Gene cloning, protein purification, and enzymatic properties of multicopper oxidase, from Klebsiella sp. 601

A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat//Km values of 0.49 mmol/L, 1.08 x 103 s-1, and 2.23 x 103 s-1.mmol/L-1, respectively, for DMP, 5.63 mmol/L, 6.64 x 103 s-1, and 1.18 x 103 s-1.mmol/L-1 for ABTS, and 0.023 mmol/L, 11 s-1, and 4.68 x 102 s-1.mmol/L-1 for SGZ.     

Functional characterization of the competence protein DprA/Smf in Escherichia coli

In several bacterial species that show natural transformation, dprA has been described as a competence gene. The DprA protein has been suggested to be involved in the protection of incoming DNA. However, members of the dprA gene family (also called smf) can be detected in virtually all bacterial species, which suggests that their gene products have a more general function. We examined the function of the DprA/Smf homologue of Escherichia coli. Escherichia coli dprA/smf is able to partially restore transformation in a Haemophilus influenzae dprA mutant, which shows that dprA/smf genes from competent and noncompetent species are interchangeable with respect to their involvement in natural transformation. From this, we conclude that natural transformation is probably an additional function of these genes. Subsequently, the dprA/smf gene was deleted in various recombination mutants of E. coli, and the resultant phenotype was tested. All the resultant E. coli dprA/smf mutants did not differ from their parent strains with respect to transformation, Hfr-conjugation, recombination and DNA repair. Therefore, a role of DprA/Smf in DNA recombination could not be established and the basic function of dprA/smf remains unclear.     

Transposition of the kanamycin resistance determinant (Tn601) from plasmid 5T to the genome of bacteriophage lambda and the expression of this gene after prophage induction

Tn601, determinging kanamycin resistance of Escherichia coli, has been transposed into the bacteriophage lambda genome from R6 plasmid. After curing lambda gtc1857 (Tn601) lysogenes on the kanamycin containing medium, the clones with stable and unstable integrations of the Tn6-1 into the chromosome were obtained. After the lysogenization of these clones with the phage lambda att80c1857S7, the phages lambda att80c1857S7 (Tn601) were obtained. These phages contained the Tn601 from the sites of stable or unstable integrations. The frequency of the Tn601 transposition from the sites of unstable integration was 10(-7), that was two order of magnitude higher than the frequency of the Tn601 transpostion from the site of stable integration. Temperature induction of the lambda att80c1857 (Tn601) prophage resulted in 10--15 times increase of the yeild of aminoglycoside-3'-phosphotransferase I, the enzyme coded by the aphA gene of the Tn601.     

YcdY protein of Escherichia coli, an atypical member of the TorD chaperone family

The TorD family of specific chaperones is divided into four subfamilies dedicated to molybdoenzyme biogenesis and a fifth one, exemplified by YcdY of Escherichia coli, for which no defined partner has been identified so far. We propose that YcdY is the chaperone of YcdX, a zinc protein involved in the swarming motility process of E. coli, since YcdY interacts with YcdX and increases its activity in vitro.     

E1 protein of human papillomavirus is a DNA helicase/ATPase.

Replication of human papillomavirus (HPV) DNA requires the viral proteins E1 and E2. Amino acid similarities to SV40 large-T antigen had suggested that E1 is a DNA helicase/ATPase involved in initiating viral DNA replication, and this has recently been shown for bovine papillomavirus type 1 (BPV-1) E1 protein. However, in vitro analysis of HPV E1 has been hampered by the inability to produce purified protein using heterologous expression systems. We have succeeded in demonstrating ATPase and DNA helicase activities in purified HPV E1, expressed in E. coli as a maltose-binding protein fusion (MBP-E1), for the first time. As further confirmation that the ATPase and DNA helicase activities are due to E1 and not contaminating E. coli enzymes, we have shown that a fusion protein containing an amino acid change (E1 Pro-479 to Ser), predicted to inactivate ATP-binding, has impaired activities. We have carried out a structure prediction analysis which suggests that E1 may form two domains: a relatively open N-terminal domain (residues 1-125), and a highly structured C-terminal domain (170-649), with an intermediate region (125-170) predicted to form an inter-domain linker. This is consistent with the proteolytic susceptibility of MBP-E1 at a site 15-20 kD from the N-terminus of E1, and the accumulation of a 58 kD C-terminal fragment of E1. We speculate that the N-terminal domain is involved in DNA-binding, while the C-terminal 58 kD may constitute a distinct enzymatic domain. HPV E1 is of interest as a therapeutic target and the availability of pure enzyme will be invaluable in the search for antiviral compounds.     

Use of peptide antibodies to probe for the mitoxantrone resistance-associated protein MXR/BCRP/ABCP/ABCG2

Previous kinetic characterization of Escherichia coli fructose 1,6-bisphosphatase (FBPase) was performed on enzyme with an estimated purity of only 50%. Contradictory kinetic properties of the partially purified E. coli FBPase have been reported in regard to AMP cooperativity and inactivation by fructose-2,6-bisphosphate. In this investigation, a new purification for E. coli FBPase has been devised yielding enzyme with purity levels as high as 98%. This highly purified E. coli FBPase was characterized and the data compared to that for the pig kidney enzyme. Also, a homology model was created based upon the known three-dimensional structure of the pig kidney enzyme. The kcat of the E. coli FBPase was 14.6 s(-1) as compared to 21 s(-1) for the pig kidney enzyme, while the K(m) of the E. coli enzyme was approximately 10-fold higher than that of the pig kidney enzyme. The concentration of Mg2+ required to bring E. coli FBPase to half maximal activity was estimated to be 0.62 mM Mg2+, which is twice that required for the pig kidney enzyme. Unlike the pig kidney enzyme, the Mg2+ activation of the E. coli FBPase is not cooperative. AMP inhibition of mammalian FBPases is cooperative with a Hill coefficient of 2; however, the E. coli FBPase displays no cooperativity. Although cooperativity is not observed, the E. coli and pig kidney enzymes show similar AMP affinity. The quaternary structure of the E. coli enzyme is tetrameric, although higher molecular mass aggregates were also observed. The homology model of the E. coli enzyme indicated slight variations in the ligand-binding pockets compared to the pig kidney enzyme. The homology model of the E. coli enzyme also identified significant changes in the interfaces between the subunits, indicating possible changes in the path of communication of the allosteric signal.     

Uptake of LPS/E. coli/latex beads via distinct signalling pathways in medfly hemocytes: the role of MAP kinases activation and protein secretion

In response to LPS/E. coli treatment, extracellular signal-regulated kinase (ERK) is activated in medfly hemocytes. To explore the molecular mechanisms underlying LPS/E. coli/latex beads endo- and phagocytosis, we studied the signalling pathways leading to p38 and c-jun N-terminal kinase (JNK) activation. JNK and p38-like proteins were initially identified within medfly hemocytes. Flow cytometry analysis revealed that mitogen-activated protein kinases (MAPK) are required for phagocytosis. Inhibition of specific MAPK signalling pathways, with manumycin A, toxin A, cytochalasin D and latrunculin A, revealed activation of p38 via Ras/Rho/actin remodelling pathway and activation of JNK that was independent of actin cytoskeleton reorganization. ERK and p38 pathways, but not JNK, appeared to be involved in LPS-dependent hemocyte secretion, whereas all MAPK subfamilies seemed to participate in E. coli-dependent secretion. In addition, flow cytometry experiments in hemocytes showed that the LPS/E. coli-induced release was a prerequisite for LPS/E. coli uptake, whereas latex bead phagocytosis did not depend on hemocyte secretion. This is a novel aspect, as in mammalian monocytes/macrophages LPS/E. coli-triggered release has not been yet correlated with phagocytosis. It is of interest that these data suggest distinct mechanisms for the phagocytosis of E. coli and latex beads in medfly hemocytes.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes.

Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.     

Escherichia coli single-stranded DNA binding protein stimulates the DNA deoxyribophosphodiesterase activity of exonuclease I.

The E. coli single-stranded binding protein (SSB) has been demonstrated in vitro to be involved in a number of replicative, DNA renaturation, and protective functions. It was shown previously that SSB can interact with exonuclease I to stimulate the hydrolysis of single-stranded DNA. We demonstrate here that E. coli SSB can also enhance the DNA deoxyribophosphodiesterase (dRpase) activity of exonuclease I by stimulating the release of 2-deoxyribose-5-phosphate from a DNA substrate containing AP endonuclease-incised AP sites, and the release of 4-hydroxy-2-pentenal-5-phosphate from a DNA substrate containing AP lyase-incised AP sites. E. coli SSB and exonuclease I form a protein complex as demonstrated by Superose 12 gel filtration chromatography. These results suggest that SSB may have an important role in the DNA base excision repair pathway.     

Puromycin enhances 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster

The effect of cycloheximide and puromycin on 20-hydroxyecdysone-induced protein synthesis in wing discs of Drosophila melanogaster has been studied by one-dimensional and two-dimensional SDS polyacrylamide electrophoresis. It is found that puromycin, but not cycloheximide, when applied simultaneously with the hormone enhanced the hormone-induced synthesis of the early and late proteins. However, when puromycin was applied after hormone treatment, only the late proteins were induced. The possible implication of these observations is discussed.     

A conserved region within the Bordetella pertussis autotransporter BrkA is necessary for folding of its passenger domain

Autotransporter secretion represents a unique mechanism that Gram-negative bacteria employ to deliver proteins to their cell surface. BrkA is a Bordetella pertussis autotransporter protein that mediates serum resistance and contributes to adherence of the bacterium to host cells. BrkA is a 103 kDa protein that is cleaved to form a 73 kDa alpha-domain and a 30 kDa beta domain. The alpha domain, also referred to as the passenger domain, is responsible for the effector functions of the protein, whereas the beta domain serves as a transporter. In an effort to characterize BrkA secretion, we have shown that BrkA has a 42 amino acid signal peptide for transit across the cytoplasmic membrane, and a translocation unit made up of a short linker region fused to the beta-domain to ferry the passenger domain to the bacterial surface through a channel formed by the beta-domain. In this report, we provide genetic, biochemical and structural evidence demonstrating that a region within the BrkA passenger (Glu601-Ala692) is necessary for folding the passenger. This region is not required for surface display in the outer membrane protease OmpT-deficient Escherichia coli strain UT5600. However, a BrkA mutant protein bearing a deletion in this region is susceptible to digestion when expressed in E. coli strains expressing OmpT suggesting that the region is required to maintain a stable structure. The instability of the deletion mutant can be rescued by surface expressing Glu601-Ala692in trans suggesting that this region is acting as an intramolecular chaperone to effect folding of the passenger concurrent with or following translocation across the outer membrane.     

Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter

To increase yields of calf prochymosin (PC) produced in Escherichia coli, PC cDNA was cloned in a plasmid vector under control of the trp promoter. The hybrid plasmid pCR501 constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region. E. coli C600 harboring this plasmid produces approx. 300 000 molecules of PC per cell. This is about a tenfold increase above the amount obtained using lacUV5 promoter [Nishimori et al., Gene 19 (1982) 337-344]. A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501. In pCR601 the trp attenuator is deleted. Another plasmid, pCR701, in which construction of a sequence coding for fMet-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate. Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.     

Acquirement and characterization of a carotenoid mutant (GM309) of Rhodobacter sphaeroides 601

A green mutant was obtained among the chemically induced mutants of Rhodo-bacter sphaeroides 601 (RS601) and named GM309. A blue shift of 20 nm of the carotenoid absorption spectrum was found in the light-harvesting complex II (LH2) of GM309. Different from LH2 of RS601, it was found that the carotenoids in GM309-LH2 changed to be neurosporene by mutation. Neurosporene lacks a conjugate double bond, compared with the spheroidene in RS601-LH2 which has ten conjugate double bonds. As shown by absorption and circular di-chroism spectroscopy, the overall structure of GM309-LH2 is little affected by this change. From fluorescence emission spectra, it is found that GM309-LH2 can transfer energy from carotenoids to Bchl-B850 without any change in efficiency. But the efficiency of energy transfer from B800 to B850 in GM309-LH2 is decreased to be 42% of that of the native. This work would provide a novel method to investigate the mechanism of excitation energy transfer in LH2.     

Identification of the calmodulin-binding domain of neuron-specific protein kinase C substrate protein CAP-22/NAP-22. Direct involvement of protein myristoylation in calmodulin-target protein interaction

Various proteins in the signal transduction pathways as well as those of viral origin have been shown to be myristoylated. Although the modification is often essential for the proper functioning of the modified protein, the mechanism by which the modification exerts its effects is still largely unknown. Brain-specific protein kinase C substrate, CAP-23/NAP-22, which is involved in the synaptogenesis and neuronal plasticity, binds calmodulin, but the protein lacks any canonical calmodulin-binding domain. In the present report, we show that CAP-23/NAP-22 isolated from rat brain is myristoylated and that the modification is directly involved in its interaction with calmodulin. Myristoylated and non-myristoylated recombinant proteins were produced in Escherichia coli, and their calmodulin-binding properties were examined. Only the former bound to calmodulin. Synthetic peptides based on the N-terminal sequence showed similar binding properties to calmodulin, only when they were myristoylated. The calmodulin-binding site narrowed down to the myristoyl moiety together with a nine-amino acid N-terminal basic domain. Phosphorylation of a single serine residue in the N-terminal domain (Ser5) by protein kinase C abolished the binding. Furthermore, phosphorylation of CAP-23/NAP-22 by protein kinase C was also found myristoylation-dependent, suggesting the importance of myristoylation in protein-protein interactions.     

Construction of a new shuttle expression vector for Bacillus subtilis and Escherichia coli by using a polycistronic system

A shuttle vector has been constructed by fusing the Bacillus subtilis trimethoprim-resistance-carrying (TpR) plasmid pNC601 with the Escherichia coli plasmid pBR322. The resultant plasmid pNBL1 can replicate in both B. subtilis and E. coli, conferring Tp resistance on both cells and ampicillin resistance (ApR) on E. coli. The B. subtilis dihydrofolate reductase operon (dfr) on pNC601 and therefore on pNBL1 consists of the thymidylate synthase B gene (thyB) and the TpR-dihydrofolate reductase gene lacking the C-terminal seven codons (designated as drfA' as compared with the complete dfrA gene). A direct-expression vector pNBL3 has been constructed by inserting synthetic oligodeoxynucleotides containing a Bacillus ribosome-binding site (RBS) and the ATG codon downstream from dfrA' on pNBL1. When the E. coli lacZ gene was placed downstream from the dfrA' gene in pNBL3, efficient synthesis of beta-galactosidase was observed in both cells, showing that the polycistronic expression system is suitable for directing expression of heterologous genes. Translational efficiency of the lacZ gene on pNBL3 was further examined in B. subtilis by changing the sequence upstream from lacZ. Unlike the results previously reported [Sprengel et al., Nucleic Acids Res. 13 (1985) 893-909], when RBS was present, the high level of lacZ expression was preserved irrespective of spacing between the stop codon of the upstream dfrA' gene and the start codon of the downstream lacZ gene. However, in the absence of RBS, the spacing between both genes affected lacZ expression. That is, translational coupling of dfrA'-lacZ was observed, although the translational efficiency was very low.     

Concentration-dependent collateral sensitivity of cisplatin-resistant gastric cancer cell sublines

The cisplatin-resistant gastric cancer cell sublines, SNU-601/Cis2 and /Cis10, were 49 and >530 times more resistant to cisplatin, respectively, compared with the drug-sensitive cells, SNU-601/WT. The SNU-601/Cis2 showed cross-resistance to carboplatin, heptaplatin, doxorubicin, mitomycin C, and 5-fluorouracil compared with the SNU-601/WT whereas the SNU-601/Cis10 displayed collateral sensitivity to these drugs with the exception of cisplatin compared with the SNU-601/Cis2, suggesting that the cross-resistance and collateral sensitivity of cisplatin-resistant gastric cancer cells are dependent upon cisplatin concentrations. Altered expression of the antioxidant and transporter genes (metallothionein, catalase, superoxide dismutases, P-glycoprotein, and the breast cancer resistance protein) was involved in these phenotypes of the cisplatin-resistant gastric cancer cell lines.