Two distinct regions of the large serine recombinase TnpX are required for DNA binding and biological function

The large serine recombinase, TnpX, from the Clostridium perfringens integrative mobilizable element Tn 4451 , consists of three domains and has two known DNA binding regions. In this study random and site-directed mutagenesis was used to identify other regions of TnpX that were required for biological activity. Genetic and biochemical analysis of these mutants led to the identification of important TnpX residues in the N-terminal catalytic pocket. In addition, another region of TnpX (aa 243–261), which is conserved within large serine recombinases, was shown to be essential for both excision and insertion. Mutation of charged residues within this region led to a loss of biological activity and aberrant DNA binding. This phenotype was mediated by interaction with the distal DNA binding region (aa 598–707). In these mutants, removal of residues 598–707 resulted in loss of DNA binding, despite the presence of the primary DNA binding region (aa 533–583). Analysis of mutations within the aa 243–261 region indicated that different protein conformations were involved in the insertion and the excision reactions. In summary, we have shown that TnpX is a complex protein that has multiple intra- and intermolecular interaction sites, providing insight into the structural and functional complexity of this important enzyme family.     

Structural determinants of factor IX(a) binding in nitrophorin 2, a lipocalin inhibitor of the intrinsic coagulation pathway

Nitrophorin 2 (NP2) is a salivary lipocalin from Rhodnius prolixus that binds with coagulation factors IX (fIX) and IXa (fIXa). Binding of NP2 with fIXa results in potent inhibition of the intrinsic factor Xase complex. A panel of site-directed surface mutants of NP2 was generated to locate determinants of high affinity fIX(a) binding. The locations of the mutations were based on comparisons with the related, but less potent, inhibitor nitrophorin 3 (NP3). Three point mutants (K21A, K92A, and V94A) were found that clearly reduced the inhibitory potency as measured by the activity of a reconstituted factor Xase system. Binding of NP2 with fIXa and fIX as measured by surface plasmon resonance and isothermal titration calorimetry was reduced in a similar manner. Of the three mutants, two (K92A and V94A) were located on the loop connecting beta-strands E and F of the lipocalin beta-barrel. The largest changes were seen with the K92A mutation, which lies at the apex of the loop, with a smaller effect being seen with mutation of Val(94). Combination of four E-F loop mutations (K92A, A93K, V94A, E97A) in a single mutant reduced the inhibitory potency and binding to levels similar to those seen with NP3 without affecting heme or histamine binding.     

Two arginine repressors regulate arginine biosynthesis in Lactobacillus plantarum

The repression of the carAB operon encoding carbamoyl phosphate synthase leads to Lactobacillus plantarum FB331 growth inhibition in the presence of arginine. This phenotype was used in a positive screening to select spontaneous mutants deregulated in the arginine biosynthesis pathway. Fourteen mutants were genetically characterized for constitutive arginine production. Mutations were located either in one of the arginine repressor genes (argR1 or argR2) present in L. plantarum or in a putative ARG operator in the intergenic region of the bipolar carAB-argCJBDF operons involved in arginine biosynthesis. Although the presence of two ArgR regulators is commonly found in gram-positive bacteria, only single arginine repressors have so far been well studied in Escherichia coli or Bacillus subtilis. In L. plantarum, arginine repression was abolished when ArgR1 or ArgR2 was mutated in the DNA binding domain, or in the oligomerization domain or when an A123D mutation occurred in ArgR1. A123, equivalent to the conserved residue A124 in E. coli ArgR involved in arginine binding, was different in the wild-type ArgR2. Thus, corepressor binding sites may be different in ArgR1 and ArgR2, which have only 35% identical residues. Other mutants harbored wild-type argR genes, and 20 mutants have lost their ability to grow in normal air without carbon dioxide enrichment; this revealed a link between arginine biosynthesis and a still-unknown CO2-dependent metabolic pathway. In many gram-positive bacteria, the expression and interaction of different ArgR-like proteins may imply a complex regulatory network in response to environmental stimuli.     

Isolation of Bacillus subtilis transformation-deficient mutants and mapping of competence genes

We have isolated and characterized 48 Bacillus subtilis competence-deficient mutants. The mutants, obtained by nitrosoguanidine mutagenesis or by insertional mutagenesis with transposon Tn917, had a reduced transformation frequency and a wild-type transduction frequency. The com mutations were mapped by PBS1 transduction and at least four new com genes have been identified. The mutants were also characterized for their capacity to bind and take up the transforming DNA.     

Development of a transformation system for the flavinogenic yeast Candida famata

Riboflavin-overproducing mutants of the flavinogenic yeast Candida famata are used for industrial riboflavin production. This paper describes the development of an efficient transformation system for this species. Leucine-deficient mutants have been isolated from C. famata VKM Y-9 wild-type strain. Among them leu2 mutants were identified by transformation to leucine prototrophy with plasmids YEp13 and PRpL2 carrying the Saccharomyces cerevisiae LEU2 gene. DNA fragments (called CfARSs) conferring increased transformation frequencies and extrachromosomal replication were isolated from a C. famata gene library constructed on the integrative vector containing the S. cerevisiae LEU2 gene as a selective marker. The smallest cloned fragment (CfARS16) has been sequenced. This one had high adenine plus thymine (A+T) base pair content and a sequence homologous to the S. cerevisiae ARS Consensus Sequence. Methods for spheroplast transformation and electrotransformation of the yeast C. famata were optimized. They conferred high transformation frequencies (up to 10(5) transformants per microg DNA) with a C. famata leu2 mutant using replicative plasmids containing the S. cerevisiae LEU2 gene as a selective marker. Riboflavin-deficient mutants were isolated from the C. famata leu2 strain and their biochemical identification was carried out. Using the developed transformation system, several C. famata genomic fragments complementing mutations of structural genes for riboflavin biosynthesis (coding for GTP cyclohydrolase, reductase, dihydroxybutanone phosphate synthase and riboflavin synthase, respectively) have been cloned.     

On the functional role of coupling factor B in the mitochondrial H+ -ATPase

Evidence for the presence of a functionally important vicinal dithiol in mitochondrial coupling factor B (FB) has been presented earlier (Sanadi, D. R. (1982) Biochim. Biophys. Acta 683, 39-56). FB was completely inactivated by 38 micron of copper o-phenanthroline or 0.63 mM iodosobenzoate, and the kinetics were consistent with intramolecular disulfide formation as were polyacrylamide gel patterns which showed that FB which had been treated with copper o-phenanthroline had a different mobility from that of untreated FB. ATP-Pi exchange activity and ATP-induced binding of bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol (oxonol VI) to H+ -ATPase were also inhibited by the thiol oxidizing reagents, although oligomycin-sensitive ATPase activity was unaffected. F0 isolated from H+ -ATPase rebinds purified F1 with the restoration of ATP-induced oxonol-binding activity. Prior treatment of F0 (but not of F1) with copper o-phenanthroline abolished the oxonol-binding activity of reconstituted F0-F1. 115Cd binds tightly to H+ -ATPase and the bound protein can be recovered by gel electrophoresis in phosphate buffer in the presence of sodium dodecyl sulfate at a position corresponding to FB. Prior treatment of the H+ -ATPase with copper o-phenanthroline abolished 115Cd binding. The results indicate that the major effect of these inhibitors is on FB dithiol and leave little doubt that Cd2+ is indeed bound to a vicinal dithiol group.     

Binding of polycyclic aromatic hydrocarbons to DNA of cells in culture: a rapid method for its analysis using hydroxylapatite column chromatography.

A rapid procedure to study the interaction of carcinogens with DNA in cultured cells has been developed. The cells, which are labeled with 7,12-[3H]dimethylbenz[a] anthracene ([3H]DMBA), are lysed with 0.24 M phosphate buffer (pH 6.8), 1% sodium dodecyl sulfate (SDS), 8 M urea and 0.01 M ethylenediamine-tetraacetate (EDTA) and sonicated. The cell lysates are fractionated on columns of hydroxylapatite. Proteins and RNA are removed with 8 M urea in 0.24 M phosphate buffer (pH 6.8). DMBA-bound DNA is eluted with 0.4 M phosphate buffer (pH 6.8). DMBA-DNA isolated by this procedure is virtually free from proteins and RNA. Thermal stability, ultraviolet spectra and the density of DNA is not altered by DMBA binding. The uptake of DMBA by mouse epidermal cells is rapid and the binding of DMBA to DNA is linear for the first 8 h of exposure. DMBA binds to DNA in all phases of the cell cycle. However, the highest binding occurs immediately following maximum DNA synthesis.     

Mutational analysis of the RecA protein L1 region identifies this area as a probable part of the co-protease substrate binding site

Previous mutational analysis of the L1 region of the RecA protein suggested that Gly-157 and Glu-158 are 'hot-spots' for the occurrence of constitutive LexA co-protease mutants (coprt^c). In the present study, we clearly establish that position 157 is a hot-spot for the occurrence of such mutants, as 12 of 14 and 10 of 14 substitutions result in this phenotype for UmuD and LexA cleavage respectively. The frequency of such mutations at position 158 is somewhat lower, 8 of 13 and 5 of 13 for UmuD and LexA respectively. Comparison of the UmuD vs. LexA co-protease activity for all single mutants with substitutions at positions 154, 155, 156, 157 and 158 (47 in total) reveals that, although there is good agreement among most mutants regarding their ability to cleave both LexA and UmuD, there are two in particular (Glu-154→Asp and Glu-154→Gln) that show a clear preference for cleavage of UmuD. We also show that three second-site mutations that completely suppress coprt^c activity toward LexA have little or no effect on the coprt^c activity of the primary mutant toward UmuD. In addition, we observe a high frequency of second-site suppressor mutations, suggesting a functional interaction among side-chains in this region. Together, these results support the idea that the L1 region of RecA makes up part of the co-protease substrate-binding site.     

Thermodynamic studies on anion binding to apotransferrin and to recombinant transferrin N-lobe half molecules

Equilibrium constants for the binding of anions to apotransferrin, to the recombinant N-lobe half transferrin molecule (Tf/2N), and to a series of mutants of Tf/2N have been determined by difference UV titrations of samples in 0.1 M Hepes buffer at pH 7.4 and 25 degrees C. The anions included in this study are phosphate, sulfate, bicarbonate, pyrophosphate, methylenediphosphonic acid, and ethylenediphosphonic acid. There are no significant differences between anion binding to Tf/2N and anion binding to the N-lobe of apotransferrin. The binding of simple anions like phosphate appears to be essentially equivalent for the two apotransferrin binding sites. The binding of pyrophosphate and the diphosphonates is inequivalent, and the studies on the recombinant Tf/2N show that the stronger binding is associated with the N-terminal site. Anion binding constants for phosphate, pyrophosphate, and the diphosphonates with the N-lobe mutants K206A, K296A, and R124A have been determined. Anion binding tends to be weakest for the K296A mutant, but the variation in log K values among the three mutants is surprisingly small. It appears that the side chains of K206, K296, and R124 all make comparable contributions to anion binding. There are significant variations in the intensities of the peaks in the difference UV spectra that are generated by the titrations of the mutant apoproteins with these anions. These differences appear to be related more to variations in the molar extinction coefficients of the anion-protein complexes rather than to differences in binding constants.     

Interaction of rat liver dexamethasone-receptor complexes with DNA]

Rat liver glucocorticoid-receptor complexes (GRC) acquire the ability to bind to DNA in a high affinity manner after activation by heating or precipitation with (NH)2SO4. DNA is practically non-saturable by GRC in low salt buffers as well as in 0.15 M NaCl-containing buffer, although in the latter case the binding decreases approximately 3--5 times. GRC bind to homo- and heterologous prokaryotic DNA in a similar way; in both cases an addition of KCl (up to 0.15 M) to the medium is followed by the same decrease of the binding. This data suggest that the association of GRC with DNA observed in vitro is not accompanied by "recognition" of any certain DNA site. Besides DNA, activated GRC can associate with other polymers, charged positively (DEAE-cellulose) or negatively (RNA, polyvinylsulfate). GRC interact very weakly with neutral compounds of the cellulose type but are strongly adsorbed on hydroxyapatite. Hence the activated GRC can be considered as an amphoteric protein. Salt solutions provoke dissociation of the GRC-DNA triple complexes: a complete dissociation is observed in the presence of 0,4 M NaCl or 0,4 M sodium phosphate buffer (pH 6,9). Sodium phosphate buffer also elutes GRC from other sorbents such as DEAE-cellulose or hydroxyapatite. No significant dissociation of the GRC-DNA complexes is observed at sucrose concentration up to 2 M. The data obtained are indicative of an essential role of electrostatic forces for the interaction of GRC with DNA. The non-ionic detergent Triton X-100 at a concentration as low as 0,05% completely destroys the GRC-DNA triple complexes. The models explicating the selectivity of the genome activation by GRC without their "recognition" of any specific DNA sequences are proposed.     

Induced Natural Transformation of Acinetobacter calcoaceticus in Soil Microcosms

Factors affecting natural transformation of Acinetobacter calcoaceticus BD413 with homologous chromosomal DNA in a silt loam soil microcosm were investigated. Inducible transformation of declining populations of noncompetent A. calcoaceticus cells was detectable for up to 6 days when a simple carbon source, salts, and freshly added DNA were used. In two different experimental setups, the residence time in soil of induced cells could be increased to either 11 or 24 h before DNA addition without reduced transformation frequency; 200-to 1,000-fold fewer transformants were observed following the addition of water. These observations suggest that A. calcoaceticus remains transformable for several hours after its activation by nutrients in soil. Increasing the levels of phosphate salts significantly enhanced the numbers of transformants without increasing the recipient counts correspondingly. Variable levels of ammonium or divalent cations (Mg(sup2+) and Ca(sup2+)) did not have a similar major influence. Soil moisture content significantly affected the transformation frequency of A. calcoaceticus cells, with a general tendency of higher frequencies in drier soil. A minimal frequency was observed at around 35% soil moisture. The data indicate that A. calcoaceticus cells in soil which cannot be detectably transformed are easily induced by nutrients to undergo natural transformation with chromosomal DNA. Access to nutrients seems to be critical for the development and maintenance of competence in soil, which is also affected by abiotic factors like moisture level and phosphate salt concentration.     

Phosphate adsorption and availability plant of phosphate

Summary The effects of phosphate buffer capacity on the plant-availability of labile soil phosphate, when measured as intensity (I) or quantity (Q), are described and tested using results from a greenhouse experiment on 24 Sherborne soils. In multiple regression studies, phosphate buffer capacity with I or Q measurements as independent variables accounted for up to 94% of the variance in P uptake by ryegrass, the maximum buffer capacity being generally more useful than the equilibrium buffer capacity.When the quantity of soil P is measured (Q), its availability (i.e. ease of desorption) to plant roots is inversely related to the Langmuir bonding energy parameter and the buffer capacity. When the intensity of soil P is measured (I), its availability (i.e. resistance to change) is directly related to the adsorption and buffer capacities. The levels of Q or I, therefore, which are optimal for plant uptake vary with the buffer capacity of the soil. There is little or no correlation between the adsorption capacity and the bonding energy in many soils and consequently phosphate buffer capacity is only poorly correlated with the total adsorption capacity.     

Energetic differences between the specific binding of a 40 bp DNA duplex and the lac promoter to lac repressor protein

The energetics of LRP binding to a 104 bp lac promoter determined from ITC measurements were compared to the energetics of binding to a shorter 40 bp DNA duplex with the 21 bp promoter binding site sequence. The promoter binding affinity of 2.47 +/- 0.0 1x 10(7) M(-1) was higher than the DNA binding affinity of 1.81 +/- 0.67 x 10(7) M(-1) while the binding enthalpy of -804 +/- 41 kJ mol(-1) was lower than that of the DNA binding enthalpy of -145 +/- 16 kJ mol(-1) at 298.15 K. Both the promoter and DNA binding reactions were exothermic in phosphate buffer but endothermic in Tris buffer that showed the transfer of four protons to LRP in the former reaction but only two in the latter. A more complicated dependence of these parameters on temperature was observed for promoter binding. These energetic differences are attributable to additional LRP-promoter interactions from wrapping of the promoter around the LRP.     

Plasmid mediated mutagenesis of a cellular gene in transfected eukaryotic cells.

NIH3T3 cells are widely used in transformation assays and readily take up transfected DNA. A system has been devised using NIH3T3 cells to measure the mutagenic effect of transfected DNA on recipient cell genes. NIH3T3 cells can be mutated to 6-thioguanine resistance at a frequency which suggests that at least a portion of the cells have only one functional copy of the HGPRT gene. They have a low spontaneous background mutation frequency (approximately 1 X 10(-7)). Transfection of three different plasmids into NIH3T3 cells induced 6-thioguanine resistant mutants at frequencies ranging from 3 to 11 fold above background. The mutant phenotype is stable and reversion frequencies of several mutants are less than or equal to 1 X 10(-7). Southern blot analysis of the HGPRT gene in several mutants showed that 4 of 26 mutants (15.4%) had detectable alterations in the structure of the HGPRT gene. Interestingly 3 of the 4 mutants showing rearrangements were obtained by transfection of the HSV-2 morphological transforming region.     

Genetic transformation of Bacillus brevis 47, a protein-secreting bacterium, by plasmid DNA.

A method has been developed for introducing plasmid DNA into Bacillus brevis 47, a protein-secreting bacterium. Treatment of B. brevis 47 cells with 50 mM Tris-hydrochloride buffer of alkaline pH was effective for inducing DNA uptake competence. In the presence of polyethylene glycol, the Tris-treated cells incorporated plasmid DNA with a frequency of 10(-4) (transformants per viable cell) when 1 microgram of plasmid DNA was added to 10(9) Tris-treated cells. The pH of Tris-hydrochloride buffer as well as the concentration and molecular weight of the polyethylene glycol affected the transformation frequency. The growth phase of B. brevis 47 cells strongly influenced the frequency. Two plasmids, pHW1 and pUB110, have been introduced into B. brevis 47 by this method. The mechanism of induction of competence for DNA uptake in connection with removal of the outer two protein layers of the cell wall by treatment of B. brevis 47 cells with Tris-hydrochloride buffer is discussed.     

Integration Efficiency in DNA-Induced Transformation of Pneumococcus. I. a Method of Transformation in Solid Medium and Its Use for Isolation of Transformation-Deficient and Recombination-Modified Mutants

A method of transformation on solid medium especially adapted for pneumococcus has been developed. Under specific conditions, all colonies that are allowed to grow in the presence of transforming DNA for six hours give rise to transformed bacteria. Combined with replica plating this technique has been used to isolate mutants modified with regard to recombination. Most of the mutants found are transformation-defective and show a large diversity in their response to ultraviolet light. Some of these mutants have lost their ability to take up transforming DNA. One shows a reduced yield of transformants for a given quantity of DNA taken up. Mutants that manifest altered behavior with regard to marker efficiencies have also been isolated. One of these exhibits a decrease in the transformation efficiency of only the high efficiency markers and two mutants show a decrease in the transformation efficiency of the low efficiency markers.     

Plasmid transformation of Streptococcus sanguis (Challis) occurs by circular and linear molecules

Summary Transformation of Streptococcus sanguis (Challis) by antibiotic resistance plasmids has shown that (a) competente developed with identical kinetics for chromosomal and plasmid DNA; (b) dependence of transformant yield on plasmid DNA concentration was second order; (c) open circular plasmid DNA transformed Challis, although at reduced frequency; (d) linearization of plasmid DNA by restriction enzymes cutting at unique sites inactivated the transforming capacity; (e) transforming activity was restored when linear plasmid molecules generated by different restriction enzymes were mixed; (f) restoration of transforming activity depended on the distance between the linearizing cuts, i.e. on the presence of sufficiently long overlapping homologous sequences; (g) when linear deletion mutants were mixed with linear parental plasmids the smaller plasmid was restored with significantly higher frequency.Based on these data, a model for plasmid transformation of Challis is proposed according to which circular plasmid is linearized during binding and uptake. One DNA strand enters the cell and restoration of circular plasmids inside the cell occurs by annealing of complementary single strands from two different donor molecules. Implications of this model for recombinant DNA experiments in streptococci are discussed.     

A mutation in alpha helix 3 of CA renders human immunodeficiency virus type 1 cyclosporin A resistant and dependent: rescue by a second-site substitution in a distal region of CA

The replication of many isolates of human immunodeficiency virus type 1 (HIV-1) is enhanced by binding of the host cell protein cyclophilin A (CypA) to the viral capsid protein (CA). The immunosuppressive drug cyclosporine A (CsA) and its nonimmunosuppressive analogs bind with high affinity to CypA and inhibit HIV-1 replication. Previous studies have identified two mutations, A92E and G94D, in the CypA-binding loop of CA that confer the ability of HIV-1 to replicate in the presence of CsA. Interestingly, CsA stimulates the replication of HIV-1 mutants containing either the A92E or G94D substitution in some human cell lines. Here, we show that substitution of alanine for threonine at position 54 of CA (T54A) also confers HIV-1 resistance to and dependence on CsA. Like the previously identified CsA-resistant/dependent mutants, infection by the T54A mutant was stimulated by CsA in a target cell-specific manner. RNA interference-mediated reduction of CypA expression enhanced the permissiveness of HeLa cells to infection by the T54A mutant. A suppressor mutation, encoding a substitution of threonine for alanine at position 105 of CA (A105T), was identified through adaptation of the T54A mutant virus for growth in CEM cells. A105T rescued the impaired single-cycle infectivity and replication defects of both T54A and A92E mutants. These results indicate that CA determinants outside the CypA-binding loop can modulate the dependence of HIV-1 infection on CypA.