Identity of the 17-kilodalton protein, a DNA-binding protein from Escherichia coli, and the firA gene product.

The 17-kilodalton protein, a DNA-binding protein encoded by the skp gene of Escherichia coli, was found to be identical to histonelike protein I, the product of the firA gene. This conclusion was reached after chromosomal localization, using the recently constructed high- and low-resolution E. coli restriction maps, and by direct comparison of the N-terminal amino acid sequence of histonelike protein I and the 17-kilodalton protein.     

Rapid site-specific DNA inversion in Escherichia coli mutants lacking the histonelike protein H-NS.

Escherichia coli pilG mutants are thought to have a dramatically higher DNA inversion rate as measured by the site-specific DNA inversion of the type 1 pili pilA promoter. DNA sequence of the pilG gene confirmed its identity to the gene encoding the bacterial histonelike protein H-NS. Unlike other histonelike protein complexes that enhance site-specific DNA recombination, the H-NS protein inhibited this process. This inhibition was indicated by the increased inversion rate of the pilA promoter region effected by two different mutant pilG alleles. One of these alleles, pilG1, conferred a mutant phenotype only at low temperature attributable to a T-to-G transversion in the -35 sequence of the pilG promoter. The other allele, pilG2-tetR, was an insertion mutation in the pilG coding region that conferred the mutant phenotype independent of temperature. We measured an approximately 100-fold-increased pilA promoter inversion rate in the mutant by exploiting the temperature-dependent expression of pilG1 and using a novel rapid-population-sampling method. Contrary to one current view on how the H-NS protein might act to increase DNA inversion rate, we found no evidence to support the hypothesis that DNA supercoiling affected pilA promoter inversion.     

Evidence that a eukaryotic-type serine/threonine protein kinase from Mycobacterium tuberculosis regulates morphological changes associated with cell division.

A eukaryotic-type protein serine/threonine kinase, PknA, was cloned from Mycobacterium tuberculosis strain H37Ra. Sequencing of the clone indicated 100% identity with the published pknA sequence of M. tuberculosis strain H37Rv. PknA fused to maltose-binding protein was expressed in Escherichia coli; it exhibited a molecular mass of approximately 97 kDa. The fusion protein was purified from the soluble fraction by affinity chromatography using amylose resin. In vitro kinase assays showed that the autophosphorylating ability of PknA is strictly magnesium/manganese-dependent, and sodium orthovanadate can inhibit this activity. Phosphoamino-acid analysis indicated that PknA phosphorylates at serine and threonine residues. PknA was also able to phosphorylate exogenous substrates, such as myelin basic protein and histone. A comparison of the nucleotide-derived amino-acid sequence of PknA with that of functionally characterized prokaryotic serine/threonine kinases indicated its possible involvement in cell division/differentiation. Protein--protein interaction studies revealed that PknA is capable of phosphorylating at least a approximately 56-kDa soluble protein from E. coli. Scanning electron microscopy showed that constitutive expression of this kinase resulted in elongation of E. coli cells, supporting its regulatory role in cell division.     

High-level production of bacillus subtilis glycine oxidase by fed-batch cultivation of recombinant Escherichia coli Rosetta (DE3)

A fed-batch process for the high cell density cultivation of Escherichia coli Rosetta (DE3) and the production of the recombinant protein glycine oxidase (GOX) from Bacillus subtilis was developed. GOX is a deaminating enzyme that shares substrate specificity with d-amino acid oxidase and sarcosine oxidase and has great biotechnological potential. The B. subtilis gene coding for GOX was expressed in E. coli Rosetta under the strong inducible T7 promotor of the pET28a vector. Exponential feeding based on the specific growth rate and a starvation period for acetate utilization was used to control cell growth, acetate production, and reconsumption and glucose consumption during fed-batch cultivation. Expression of GOX was induced at three different cell densities (20, 40, and 60 g . L(-1)). When cells were induced at intermediate cell density, the amount of GOX produced was 20 U . g(-1) cell dry weight and 1154 U . L(-1) with a final intracellular protein concentration corresponding to approximately 37% of the total cell protein concentration. These values were higher than those previously published for GOX expression and also represent a drastic decrease of 26-fold in the cost of the culture medium.     

Use of fed-batch cultivation for achieving high cell densities for the pilot-scale production of a recombinant protein (phenylalanine dehydrogenase) in Escherichia coli

A fed-batch process for the high cell density cultivation of E. coli TG1 and the production of the recombinant protein phenylalanine dehydrogenase (PheDH) was developed. A model based on Monod kinetics with overflow metabolism and incorporating acetate utilization kinetics was used to generate simulations that describe cell growth, acetate production and reconsumption, and glucose consumption during fed-batch cultivation. Using these simulations a predetermined feeding profile was elaborated that would maintain carbon-limited growth at a growth rate below the critical growth rate for acetate formation (mu < mu(crit)). Two starvation periods are incorporated into the feed profile in order to induce acetate utilization. Cell concentrations of 53 g dry cell weight (DCW)/L were obtained with a final intracellular product concentration of recombinant protein corresponding to approximately 38% of the total cell protein. The yield of PheDH was 129 U/mL with a specific activity of 1.2 U/mg DCW and a maximum product formation rate of 0.41 U/mg DCW x h. The concentration of aectate was maintained below growth inhibitory levels until 3 h before the end of the fermentation when the concentration reached a maximum of 10.7 g/L due to IPTG induction of the recombinant protein.     

Overexpression of a recombinant wild-type and His-tagged Bacillus subtilis glycine oxidase in Escherichia coli.

We have cloned the gene coding for the Bacillus subtilis glycine oxidase (GO), a new flavoprotein that oxidizes glycine and sarcosine to the corresponding alpha-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expression vector pT7.7 we produced a recombinant plasmid (pT7-GO). The pT7-GO encodes a fully active fusion protein with six additional residues at the N-terminus of GO (MARIRA). In BL21(DE3)pLysS Escherichia coli cells, and under optimal isopropyl thio-beta-D-galactoside induction conditions, soluble and active chimeric GO was expressed up to 1.14 U g(-1) of cell (and a fermentation yield of 3.82 U x L(-1) of fermentation broth). An N-terminal His-tagged protein (HisGO) was also successfully expressed in E. coli as a soluble protein and a fully active holoenzyme. HisGO represents approximately 3.9% of the total soluble protein content of the cell. The His-tagged GO was purified in a single step by nickel-chelate chromatography to a specific activity of 1.06 U x mg(-1) protein at 25 degrees C and with a yield of 98%. The characterization of the purified enzyme showed that GO is a homotetramer of approximately 180 kDa with the spectral properties typical of flavoproteins. GO exhibits good thermal stability, with a Tm of 46 degrees C after 30 min incubation; its stability is maximal in the 7.0-8.5 pH range. A comparison of amino-acid sequence and substrate specificity indicates that GO has similarities to other flavoenzymes acting on primary amines and on D-amino acids.     

Multicopy suppression: an approach to understanding intracellular functioning of the protein export system.

Escherichia coli genes were cloned onto a multicopy plasmid and selected by the ability to restore growth and protein export defects caused by a temperature-sensitive secY or secA mutation. When secA51 was used as the primary mutation, only clones carrying groE, which specifies the chaperonin class of heat shock protein, were obtained. Selection using secY24 yielded three major classes of genes. The first class encodes another heat shock protein, HtpG; the most frequently obtained second class encodes a neutral histonelike protein, H-NS; and the third class, msyB, encodes a 124-residue protein of which 38 residues are acidic amino acids. Possible mechanisms of suppression as well as the significance and limitations of the multicopy suppression approach are discussed.     

Expression and characterization of the ponA (ORF I) gene of Haemophilus influenzae: functional complementation in a heterologous system.

The coding sequence of the Haemophilus influenzae ORF I gene was amplified by PCR and cloned into different Escherichia coli expression vectors. The ORF I-encoded protein was approximately 90 kDa and bound 3H-benzyl-penicillin and 125I-cephradine. This high-molecular-weight penicillin-binding protein (PBP) was also shown to possess transglycosylase activity, indicating that the ORF I product is a bifunctional PBP. The ORF I protein was capable of maintaining the viability of E. coli delta ponA ponB::spcr cells in transcomplementation experiments, establishing the functional relevance of the significant amino acid homology seen between E. coli PBP 1A and 1B and the H. influenzae ORF I product. In addition, the physiological functioning of the H. influenzae ORF I (PBP 1A) product in a heterologous species established the ability of the enzyme not only to recognize the E. coli substrate but also to interact with heterologous cell division proteins. The affinity of the ORF I product for 3H-benzylpenicillin and 125I-cephradine, the MIC of beta-lactams for E. coli delta ponA ponB::spcr expressing the ORF I gene, and the amino acid alignment of the PBP 1 family of high-molecular-weight PBPs group the ORF I protein into the PBP 1A family of high-molecular-weight PBPs.     

Sequencing, expression, and genetic characterization of the Helicobacter pylori ftsH gene encoding a protein homologous to members of a novel putative ATPase family.

In this study, we isolated and sequenced a Helicobacter pylori gene, designated ftsH, coding for a 632-amino-acid protein which displayed striking similarity throughout its full length to FtsH proteins identified in Escherichia coli, Lactococcus lactis, and Bacillus subtilis. H. pylori FtsH also possessed approximately 200-amino-acid region containing a putative ATPase module which is conserved among members of the AAA protein family (AAA, ATPase associated with diverse cellular activities). The H. pylori ftsH product was overexpressed in E. coli and reacted immunologically with an anti-E. coli FtsH serum (T. Tomoyasu, K. Yamanaka, K. Murata, T. Suzaki, P. Bouloc, A. Kato, H. Niki, S. Hiraga, and T. Ogura, J. Bacteriol. 175:1352-1357, 1993). FtsH was also shown to be present in the membrane fraction of H. pylori, suggesting that it is membrane bound. Disruption of the ftsH gene led to the loss of viability of H. pylori, demonstrating that this gene is essential for cell growth. Overproduction of both H. pylori FtsH and E. coli FtsH together tremendously reduced the growth rate of the E. coli host cells, whereas the growth of the E. coli cells carrying the wild-type E. coli ftsH operon on the chromosome was not significantly affected by overproduction of H. pylori FtsH itself. This result suggests that the abnormal growth of cells results from interaction between H. pylori FtsH and E. coli FtsH.     

Primary structure and mapping of the hupA gene of Salmonella typhimurium.

In bacteria, the complex nucleoid structure is folded and maintained by negative superhelical tension and a set of type II DNA-binding proteins, also called histonelike proteins. The most abundant type II DNA-binding protein is HU. Southern blot analysis showed that Salmonella typhimurium contained two HU genes that corresponded to Escherichia coli genes hupA (encoding HU-2 protein) and hupB (encoding HU-1). Salmonella hupA was cloned, and the nucleotide sequence of the gene was determined. Comparison of hupA of E. coli and S. typhimurium revealed that the HU-2 proteins were identical and that there was high conservation of nucleotide sequences outside the coding frames of the genes. A 300-member genomic library of S. typhimurium was constructed by using random transposition of MudP, a specialized chimeric P22-Mu phage that packages chromosomal DNA unidirectionally from its insertion point. Oligonucleotide hybridization against the library identified one MudP insertion that lies within 28 kilobases of hupA; the MudP was 12% linked to purH at 90.5 min on the standard map. Plasmids expressing HU-2 had a surprising phenotype; they caused growth arrest when they were introduced into E. coli strains bearing a himA or hip mutation. These results suggest that IHF and HU have interactive roles in bacteria.     

Involvement of integration host factor (IHF) in maintenance of plasmid pSC101 in Escherichia coli: characterization of pSC101 mutants that replicate in the absence of IHF

Escherichia coli mutants defective in the stable maintenance of plasmid pSC101 have been isolated following Tn10 insertion mutagenesis. One class of mutations affecting pSC101 replication was located in the genes himA and himD (hip), which encode the two subunits of integration host factor (IHF), a small histonelike DNA-binding protein that has multiple cellular functions. Mutants of pSC101 that could replicate in the absence of IHF were isolated and characterized; four independent mutational alterations were found to affect the third codon of the pSC101 rep gene, resulting in the replacement of glutamic acid by lysine. The compensating alteration appears to function by altering the activity of the pSC101 rep protein in him mutants.     

Two roles for transforming growth factor beta 1 in colon enterocytic cell differentiation.

The role of transforming growth factor beta 1 (TGF-beta 1) in enterocytic differentiation was examined by treating two undifferentiated HT29 colon carcinoma sublines, U4 and U9, with hexamethylene bisacetamide to up-regulate their level of TGF-beta 1 mRNA expression. Although both lines after treatment secreted approximately equal levels of biologically active TGF-beta 1, only U4H cells were found to undergo enterocytic differentiation when cultured postconfluence on collagen I-coated transwells, forming polarized monolayer cells with an apical brush border, whereas U9H cells remained multilayered and undifferentiated. Enterocytic U4H cells exhibited four times as much cell surface expression of the collagen I-binding protein alpha 2-integrin, twice as much of the accessory collagen-binding protein carcinoembryonic antigen, and almost twice as much binding to collagen I films as undifferentiated U9H cells. TGF-beta 1 treatment doubled U4 cell collagen I binding, increased expression of alpha 2-integrin 4-fold, but increased carcinoembryonic antigen expression only marginally. U4H cells displayed cell cycle regulation by arresting reversibly at a restriction point in G1 when placed in the postconfluent culture conditions which initiated enterocytic differentiation. In contrast, undifferentiated U9H cells exhibited no restriction point but arrested throughout G1. TGF-beta 1 blocked synchronized U4H cells in G1, whereas it stimulated the growth of U9H cells. Thus, TGF-beta 1 has two roles in enterocytic differentiation: to increase levels of collagen I adhesion proteins and to block enterocytic cells in G1 so that they can differentiate.     

具超强富集U(Ⅵ)能力工程菌E.coli的构建

以鼠伤寒沙门氏菌基因组DNA为模板,PCR扩增得到非特异性酸性磷酸酶基因（phoN）,将其克隆到pMD18T-Vector中。用Spe I、Nde I限制性内切酶对重组转移载体T-VectorphoN与穿梭载体pRADZ3分别进行双酶切,再将phoN片段和穿梭载体pRADZ3中的大片段通过T4DNA连接酶连接。经PCR与双酶切双重鉴定,证实重组穿梭载体pRADZ3phoN构建成功。转化Escherichia coli DH5α感受态细胞,使其在正常情况下表达PhoN蛋白,经Western blot 证实phoN基因在DH5α中成功表达。利用含pRADZ3phoN的工程菌进行富集U(Ⅵ)实验,结果表明该工程菌对U(Ⅵ)的富集量较宿主菌提高约4倍,达46.16mg/g,去除率为92.32%。     

Isolation and characterization of nonhistone chromosomal protein C-14 which stimulates RNA synthesis.

The nonhistone chromatin protein, C-14, was extracted from chromatin of Novikoff hepatoma ascites cells and isolated in high purity as shown by its migration as a single dense spot on two-dimensional polyacrylamide gels. Its mobility on sodium dodecyl sulfate gels is consistent with a molecular weight of approximately 70 000. The amino acid composition shows that protein C-14 has an acidic:basic amino acid ratio of 1.8. Its amino terminal amino acid is lysine. Protein C-14 stimulated the incorporation of [3H]UMP into RNA by approximately 30% when added to naked DNA and homologous RNA polymerase I. A 30% stimulation of [3H]UMP incorporation into RNA was also found when protein C-14 was added to an E. coli RNA polymerase system containing either E. coli or Novikoff hepatoma DNA.     

Interference of mRNA function by sequence-specific endoribonuclease PemK

In Escherichia coli, programmed cell death is mediated through the system called "addiction module," which consists of a pair of genes encoding a stable toxin and a labile antitoxin. The pemI-pemK system is an addiction module present on plasmid R100. It helps to maintain the plasmid by post-segregational killing in E. coli population. Here we demonstrate that purified PemK, the toxin encoded by the pemI-pemK addiction module, inhibits protein synthesis in an E. coli cell-free system, whereas the addition of PemI, the antitoxin against PemK, resumes the protein synthesis. Further studies reveal that PemK is a sequence-specific endoribonuclease that cleaves mRNAs to inhibit protein synthesis, whereas PemI blocks the endoribonuclease activity of PemK. PemK cleaves only single-stranded RNA preferentially at the 5' or 3' side of the A residue in the "UAH" sequences (where H is C, A, or U). Upon induction, PemK cleaves cellular mRNAs to effectively block protein synthesis in E. coli. The pemK homologue genes have been identified on the genomes of a wide range of bacteria. We propose that PemK and its homologues form a novel endoribonuclease family that interferes with mRNA function by cleaving cellular mRNAs in a sequence-specific manner.     

Methylcobalamin:homocysteine methyltransferase from Methanobacterium thermoautotrophicum. Identification as the metE gene product.

Methanobacterium thermoautotrophicum is a methane-forming archaeon that grows on H2 and CO2 as sole carbon and energy source. Cell extracts of the methanogen were found to contain methylcobalamin: homocysteine methyltransferase activity which was purified 3000-fold to a specific activity of approximately 500 U.mg-1 protein. SDS/PAGE revealed the presence of a polypeptide with an apparent molecular mass of 34 kDa. Via its N-terminal amino acid sequence, the 34-kDa polypeptide was identified as the metE gene product. The metE gene was heterologously expressed in Escherichia coli. The overproduced protein was recovered in the inclusion body fraction and was found to be inactive. The protein could be partially solubilized by unfolding in 8 M urea and then refolding. The solubilized protein had a specific activity of 450 U.mg-1. It exhibited first-order kinetics with respect to methylcobalamin concentration and Michaelis-Menten kinetics with respect to L-homocysteine concentration (apparent Km 0.1 mM). The enzyme was specific for L-homocysteine as methyl acceptor. Methylcobalamin could be substituted with methylcobinamide as methyl donor.     

Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli

A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.     

Coevolution of bacteriophage PP01 and Escherichia coli O157:H7 in continuous culture

The interaction between Escherichia coli O157:H7 and its specific bacteriophage PP01 was investigated in chemostat continuous culture. Following the addition of bacteriophage PP01, E. coli O157:H7 cell lysis was observed by over 4 orders of magnitude at a dilution rate of 0.876 h(-1) and by 3 orders of magnitude at a lower dilution rate (0.327 h(-1)). However, the appearance of a series of phage-resistant E. coli isolates, which showed a low efficiency of plating against bacteriophage PP01, led to an increase in the cell concentration in the culture. The colony shape, outer membrane protein expression, and lipopolysaccharide production of each escape mutant were compared. Cessation of major outer membrane protein OmpC production and alteration of lipopolysaccharide composition enabled E. coli O157:H7 to escape PP01 infection. One of the escape mutants of E. coli O157:H7 which formed a mucoid colony (Mu) on Luria-Bertani agar appeared 56 h postincubation at a dilution rate of 0.867 h(-1) and persisted until the end of the experiment (approximately 200 h). Mu mutant cells could coexist with bacteriophage PP01 in batch culture. Concentrations of the Mu cells and bacteriophage PP01 increased together. The appearance of mutant phage, which showed a different host range among the O157:H7 escape mutants than wild-type PP01, was also detected in the chemostat culture. Thus, coevolution of phage and E. coli O157:H7 proceeded as a mutual arms race in chemostat continuous culture.