A high yielding mutant of mycobacteriophage L1 and its application as a diagnostic tool.

L1 is a lysogenic phage of mycobacteria, which along with L5 and D29 constitute a closely linked family of homoimmune mycobacteriophages. These phages can be potentially used for genetic engineering of mycobacteria and diagnosis of mycobacterial infection. The effectiveness of such phage based systems depends on the efficiency with which they infect and grow within target cells. While working with phage L1c1ts which is a temperature sensitive mutant of phage L1, we observed that high yielding phage stocks were generated by repeated passage through the host, Mycobacterium smegmatis. A plaque purified mutant L1-P2, obtained from one such high yielding stock, when analyzed further was found to infect host cells with increased efficiency. The DNA obtained from L1-P2 was examined by restriction digestion, and it was observed that spontaneous loss of DNA fragment from the right arm, which encodes early regulatory factors, had occurred. It has been further demonstrated that the high yielding property of the mutant phage could be utilized to increase the sensitivity of mycobacteriophage-based detection systems.

Transposition-induced structural instability of Escherichia coli-mycobacteria shuttle vectors.

Escherichia coli-mycobacteria shuttle vectors, derived from pAL5000 (a mycobacterial plasmid) and pUC19, were frequently found to undergo structural alterations due to transposition of IS1096, a Mycobacterium smegmatis transposable element, at a cluster of sites located within a small region of 60 bp, immediately upstream of a kanamycin resistance gene present in these vectors. The structural alterations led to deletion of large regions of the vector which, in several cases, were found to extend into the ORF2 (RepB) coding sequences of the pAL5000 replication region without affecting its replication capability. This suggests that the entire ORF2 coding sequences of the pAL5000 replication region may not be essential for replication of pAL5000-derived vectors. The deletion derivatives, which contain the minimal sequences required for replication and selection in mycobacteria, were found to be structurally stable and therefore these could be potentially used as stable vector systems for the transformation of mycobacteria.

2H and 13C nuclear magnetic resonance study of N-palmitoylgalactosylsphingosine (cerebroside)/cholesterol bilayers.

13C- and 2H-NMR experiments were used to examine the phase behavior and dynamic structures of N-palmitoylgalactosylsphingosine (NPGS) (cerebroside) and cholesterol (CHOL) in binary mixtures. 13C spectra of 13C=O-labeled and 2H spectra of [7,7-2H2] chain-labeled NPGS as well as 3 alpha-2H1 CHOL indicate that cerebroside and CHOL are immiscible in binary mixtures at temperatures less than 40 degrees C. In contrast, at 40 degrees C < t < or = T(C) (NPGS), up to 50 mol% CHOL can be incorporated into melted cerebroside bilayers. In addition, 13C and 2H spectra of melted NPGS/CHOL bilayers show a temperature and cholesterol concentration dependence. An analysis of spectra obtained from the melted 13C=O NPGS bilayer phase suggests that the planar NH-C=O group assumes an orientation tilted 40 degrees-55 degrees down from the bilayer interface. The similarity between the orientation of the amide group relative to the bilayer interface in melted bilayers and in the crystal structure of cerebroside suggests that the overall crystallographic conformation of cerebroside is preserved to a large degree in hydrated bilayers. Variation of temperature from 73 degrees to 86 degrees C and CHOL concentration from 0 to 51 mol% results in small changes in this general orientation of the amide group. 2H spectra of chain-labeled NPGS and labeled CHOL in NPGS/CHOL bilayer demonstrate that molecular exchange between the gel and liquid-gel (LG) phases is slow on the 2H time scale, and this facilitates the simulation of the two component 2H spectra of [7,7-2H2]NPGS/CHOL mixtures. Simulation parameters are used to quantitate the fractions of gel and LG cerebroside. The quadrupole splitting of [7,7-2H2]NPGS/CHOL mixtures and 2H simulations allows the LG phase bilayer fraction to be characterized as an equimolar mixture of cerebroside and CHOL.

A 13C and 2H nuclear magnetic resonance study of phosphatidylcholine/cholesterol interactions: characterization of liquid-gel phases.

A detailed study on the structure, dynamics, and thermodynamic behavior of phosphatidylcholine/cholesterol (PC/CHOL) mixtures was undertaken using differential scanning calorimetry (DSC) and solid-state nuclear magnetic resonance (NMR) spectroscopy. DSC thermograms of mixtures of cholesterol (CHOL) with 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC), 1,2-distearoyl-sn-phosphatidylcholine (DSPC), and 1,2-diarachidoyl-sn-phosphatidylcholine (DAPC) showed a broadening of the first-order gel-->liquid crystalline transition and a decrease in the transition enthalpy, indicating a gradual loss of cooperativity for high CHOL concentrations. DPPC and DSPC were labeled with 13C at the carbonyl group of the sn-2 chain and 2H was introduced into the middle of the sn-2 chain at the 6- and 12-position for DPPC and DSPC, respectively. The 13C and 2H NMR spectra of each labeled lipid were studied as a function of temperature and CHOL concentration. The residual quadrupole splitting in the 2H NMR spectra, delta nu Q perpendicular, was analyzed as a function of temperature and composition. For CHOL concentrations less than 30 mol %, a precipitous change in delta nu Q perpendicular occurs near the chain melting temperature of the phospholipid. Further increases in CHOL concentration broaden the transition and shift the midpoint to higher temperature, indicating the presence of a new phase at higher CHOL contents. Moreover, at a given temperature, delta nu Q perpendicular increases with increasing cholesterol content, which indicates a more ordered structure. The 13C NMR spectra in the gel state consisted of a superposition of two components which can be attributed to both gel-like and fluid phospholipid domains in the bilayer. This two-component spectrum can be simulated quantitatively with a two-parameter chemical exchange model, which permits the fraction of each form and the exchange rate to be determined as a function of temperature and composition. At high CHOL contents the line width of the fluid component broadens, suggesting an increase in the exchange rate between the domains. These results were interpreted in terms of a temperature composition diagram with one region L beta', two regions LGI and LGII, and one liquid crystalline region L alpha, with LG denoting "liquid-gel" type phases. Liquid-gel phases correspond to phases with increased order in the hydrocarbon chains (in comparison to that of the pure PC bilayer in the L alpha phase) combined with fast limit axial diffusion that averages the 13C NMR spectrum to a "fluidlike" line.(ABSTRACT TRUNCATED AT 400 WORDS)

Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector.

We have constructed a promoter selection vector for mycobacteria to analyze the sequences involved in mycobacterial transcriptional regulation. The vector pSD7 contains extrachromosomal origins of replication from Escherichia coli as well as from Mycobacterium fortuitum and a kanamycin resistance gene for positive selection in mycobacteria. The promoterless chloramphenicol acetyltransferase (CAT) reporter gene has been used to detect mycobacterial promoter elements in a homologous environment and to quantify their relative strengths. Using pSD7, we have isolated 125 promoter clones from the slowly growing pathogen Mycobacterium tuberculosis H37Rv and 350 clones from the fast-growing saprophyte Mycobacterium smegmatis. The promoters exhibited a wide range of strengths, as indicated by their corresponding CAT reporter activities (5 to 2,500 nmol/min/mg of protein). However, while most of the M. smegmatis promoters supported relatively higher CAT activities ranging from 100 to 2,500 nmol/min/mg of protein, a majority of those from M. tuberculosis supported CAT activities ranging from 5 to only about 100 nmol/min/mg of protein. Our results indicate that stronger promoters occur less frequently in the case of M. tuberculosis compared with M. smegmatis. To assess the extent of divergence of mycobacterial promoters vis-à-vis those of E. coli, the CAT activities supported by the promoters in E. coli were measured and compared with their corresponding activities in mycobacteria. Most of the mycobacterial promoter elements functioned poorly in E. coli. The homologous selection system that we have developed has thus enabled the identification of mycobacterial promoters that apparently function optimally only in a native environment.

The perturbation of model membranes by (-)-delta 9-tetrahydrocannabinol. Studies using solid-state 2H- and 13C-NMR.

The effects of (-)-delta 9-tetrahydrocannabinol (delta 9-THC) on model phospholipid membranes were studied using solid-state 2H and 13C nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine with specific 2H- and 13C-labels as endogenous probes at the C7, methylene and the carbonyl groups, respectively, of the sn-2 chain were used to study the conformational and dynamic properties of the bilayer as a function of temperature and drug concentration. The drug molecule decreases the phase transition temperature of the bilayer in a concentration dependent manner up to 20 molar percent when full saturation has occurred. The 2H spectra show that delta 9-THC broadens the phase transition during which the spectra acquire a characteristic shape of a two-component system exchanging at an intermediate rate (approximately 10(6) s-1) with some liquid crystalline features. Such spectra provide information related to the melting of the phospholipid chains. At intermediate temperatures, the 13C spectra show a gel-like and a liquid-crystalline-like exchanging components and provide information about a conformational change at the phospholipid glycerol backbone occurring at or near the pretransition. The spectral composition and rate of exchange are both dependent on drug concentration. We have carried out computer simulations of the 13C spectra and obtained conformational information related to the phase transition process in the bilayer from gel to liquid crystal. Our studies show that delta 9-THC has a stronger effect on the sn-2 carbonyl near the bilayer interface than on the lipid chains and serve to describe the membrane perturbing effects of cannabinoids in molecular terms.

Solid-state 13C NMR study of tyrosine protonation in dark-adapted bacteriorhodopsin.

Solid-state 13C MAS NMR spectra were obtained for dark-adapted bacteriorhodopsin (bR) labeled with [4'-13C]Tyr. Difference spectra (labeled minus natural abundance) taken at pH values between 2 and 12, and temperatures between 20 and -90 degrees C, exhibit a single signal centered at 156 ppm, indicating that the 11 tyrosines are protonated over a wide pH range. However, at pH 13, a second line appears in the spectrum with an isotropic shift of 165 ppm. Comparisons with solution and solid-state spectra of model compounds suggest that this second line is due to the formation of tyrosinate. Integrated intensities indicate that about half of the tyrosines are deprotonated at pH 13. This result demonstrates that deprotonated tyrosines in a membrane protein are detectable with solid-state NMR and that neither the bR568 nor the bR555 form of bR present in the dark-adapted state contains a tyrosinate at pH values between 2 and 12. Deprotonation of a single tyrosine in bR568 would account for 3.6% of the total tyrosine signal, which would be detectable with the current signal-to-noise ratio. We observe a slight heterogeneity and subtle line-width changes in the tyrosine signal between pH 7 and pH 12, which we interpret to be due to protein environmental effects (such as changes in hydrogen bonding) rather than complete deprotonation of tyrosine residue(s).

Anisotropic 2H NMR spin-lattice relaxation in L alpha-phase cerebroside bilayers.

A series of 2H NMR inversion recovery experiments in the L alpha phase of the cerebroside N-palmitoylgalactosylsphingosine (NPGS) have been performed. In these liquid crystalline lipid bilayers we have observed substantial anisotropy in the spin-lattice relaxation of the CD2 groups in the acyl chains. The form and magnitude of the anisotropy varies with position in the chain, being positive in the upper region, decreasing to zero at the 4-position, and reversing sign at the lower chain positions. It is also shown that addition of cholesterol to the bilayer results in profound changes in the anisotropy. These observations are accounted for by a simple motional model of discrete hops among nine sites, which result from the coupling of two modes of motion--long-axis rotational diffusion and gauche-trans isomerization. This model is employed in quantitative simulations of the spectral line shapes and permits determination of site populations and motional rates. These results, plus preliminary results in sphingomyelin and lecithin bilayers, illustrate the utility of T1 anisotropy measurements as a probe of dynamics in L alpha-phase bilayers.

Characterization of the L lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction.

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition (L kappa----L lambda) at temperatures similar to the L beta'----(P beta')----L alpha transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. The L kappa phase shows typical gel-phase X-ray diffraction patterns. In contrast, the L lambda-phase diffraction patterns indicate disordered hydrocarbon chains. 2H NMR of specifically 2H chain-labeled DPPC confirmed that the acyl chains are disordered in the L lambda phase over their entire lengths. 2H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and 13C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicating very little motion at the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. We postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluidity of the hydrophobic region in the L lambda phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.

Anisotropic 2H-nuclear magnetic resonance spin-lattice relaxation in cerebroside- and phospholipid-cholesterol bilayer membranes.

The axially symmetric powder pattern 2H-nuclear magnetic resonance (NMR) lineshapes observed in the liquid crystalline phase of pure lipid or lipid/cholesterol bilayers are essentially invariant to temperature, or, equivalently, to variations in the correlation times characterizing C-2H bond reorientations. In either of these melted phases, where correlation times for C-2H bond motions are shorter than 10(-7) s, information on the molecular dynamics of the saturated hydrocarbon chain would be difficult to obtain using lineshape analyses alone, and one must resort to other methods, such as the measurement of 2H spin-lattice relaxation rates, in order to obtain dynamic information. In pure lipid bilayers, the full power of the spin-lattice relaxation technique has yet to be realized, since an important piece of information, namely the orientation dependence of the 2H spin-lattice relaxation rates is usually lost due to orientational averaging of T1 by rapid lateral diffusion. Under more favorable circumstances, such as those encountered in the lipid/cholesterol mixtures of this study, the effects of orientational averaging by lateral diffusion are nullified, due to either a marked reduction (by at least an order of magnitude) in the diffusion rate, or a marked increase in the radii of curvature of the liposomes. In either case, the angular dependence of 2H spin-lattice relaxation is accessible to experimental study, and can be used to test models of molecular dynamics in these systems. Simulations of the partially recovered lineshapes indicate that the observed T1 anisotropies are consistent with large amplitude molecular reorientation of the C-2H bond among a finite number of sites. Furthermore, from the observed orientation dependence of the 2H spin-lattice relaxation rates, we conclude that order director fluctuations cannot provide the dominant relaxation pathway for acyl chain deuterons.

Nuclear magnetic resonance and calorimetric study of the structure, dynamics, and phase behavior of uranyl ion/dipalmitoylphosphatidylcholine complexes.

The interaction of UO2(2+) with dipalmitoylphosphatidylcholine (DPPC) has been studied as a function of temperature and composition using nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC), and monolayer studies. Computer simulations of the 31P-NMR powder spectra of DPPC dispersions in the presence of various concentrations of UO2(2+) are consistent with the binding stoichiometry of [UO2(2+)]/[DPPC] = 1:4 at [UO2(2+)]/[DPPC] less than 0.3. This complex undergoes a phase transition to the liquid crystalline phase at T'm = 50 +/- 3 degrees C with a breadth delta T'm = 7 +/- 3 degrees C. This broad transition gradually disappears at higher UO2(2+) concentrations, suggesting the presence of yet another UO2(2+)/DPPC complex (or complexes) whose NMR spectra are indistinguishable from those of the 1:4 UO2(2+)/DPPC species. The temperature-dependent 13C powder spectra of 2(1-13C) DPPC dispersions in the presence of 1.2 mol ratio of UO2(2+) show that this higher order complex (complexes) also undergoes a phase transition to the liquid crystalline state at T'm +/- = 58 +/- 3 degrees C with a breadth delta T"m = 15 +/- 5 degrees C. The NMR spectra indicate that exchange among these various UO2(2+)/DPPC complexes is slow. In addition, computer simulations of the 31P-, 13C-, and 2H-NMR powder spectra show that axial diffusion of the DPPC molecules about their long axes is quenched by addition of UO2(2+) and acyl chain isomerization is the dominant motional mode. The isomerization is best described as two-site hopping of the greater than C-D bond at a rate of approximately 10(6) s-1, a motional mode which is expected for a kink diffusion.

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 1. M412 and L550 intermediates.

The role of tyrosines in the bacteriorhodopsin (bR) photocycle has been investigated by using Fourier transform infrared (FTIR) and UV difference spectroscopies. Tyrosine contributions to the BR570----M412 FTIR difference spectra recorded at several temperatures and pH's were identified by isotopically labelling tyrosine residues in bacteriorhodopsin. The frequencies and deuterium/hydrogen exchange sensitivities of these peaks and of peaks in spectra of model compounds in several environments suggest that at least two different tyrosine groups participate in the bR photocycle during the formation of M412. One group undergoes a tyrosinate----tyrosine conversion during the BR570----K630 transition. A second tyrosine group deprotonates between L550 and M412. Low-temperature UV difference spectra in the 220--350-nm region of both purple membrane suspensions and rehydrated films support these conclusions. The UV spectra also indicate perturbation(s) of one or more tryptophan group(s). Several carboxyl groups appear to undergo a series of protonation changes between BR570 and M412, as indicated by infrared absorption changes in the 1770--1720-cm-1 region. These results are consistent with the existence of a proton wire in bacteriorhodopsin that involves both tyrosine and carboxyl groups.

Tyrosine and carboxyl protonation changes in the bacteriorhodopsin photocycle. 2. Tyrosines-26 and -64.

Low-temperature Fourier transform infrared (FTIR) and UV difference spectroscopies combined with selective tyrosine nitration and tyrosine isotopic labeling have been used to investigate the participation of tyrosines-26 and -64 in the bacteriorhodopsin (bR) photocycle. Nitration of Tyr-26 has no detectable effect on the FTIR or UV difference spectra of the BR570----K630 or BR570----M412 transitions. In contrast, nitration of Tyr-64 causes changes in both the FTIR and UV spectra of these transitions. However, this nitration does not alter tyrosine peaks in the FTIR difference spectra which have previously been associated with the protonation of a tyrosinate by K630 and the deprotonation of a tyrosine by M412 [Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. Instead, Tyr-64 nitration appears to affect other tyrosine peaks. These results and changes in UV difference spectra upon Tyr-64 nitration are consistent with the deprotonation of Tyr-64 by M412 as concluded previously [Scherrer, P., & Stoeckenius, W. (1985) Biochemistry 24, 7733-7740]. Effects on chromophore vibrations caused by Tyr-64 nitration are unaltered upon reducing the nitrotyrosine to aminotyrosine with sodium dithionite. Finally, nitro-Tyr-64 causes a shift in the frequency of a positive peak at 1739 cm-1 in the BR570----M412 FTIR difference spectrum which reflects the protonation of a carboxyl-containing residue [Engelhard, M., Gerwert, K., Hess, B., Kreutz, W., & Siebert, F. (1985) Biochemistry 24, 400-407; Roepe, P., Ahl, P. L., Das Gupta, S. K., Herzfeld, J., & Rothschild, K. J. (1987) Biochemistry (preceding paper in this issue)]. The shift does not occur for samples containing amino-Tyr-64. These data suggest that Tyr-64 may interact with this carboxyl group.

Magnetic orientation of sphingomyelin-lecithin bilayers.

Phospholipid bilayers consisting of a 60:40 mixture of N-palmitoylsphingomyelin and dimyristoylphosphatidylcholine orient in a strong magnetic field. The orientation is easily observed in 31P- and 2H-nuclear magnetic resonance spectra where the intensity of the perpendicular edges of the powder lineshapes are enhanced. The lineshapes indicate that the long axis of the molecule is perpendicular to the magnetic field.

Studies on the interaction of anesthetic steroids with phosphatidylcholine using 2H and 13C solid state NMR.

The effects of the anesthetic steroid alphaxalone and its inactive analog delta 16-alphaxalone on model phospholipid membranes were studied using 13C and 2H solid-state nuclear magnetic resonance spectroscopy. Aqueous multilamellar dispersions of dipalmitoylphosphatidylcholine (DPPC) with specific 13C and 2H labels as endogenous probes at the carbonyl and the C-7 methylene groups, respectively, of the sn-2 chain were used to study the conformational and dynamical properties of the bilayer as a function of temperature. There were no significant changes between the 13C and 2H spectra of the DPPC preparation containing the inactive steroid and that of DPPC with no drug. However, the physiologically active steroid produces significant spectral 2H and 13C changes. These changes include a reduction of the main phase transition temperature and a broadening of that transition. Alphaxalone also increases the relative number of gauche conformers in the liquid-crystalline phase of DPPC and increases the rate of axial diffusion in both the gel and liquid-crystalline phase. The thermotropic properties of the above preparations, as monitored by differential scanning calorimetry, were congruent with the spectroscopic data.

Evidence for a tyrosine protonation change during the primary phototransition of bacteriorhodopsin at low temperature.

Isotopically labeled tyrosines have been selectively incorporated into bacteriorhodopsin (bR). A comparison of the low-temperature bR570 to K Fourier transform infrared-difference spectra of these samples and normal bR provides information about the role of tyrosine in the primary phototransition. Several tyrosine contributions to the difference spectrum are found. These results and comparison with the spectra of model compounds suggest that a tyrosinate group protonates during the bR570 to K transition. This conclusion is strongly supported by the results of UV difference spectroscopy.

Phase equilibria, molecular conformation, and dynamics in phosphatidylcholine/phosphatidylethanolamine bilayers.

Solid-state 13C and 2H NMR experiments have been used to examine the phase equilibria and dynamic structure of binary mixtures of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE). The experiments rely on changes in the 13C and 2H spectra of sn-2 13C = O labeled and 2H chain and head group labeled lipids at phase boundaries. In particular, broad powder patterns are observed in the gel state, and these patterns narrow dramatically in the liquid-crystalline phase. In the two-phase region a superposition of the gel-phase and liquid-crystalline-phase spectra is observed, which results from the presence of large domains in the DPPC/DPPE system. The appearance of the liquid-crystalline component and the disappearance of the gel component permit the solidus and liquidus curves of the phase diagram to be located accurately. Furthermore, a comparison of the temperature dependence of the 13C and 2H spectra shows the phase transition mechanism to be a function of composition. This result, together with other evidence, supports the hypothesis that the gel-state lattice changes from the L beta or P beta to the L beta configuration with increasing DPPE content. Finally, a detailed examination of the composition dependence of the spectra provides evidence for nonideal mixing.