. coli to the disarmed A. tumefaciens strain, which in turn processed the T-DNA and transferred it to recipient plant cells. The mobilizable plasmid does not require a broad-host-range replication origin for this process to occur, thus reducing its size and genetic complexity. Tumorigenesis efficiency was further enhanced by incubation of the bacterial strains on medium optimized for bacterial conjugation prior to inoculation of leaf explants. These techniques circumvent the need to construct A. tumefaciens strains containing binary vectors and could simplify the creation of transgenic plants.     

Hybrid plasmid with bacterial and fungal markers carrying the denV gene of T4 phage and restoring the UV-resistance of E. coli uvrA

The hybrid plasmid pYBP2 with bacterial (ampR), yeast (LEU2) and bacteriophage T4 (denV) genes has been constructed. The plasmid transformed Escherichia coli CSR603 uvrA recA ampS leuA phr- to ampicillin resistance, leucine independence, UV-resistance similar to the one of uvrA+ recA strain. Cell-free extracts of transformed Escherichia coli cells contain low level of ultraviolet-endonuclease activity in contrast to nontransformed cells containing no enzyme.     

Cloning and heterologous expression of an enantioselective amidase from Rhodococcus erythropolis strain MP50

The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, alpha-chlorophenylacetamide, and alpha-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.     

Ligand binding properties of bacterial hemoglobins and flavohemoglobins

Bacterial hemoglobins and flavohemoglobins share a common globin fold but differ otherwise in structural and functional aspects. The bases of these differences were investigated through kinetic studies on oxygen, carbon monoxide, and nitric oxide binding. The novel bacterial hemoglobins from Clostridium perfringens and Campylobacter jejuni and the flavohemoglobins from Bacillus subtilis and Salmonella enterica serovar Typhi have been analyzed. Examination of the biochemical and ligand binding properties of these proteins shows a clear distinction between the two groups. Flavohemoglobins show a much greater tendency to autoxidation compared to bacterial hemoglobins. The differences in affinity for oxygen, carbon monoxide, and nitric oxide between bacterial hemoglobins and flavohemoglobins are mainly due to differences in the association rate constants. The second-order rate constants for oxygen and carbon monoxide binding to bacterial hemoglobins are severalfold higher than those for flavohemoglobins. A similar trend is observed for NO association with the oxidized iron(III) form of the proteins. No major differences are observed among the values obtained for the dissociation rate constants for the two groups of bacterial proteins studied, and these constants are all rather similar to those for myoglobin. Taken together, our data suggest that differences exist between the mechanisms of ligand binding to bacterial hemoglobins and flavohemoglobins, suggesting different functions in the cell.     

Introduction of a new branchpoint in tetrapyrrole biosynthesis in Escherichia coli by co-expression of genes encoding the chlorophyll-specific enzymes magnesium chelatase and magnesium protoporphyrin methyltransferase.

The genes encoding the three Mg chelatase subunits, ChlH, ChlI and ChlD, from the cyanobacterium Synechocystis PCC6803 were all cloned in the same pET9a-based Escherichia coli expression plasmid, forming an artificial chlH-I-D operon under the control of the strong T7 promoter. When a soluble extract from IPTG-induced E. coli cells containing the pET9a-ChlHID plasmid was assayed for Mg chelatase activity in vitro, a high activity was obtained, suggesting that all three subunits are present in a soluble and active form. The chlM gene of Synechocystis PCC6803 was also cloned in a pET-based E. coli expression vector. Soluble extract from an E. coli strain expressing chlM converted Mg-protoporphyrin IX to Mg-protoporphyrin monomethyl ester, demonstrating that chlM encodes the Mg-protoporphyrin methyltransferase of Synechocystis. Co-expression of the chlM gene together with the chlH-I-D construct yielded soluble protein extracts which converted protoporphyrin IX to Mg-protoporphyrin IX monomethyl ester without detectable accumulation of the Mg-protoporphyrin IX intermediate. Thus, active Mg chelatase and Mg-protoporphyrin IX methyltransferase can be coupled in E. coli extracts. Purified ChlI, -D and -H subunits in combination with purified ChlM protein were subsequently used to demonstrate in vitro that a molar ratio of ChlM to ChlH of 1 to 1 results in conversion of protoporphyrin IX to Mg-protoporphyrin monomethyl ester without significant accumulation of Mg-protoporphyrin.     

Cytokinins do not noticeably change the methylation of adenine residues of DNA in cultured Escherichia coli B

6-Benzylaminopurine (6-BAP) (1 mg/ml) does not influence the growth of E. coli B cell cultures or the number of [8-14C] labeled N6-methyladenine (m6A) residues in the total DNA [(100.m6A/(A x m6A) = 1.7]. The growth of bacterial cells in the presence of adenine or cytokinins (6-BAP, kinetin, zeatin) (1 mg/ml) was unaccompanied by significant changes in the intracellular content of plasmid pBR 322. The mode of restriction by endonuclease Cfu I hydrolyzing the Gm6ATC site of plasmids pBR 322 from E. coli B cells grown in the presence of adenine or one of the above-mentioned cytokinins is identical. These plasmids also have identical restriction products Mbo I or Sau 3AI. Thus, the cytokinins under study do not markedly affect the methylation of adenine residues in total DNA of E. coli B cell cultures and the GATC sequence in plasmids pBR 322 isolated from these cells.