Production of Recombinant α-Galactosidases in Thermus thermophilus

A Thermus thermophilus selector strain for production of thermostable and thermoactive α-galactosidase was constructed. For this purpose, the native α-galactosidase gene (agaT) of T. thermophilus TH125 was inactivated to prevent background activity. In our first attempt, insertional mutagenesis of agaT by using a cassette carrying a kanamycin resistance gene led to bacterial inability to utilize melibiose (α-galactoside) and galactose as sole carbohydrate sources due to a polar effect of the insertional inactivation. A Gal⁺ phenotype was assumed to be essential for growth on melibiose. In a Gal⁻ background, accumulation of galactose or its metabolite derivatives produced from melibiose hydrolysis could interfere with the growth of the host strain harboring recombinant α-galactosidase. Moreover, the AgaT⁻ strain had to be Km^s for establishment of the plasmids containing α-galactosidase genes and the kanamycin resistance marker. Therefore, a suitable selector strain (AgaT⁻ Gal⁺ Km^s) was generated by applying integration mutagenesis in combination with phenotypic selection. To produce heterologous α-galactosidase in T. thermophilus, the isogenes agaA and agaB of Bacillus stearothermophilus KVE36 were cloned into an Escherichia coli-Thermus shuttle vector. The region containing the E. coli plasmid sequence (pUC-derived vector) was deleted before transformation of T. thermophilus with the recombinant plasmids. As a result, transformation efficiency and plasmid stability were improved. However, growth on minimal agar medium containing melibiose was achieved only following random selection of the clones carrying a plasmid-based mutation that had promoted a higher copy number and greater stability of the plasmid.     

Cold-Adapted β-Galactosidase from the Antarctic Psychrophile Pseudoalteromonas haloplanktis

The β-galactosidase from the Antarctic gram-negative bacterium Pseudoalteromonas haloplanktis TAE 79 was purified to homogeneity. The nucleotide sequence and the NH₂-terminal amino acid sequence of the purified enzyme indicate that the β-galactosidase subunit is composed of 1,038 amino acids with a calculated M_r of 118,068. This β-galactosidase shares structural properties with Escherichia coli β-galactosidase (comparable subunit mass, 51% amino sequence identity, conservation of amino acid residues involved in catalysis, similar optimal pH value, and requirement for divalent metal ions) but is characterized by a higher catalytic efficiency on synthetic and natural substrates and by a shift of apparent optimum activity toward low temperatures and lower thermal stability. The enzyme also differs by a higher pI (7.8) and by specific thermodynamic activation parameters. P. haloplanktis β-galactosidase was expressed in E. coli, and the recombinant enzyme displays properties identical to those of the wild-type enzyme. Heat-induced unfolding monitored by intrinsic fluorescence spectroscopy showed lower melting point values for both P. haloplanktis wild-type and recombinant β-galactosidase compared to the mesophilic enzyme. Assays of lactose hydrolysis in milk demonstrate that P. haloplanktis β-galactosidase can outperform the current commercial β-galactosidase from Kluyveromyces marxianus var. lactis, suggesting that the cold-adapted β-galactosidase could be used to hydrolyze lactose in dairy products processed in refrigerated plants.     

Selective Inhibition of Escherichia coli in the Presence of Salmonella typhimurium by Phenethyl β-D-Galactopyranoside

Phenethyl β-d-galactopyranoside (PEG) was hydrolyzed by the β-galactosidase of Escherichia coli to form the toxic product phenethyl alcohol. Salmonella typhimurium did not hydrolyze PEG. In mixed culture, the ratio of S. typhimurium to E. coli was increased by growing the organisms in lactose broth containing 2.5% PEG. The high concentration of PEG required for inhibition of E. coli can be attributed to inadequate cell permeability rather than to prevention of β-galactosidase induction.     

LAC4 Is the Structural Gene for β-Galactosidase in KLUYVEROMYCES LACTIS

Using genetic and biochemical techniques, we have determined that β-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus. The following data support this conclusion: (1) mutations in this locus result in levels of β-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac^- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac⁺ revertant, bearing lac4–14 and this unlinked suppressor, has subnormal levels of β-galactosidase activity, and the K_m for hydrolysis of o-nitrophenyl-β, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of β-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acryl-amide gel electrophoresis shows that strains bearing lac4–23 and lac4–30 contain an inactive β-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is observable in lac4–8, lac4–14 or lac4–29 mutants; (6) of all lac4 mutants, only those bearing lac4–23 or lac4–30 contain a protein that cross-reacts with anti-β-galactosidase antibody, a finding consistent with the previous result; and (7) β-galactosidase activity in several Lac⁺ revertants of strains carrying lac4–23 or lac4–30 has greatly decreased thermostability.     

Klebsiella aerogenes Strain Carrying Drug-Resistance Determinants and a lac Plasmid

In addition to carrying determinants conferring resistance to at least two antibiotics, chloramphenicol and streptomycin, a Klebsiella aerogenes strain contains a plasmid responsible for increased β-galactosidase activity. The plasmid can be transferred to Escherichia coli and Salmonella typhimurium strains. K. aerogenes segregants without the plasmid grow on lactose one-half as fast as the parent strain and contain only one-tenth to one-fifth as much β-galactosidase.     

Physiology of Escherichia coli K-12 During Conjugation: Altered Recipient Cell Functions Associated with Lethal Zygosis

The number of viable F⁻ cells decreases when Escherichia coli recipient cells are mixed with an excess of Hfr cells. Evidence is presented showing that lethal zygosis was accompanied by changes in the physiology of the recipient cells, including (i) inhibition of deoxyribonucleic acid synthesis, (ii) inhibition of β-galactosidase induction, (iii) altered transport and accumulation of galactosides, and (iv) leakage of β-galactosidase into the supernatant fluid. The results are discussed in terms of possible conjugation-associated changes that, at high Hfr to F⁻ ratios, lead to lethal zygosis.     

High-Level Production of Recombinant Human Parathyroid Hormone 1-34

Expression of the synthetic human parathyroid hormone 1-34 [hPTH(1-34)] gene by a gene fusion strategy was demonstrated. hPTH(1-34) was produced at the C terminus of the partner peptides involving amino acids 1 to 97, 1 to 117, or 1 to 139 of a modified Escherichia coli β-galactosidase by linker peptides containing oligohistidine of different lengths. The fusion proteins in the inclusion bodies were rendered soluble with urea and subjected to site-specific cleavage with the secretory type yeast Kex2 protease. Optimal expression and enzymatic processing were achieved in the fusion protein βG-117S4HPT, constructed from amino acids 1 to 117 of β-galactosidase and the linker of HHHHPGGSVKKR. The fusion protein accumulated more than 20% of the E. coli total protein. The hPTH(1-34) was purified up to 99.5% with a good yield of 0.5 g/liter of culture. The purified product was identified as intact hPTH(1-34) by amino acid analysis and N-terminal sequencing.     

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both ς²⁸-like and ς⁷⁰-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.     

pAM401-Based Shuttle Vectors That Enable Overexpression of Promoterless Genes and One-Step Purification of Tag Fusion Proteins Directly from Enterococcus faecalis

Two novel Enterococcus faecalis-Escherichia coli shuttle vectors that utilize the promoter and ribosome binding site of bacA on the E. faecalis plasmid pPD1 were constructed. The vectors were named pMGS100 and pMGS101. pMGS100 was designed to overexpress cloned genes in E. coli and E. faecalis and encodes the bacA promoter followed by a cloning site and stop codon. pMGS101 was designed for the overexpression and purification of a cloned protein fused to a Strep-tag consisting of 9 amino acids at the carboxyl terminus. The Strep-tag provides the cloned protein with an affinity to immobilized streptavidin that facilitates protein purification. We cloned a promoterless β-galactosidase gene from E. coli and cloned the traA gene of the E. faecalis plasmid pAD1 into the vectors to test gene expression and protein purification, respectively. β-Galactosidase was expressed in E. coli and E. faecalis at levels of 10³ and 10 Miller units, respectively. By cloning the pAD1 traA into pMGS101, the protein could be purified directly from a crude lysate of E. faecalis or E. coli with an immobilized streptavidin matrix by one-step affinity chromatography. The ability of TraA to bind DNA was demonstrated by the DNA-associated protein tag affinity chromatography method using lysates prepared from both E. coli and E. faecalis that overexpress TraA. The results demonstrated the usefulness of the vectors for the overexpression and cis/trans analysis of regulatory genes, purification and copurification of proteins from E. faecalis, DNA binding analysis, determination of translation initiation site, and other applications that require proteins purified from E. faecalis.     

Kinetic Studies of β-Galactosidase Induction

The kinetics of β-galactosidase induction in E. coli ML 3 have been studied. Following addition of inducer, the rate of enzyme synthesis accelerates from the uninduced to a steady-state rate. At saturating concentration of inducer the time constant (T_c) for this process is 2.5 to 3 minutes. With decreasing inducer concentration (I), increasing time constants are observed. I/I + K′ approximates I/T_c. The steady-state rate of β-galactosidase synthesis is approximated by I²/I² + K². K′ and K have been estimated for IPTG and TMG. The kinetics of β-galactosidase production after the removal of inducer by dilution or after the addition of glucose have been investigated. A transition time of 2.5 to 3 minutes is observed before enzyme synthesis slows or stops. These results are consistent with the hypothesis that the enzyme-forming unit is unstable.     

Stimulation of human DNA polymerase epsilon by MDM2

The human DNA polymerase ε catalytic subunit consists of a 140-kDa N‐terminal domain that contains the catalytic activity and a 120-kDa C-terminal domain that binds to the other subunits and to exogenous peptides, including PCNA and MDM2. We report here that recombinant human MDM2 purified from insect cells or Escherichia coli stimulated the activity of DNA polymerase ε up to 10- and 40-fold, respectively, but not those of DNA polymerase β or Klenow fragment of E.coli DNA polymerase I. Kinetic studies indicated that MDM2 increased the maximum velocity of the reaction, but did not change substrate affinities. The stimulation depended upon the interaction of the N‐terminal 166 amino acid residues of MDM2 with the C-terminal domain of the full-length catalytic subunit, since the deletion of 166 amino acids from N‐terminal of MDM2 or the removal of the C-terminal domain of DNA polymerase ε by trypsin digestion or competition for binding to it by the addition of excess C-terminal fragment eliminated the stimulation. Since DNA polymerase ε appears to be involved in DNA replication, recombination and repair synthesis, we suggest that MDM2 binding to DNA polymerase ε might be part of a reconfiguration process that allows DNA polymerase ε to associate with repair/recombination proteins in response to DNA damage.     

Role for cis-Acting RNA Sequences in the Temperature-Dependent Expression of the Multiadhesive Lig Proteins in Leptospira interrogans

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5′ untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA^fMet-mRNA ternary complex was inhibited unless a 5′ deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5′ UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5′ UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5′ UTR on expression. These results indicate that ligA and ligB expression is limited by double-stranded RNA that occludes the ribosome-binding site. At elevated temperatures, the ribosome-binding site is exposed to promote translation initiation.     

β-Galactosidase from Termination and Deletion Mutant Strains

β-Galactosidase fragments were isolated from strains of Escherichia coli with mutations in the lacZ gene. The polypeptide obtained from a termination mutant (lacZNG125) appeared to be the intact gene product, containing the first half of the β-galactosidase amino acid sequence. From an internal deletion mutant strain (lacZU163), an aggregate was obtained of several partially degraded polypeptides. Each of these was smaller than predicted from genetic data for the fragment. Introduction of the lacZU163 mutation into a protein degradation-deficient strain (Deg⁻) resulted in the protection of the amino-terminal region of the protein. Some of the BrCN peptides from the U163 polypeptides were separated and identified. From such experiments it was shown that in both Deg⁻ and Deg⁺ strains the COOH-terminal region is rapidly degraded. This indicates that the complete gene product of lacZU163 has not been detected. The use of genetically defined enzyme fragments in studying structure-function relationships and in determination of primary structure is discussed.     

Isolation and Characterization of Supercoiled Circular Deoxyribonucleic Acid from Beta-Hemolytic Strains of Escherichia coli

Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated by cesium chloride centrifugation in the presence of ethidium bromide from a naturally occurring beta-hemolytic Escherichia coli strain (SC52). The open circular forms have contour lengths of 2.25 ± 0.1 μm, 24.0 ± 0.3 μm, and 29.5 ± 0.5 μm. The beta-hemolytic character of E. coli SC52 can be transferred by conjugation to a nonhemolytic recipient strain. Analysis of the supercoiled DNA of the hemolytic recipient demonstrated that the two large supercoiled DNA molecules of E. coli SC52 are transferred during this event, too. A beta-hemolytic laboratory E. coli strain and several of its derivatives have been shown to contain at least one circular DNA molecule, slightly larger in size than those isolated from E. coli SC52 and its conjugant. The possible significance of these DNA molecules for hemolysin production and transfer is discussed.     

Plasmid Transfer into the Homoacetogen Acetobacterium woodii by Electroporation and Conjugation

Shuttle vectors (pMS3 and pMS4) which replicated in Escherichia coli and in gram-positive Acetobacterium woodii were constructed by ligating the replication origin of plasmid pAMβ1 with the E. coli cloning vector pUC19 and the tetM gene of streptococcal transposon Tn916. Electrotransformation of A. woodii was achieved at frequencies of 4.5 × 10³ transformants per μg of plasmid DNA. For conjugal plasmid transfer, the mobilizable shuttle vector pKV12 was constructed by cloning the tetM gene into pAT187. Mating of E. coli containing pKV12 with A. woodii resulted in transfer frequencies of 3 × 10^-6 to 7 × 10^-6 per donor or recipient.     

Ability of Daphnia Cell-Free Extract to Damage Escherichia coli Cells

A cell-free extract of Daphnia magna was found to lyse Escherichia coli cells as shown by leakage of the enzymes alkaline phosphatase and β-galactosidase from the bacteria. The cell-free extract was separated on Sephadex G-200, and the fractions showing an ability to lyse E. coli cels were isolated. The factor which was responsible for the lysis of the bacterial cells was probably a protein with a molecular weight of several thousands. Mg²⁺ and Ca²⁺ ions augmented the activity of the Daphnia extract on E. coli cells.     

Ultra-Low Background DNA Cloning System

Yeast-based in vivo cloning is useful for cloning DNA fragments into plasmid vectors and is based on the ability of yeast to recombine the DNA fragments by homologous recombination. Although this method is efficient, it produces some by-products. We have developed an “ultra-low background DNA cloning system” on the basis of yeast-based in vivo cloning, by almost completely eliminating the generation of by-products and applying the method to commonly used Escherichia coli vectors, particularly those lacking yeast replication origins and carrying an ampicillin resistance gene (Amp^r). First, we constructed a conversion cassette containing the DNA sequences in the following order: an Amp^r 5′ UTR (untranslated region) and coding region, an autonomous replication sequence and a centromere sequence from yeast, a TRP1 yeast selectable marker, and an Amp^r 3′ UTR. This cassette allowed conversion of the Amp^r-containing vector into the yeast/E. coli shuttle vector through use of the Amp^r sequence by homologous recombination. Furthermore, simultaneous transformation of the desired DNA fragment into yeast allowed cloning of this DNA fragment into the same vector. We rescued the plasmid vectors from all yeast transformants, and by-products containing the E. coli replication origin disappeared. Next, the rescued vectors were transformed into E. coli and the by-products containing the yeast replication origin disappeared. Thus, our method used yeast- and E. coli-specific “origins of replication” to eliminate the generation of by-products. Finally, we successfully cloned the DNA fragment into the vector with almost 100% efficiency.     

The Multifaceted Benefits of Protein Co-expression in Escherichia coli

We report here that the expression of protein complexes in vivo in Escherichia coli can be more convenient than traditional reconstitution experiments in vitro. In particular, we show that the poor solubility of Escherichia coli DNA polymerase III ε subunit (featuring 3’-5’ exonuclease activity) is highly improved when the same protein is co-expressed with the α and θ subunits (featuring DNA polymerase activity and stabilizing ε, respectively). We also show that protein co-expression in E. coli can be used to efficiently test the competence of subunits from different bacterial species to associate in a functional protein complex. We indeed show that the α subunit of Deinococcus radiodurans DNA polymerase III can be co-expressed in vivo with the ε subunit of E. coli. In addition, we report on the use of protein co-expression to modulate mutation frequency in E. coli. By expressing the wild-type ε subunit under the control of the araBAD promoter (arabinose-inducible), and co-expressing the mutagenic D12A variant of the same protein, under the control of the lac promoter (inducible by isopropyl-thio-β-D-galactopyranoside, IPTG), we were able to alter the E. coli mutation frequency using appropriate concentrations of the inducers arabinose and IPTG. Finally, we discuss recent advances and future challenges of protein co-expression in E. coli.     

Only One ATP-binding DnaX Subunit Is Required for Initiation Complex Formation by the Escherichia coli DNA Polymerase III Holoenzyme

The DnaX complex (DnaX₃δδ′χψ) within the Escherichia coli DNA polymerase III holoenzyme serves to load the dimeric sliding clamp processivity factor, β₂, onto DNA. The complex contains three DnaX subunits, which occur in two forms: τ and the shorter γ, produced by translational frameshifting. Ten forms of E. coli DnaX complex containing all possible combinations of wild-type or a Walker A motif K51E variant τ or γ have been reconstituted and rigorously purified. DnaX complexes containing three DnaX K51E subunits do not bind ATP. Comparison of their ability to support formation of initiation complexes, as measured by processive replication by the DNA polymerase III holoenzyme, indicates a minimal requirement for one ATP-binding DnaX subunit. DnaX complexes containing two mutant DnaX subunits support DNA synthesis at about two-thirds the level of their wild-type counterparts. β₂ binding (determined functionally) is diminished 12–30-fold for DnaX complexes containing two K51E subunits, suggesting that multiple ATPs must be bound to place the DnaX complex into a conformation with maximal affinity for β₂. DNA synthesis activity can be restored by increased concentrations of β₂. In contrast, severe defects in ATP hydrolysis are observed upon introduction of a single K51E DnaX subunit. Thus, ATP binding, hydrolysis, and the ability to form initiation complexes are not tightly coupled. These results suggest that although ATP hydrolysis likely enhances β₂ loading, it is not absolutely required in a mechanistic sense for formation of functional initiation complexes.