Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

The P_R promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that P_R has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of P_R showed that it uses a canonical −10 hexamer recognized by SigA, and mutants with mutations to the sequence 5′-TATAMT had the greatest activities. It does not contain a 5′-TGN-extended −10 sequence, although mutants with mutations creating an extended −10 sequence had substantially increased promoter activity. Mutations in the −35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the −35 hexamer differentially affected promoter activity, depending on the −10 and extended −10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout P_R generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter.     

In vivoandin vitroStudies of TrpR-DNA Interactions

The binding of tryptophan repressor (TrpR) to its operators was examined quantitatively usingin vitroandin vivomethods. DNA sequence requirements for 1:1 and tandem 2 :1 (TrpR : DNA) binding in various sequence contexts were studied. The results indicate that the optimal half-site sequence for recognition by one helix-turn-helix motif of one TrpR dimer is_{3′ CNTGA 5′}^{5′ GNACT 3′}, consistent with contacts observed by X-ray diffraction analysis of cocrystalline 1:1 and 2 :1 complexes. Half-sites can be paired to form a palindrome either by direct abutment, forming the nucleation site for a tandem 2 :1 complex, or with an 8-base-pair spacer, forming a 1:1 target. Dimethylsulfate (DMS) methylation-protection footprintingin vitroof 1:1 and 2 :1 complexes formed sequentially on the two unequal half-site pairs of thetrpEDCBA operator fromSerratia marcescensindicated an obligate hierarchy of site occupancy, with one half-site pair serving as the nucleation site for tandem binding. DMS footprinting ofEscherichia colioperatorsin vivoshowed that, over a wide range of intracellular TrpR concentration, thetrpEDCBA operator is occupied by three repressor dimers,aroH is occupied by two dimers, and the 1:1 binding mode is used on thetrpR operator. The coexistence of these distinct occupancy states implies that changes in protein concentration affect only the fractional occupancy of each operator rather than the binding mode, which is determined by the number of half-site sequences present in the operator region. Cooperativity of tandem complex formation measured by gel retardation using a symmetrized synthetic operator containing identical, optimal sites spaced as in natural operators was found to be modest, implying a maximum coupling free energy of ∼−2 kcal/mol. On other sequences the apparent degree of cooperativity, as well as the apparent affinity, varied with sequence and sequence context in a manner consistent with the structural models and which suggests compensation between affinity and cooperativity as a mechanism that allows tolerance of operator sequence variation.     

The Tail Sheath of Bacteriophage N4 Interacts with the Escherichia coli Receptor

Unlike other characterized phages, the lytic coliphage N4 must inject the 360-kDa virion RNA polymerase (vRNAP), in addition to its 72-kbp genome, into the host for successful infection. The process of adsorption to the host sets up and elicits the necessary conformational changes in the virion to allow genome and vRNAP injection. Infection of suppressor and nonsuppressor strains, Escherichia coli W3350 supF and E. coli W3350, with a mutant N4 isolate (N4am229) harboring an amber mutation in Orf65 yielded virions containing (N4gp65⁺) and lacking (N4gp65⁻) gp65, respectively. N4gp65⁺ but not N4gp65⁻ phage was able to adsorb to the host. Recombinant gp65 with a hexahistidine tag at the N terminus or hexahistidine and c-myc tags at the C terminus was able to complement N4gp65⁻ virions in vivo and in vitro. Immunogold detection of gp65 in vivo complemented virions revealed its localization at the N4 tail. Finally, we show both in vitro and in vivo that gp65 interacts with the previously determined N4 outer membrane receptor, NfrA.     

Transcriptional Regulation of the Vanillate Utilization Genes (vanABK Operon) of Corynebacterium glutamicum by VanR,a PadR-Like Repressor

Corynebacterium glutamicum is able to utilize vanillate, the product of lignin degradation, as the sole carbon source. The vanillate utilization components are encoded by the vanABK operon. The vanA and vanB genes encode the subunits of vanillate O-demethylase, converting vanillate to protocatechuate, while VanK is the specific vanillate transporter. The vanABK operon is regulated by a PadR-type repressor, VanR. Heterologous gene expression and variations of the vanR open reading frame revealed that the functional VanR contains 192 residues (21 kDa) and forms a dimer, as analyzed by size exclusion chromatography. In vivo, ferulate, vanillin, and vanillate induced P_vanABK in C. glutamicum, while only vanillate induced the activity of P_vanABK in Escherichia coli lacking the ferulate catabolic system. Differential scanning fluorimetry verified that vanillate is the only effector of VanR. Interaction between the P_vanABK DNA fragment and the VanR protein had an equilibrium dissociation constant (K_D) of 15.1 ± 1.7 nM. The VanR-DNA complex had a dissociation rate constant (K_d) of (267 ± 23) × 10⁻⁶ s⁻¹, with a half-life of 43.5 ± 3.6 min. DNase I footprinting localized the VanR binding site at P_vanABK, extending from +9 to +45 on the coding strand. Deletion of the nucleotides +18 to +27 inside the VanR binding site rendered P_vanABK constitutive. Fusion of the T7 promoter and the wild-type VanR operator, as well as its shortened versions, indicated that the inverted repeat AACTAACTAA(N₄)TTAGGTATTT is the specific VanR binding site. It is proposed that the VanR-DNA complex contains two VanR dimers at the VanR operator.     

Conformational and thermodynamic hallmarks of DNA operator site specificity in the copper sensitive operon repressor from Streptomyces lividans

Metal ion homeostasis in bacteria relies on metalloregulatory proteins to upregulate metal resistance genes and enable the organism to preclude metal toxicity. The copper sensitive operon repressor (CsoR) family is widely distributed in bacteria and controls the expression of copper efflux systems. CsoR operator sites consist of G-tract containing pseudopalindromes of which the mechanism of operator binding is poorly understood. Here, we use a structurally characterized CsoR from Streptomyces lividans (CsoR^Sl) together with three specific operator targets to reveal the salient features pertaining to the mechanism of DNA binding. We reveal that CsoR^Sl binds to its operator site through a 2-fold axis of symmetry centred on a conserved 5′-TAC/GTA-3′ inverted repeat. Operator recognition is stringently dependent not only on electropositive residues but also on a conserved polar glutamine residue. Thermodynamic and circular dichroic signatures of the CsoR^Sl–DNA interaction suggest selectivity towards the A-DNA-like topology of the G-tracts at the operator site. Such properties are enhanced on protein binding thus enabling the symmetrical binding of two CsoR^Sl tetramers. Finally, differential binding modes may exist in operator sites having more than one 5′-TAC/GTA-3′ inverted repeat with implications in vivo for a mechanism of modular control.     

Relation of gp70 to spontaneous cytolytic activity of mouse spleen cells

In comparing spleen cells of inbred and congenic mice for spontaneous capacity to lyse cells of the BALB/c leukemia RLc1 in vitro, we found that the activity of 129 spleen cells was more than double that of 129-G_ix ^– spleen cells. The only known difference between these two strains is that 129-G_ix ^– mice express no known demonstrable gp70 or p30, whereas 129 mice express both these MuLV-related components as mendelian traits not associated with the production of virions. We infer that MuLV-related components at the cell surface are concerned in effector-target interactions leading to cytolysis under the conditions described. — Although the congenic strains B6 (G_ix ^–) and B6-G_ix ⁺ differ likewise in expression of the type-variant G_ix-gp70, both strains express a second type-variant of gp70. The lytic activity of spleen cells of these two strains for RL1 cells was equally high, suggesting that involvement in lytic effector-target interactions is common to gp70 molecules in general. — When used as targets rather than as effectors 129 spleen cells were more sensitive to lysis than 129-G_ix ^– spleen cells. Pre-exposure to gp70, purified from R-MuLV, rendered splenic effector cells less lytic. Pre-exposure to gp70 also rendered RL1 target cells less sensitive to lysis. One explanation of these findings is that both target cells and effector cells express gp70 and also receptors for gp70 and that this is the basis of mutual cellular recognition leading to lysis in the circumstances described.     

Resonance Raman Studies of Co—O2 and O—O Stretching Vibrations in Oxy-Cobalt Hemes

Strong evidence suggests that the stretching vibration of the bound oxygen can be perturbed by an accidentally degenerate porphyrin ring mode, resulting in two split frequencies. In the Co(II)(TpivPP) (pyridine) ¹⁸O₂ complex, we demonstrate that the ν(¹⁸O—¹⁸O) mode, after being shifted from its ν(¹⁶O—¹⁶O) value at 1,156 cm^-1, undergoes a resonance interaction with the 1,080 cm^-1 porphyrin mode, giving rise to two lines at 1,067 and 1,089 cm^-1. In the O₂ complex of Co(II) mesoporphyrin IX-substituted sperm whale myoglobin, we observed a dramatic intensity increase at 1,132 cm^-1 upon ¹⁶O₂ → ¹⁸O₂ substitution, which is due to the reappearance of the 1,132-cm^-1 porphyrin mode after the removal of resonance conditions. A decrease in O₂ binding affinity, caused by the proximal base tension, corresponds to an increase in the Co—O₂ stretching frequency. The ν(Co—O₂) at 527 cm^-1 for the low affinity Co(II)(TpivPP)(1,2-Me₂Im) O₂ complex is 11 cm^-1 higher than the 516-cm^-1 value for the high affinity complex (with N-MeIm replacing 1,2-Me₂Im). However, in the corresponding iron complexes the reverse behavior is observed, i.e., the ν(Fe—O₂) decreases for the (1,2-Me₂Im) complex. There is a 24-cm^-1 difference in the Co—O₂ stretching frequencies between Co(II)(TpivPP)(N-MeIm)O₂ (at 516 cm^-1) and oxy meso CoMb (at 540 cm^-1), suggesting a protein induced distortion of the Co—O—O linkage. However, the values for ν(Fe—O₂) are nearly identical between Fe(II)(TpivPP)(N-MeIm)O₂ (at 571 cm^-1) and oxy Mb (at 573 cm^-1), indicating that O₂ binds to myoglobin in the same manner as in the sterically unhindered “picket fence” complex. Evidence is presented that suggests the presence of two dioxygen stretching frequencies due to two different conformers in each of the N-MeIm and 1,2-Me₂Im complex of oxy Co(II)(TpivPP).     

Mapping of I gene mutations which lead to repressors with increased affinity for lac operator

A genetic mapping system is used to locate mutations on the lac repressor gene (I) which lead to repressor proteins with an increased affinity for operator DNA. These tight binding repressors (I^tb) are of particular interest since their analysis should allow some conclusions on the mechanism of interaction between repressor and operator. I^tb mutations were found to map in two regions of the I gene. One is near the amino-terminal end, a region which has been shown to be essential for the DNA binding properties of the repressor. The other region in which I^tb mutations were mapped codes for approximately amino acids 255 to 295 of the repressor, a region which had so far not been considered to be essential for the DNA binding properties of the repressor protein.     

Role of cis-Acting Sites in Stimulation of the Phage λ PRM Promoter by CI-Mediated Looping

The lysogenic state of phage λ is maintained by the CI repressor. CI binds to three operators each in the right operator (O_R) and left operator (O_L) regions, which lie 2.4 kb apart. At moderate CI levels, the predominant binding pattern is two dimers of CI bound cooperatively at each regulatory region. The resulting tetramers can then interact, forming an octamer and a loop of the intervening DNA. CI is expressed from the P_RM promoter, which lies in the O_R region and is subjected to multiple regulatory controls. Of these, the most recently discovered is stimulation by loop formation. In this work, we have investigated the mechanism by which looping stimulates P_RM. We find that two cis-acting sites lying in the O_L region are involved. One site, an UP element, is required for stimulation. Based on the behavior of other promoters with UP elements located upstream of the −35 region, we suggest that a subunit of RNA polymerase (RNAP) bound at P_RM binds to the UP element located in the O_L region. In addition, adjacent to the UP element lies a binding site for integration host factor (IHF); this site plays a less critical role but is required for stimulation of the weak prm240 allele. A loop with CI at the O_L2 and O_L3 operators does not stimulate P_RM, while one with CI only at O_L2 provides some stimulation. We discuss possible mechanisms for stimulation.     

Why the Lysogenic State of Phage λ Is So Stable: A Mathematical Modeling Approach

We develop a mathematical model of the phage λ lysis/lysogeny switch, taking into account recent experimental evidence demonstrating enhanced cooperativity between the left and right operator regions. Model parameters are estimated from available experimental data. The model is shown to have a single stable steady state for these estimated parameter values, and this steady state corresponds to the lysogenic state. When the CI degradation rate (γ_cI) is slightly increased from its normal value (γ_cI 0.0 min⁻¹), two additional steady states appear (through a saddle-node bifurcation) in addition to the lysogenic state. One of these new steady states is stable and corresponds to the lytic state. The other steady state is an (unstable) saddle node. The coexistence these two globally stable steady states (the lytic and lysogenic states) is maintained with further increases of γ_cI until γ_cI 0.35 min⁻¹, when the lysogenic steady state and the saddle node collide and vanish (through a reverse saddle node bifurcation) leaving only the lytic state surviving. These results allow us to understand the high degree of stability of the lysogenic state because, normally, it is the only steady state. Further implications of these results for the stability of the phage λ switch are discussed, as well as possible experimental tests of the model.     

The rhodopsin system of the squid

Squid rhodopsin (λ_max 493 mµ)—like vertebrate rhodopsins—contains a retinene chromophore linked to a protein, opsin. Light transforms rhodopsin to lumi- and metarhodopsin. However, whereas vertebrate metarhodopsin at physiological temperatures decomposes into retinene and opsin, squid metarhodopsin is stable. Light also converts squid metarhodopsin to rhodopsin. Rhodopsin is therefore regenerated from metarhodopsin in the light. Irradiation of rhodopsin or metarhodopsin produces a steady state by promoting the reactions, See PDF for Equation Squid rhodopsin contains neo-b (11-cis) retinene; metarhodopsin all-trans retinene. The interconversion of rhodopsin and metarhodopsin involves only the stereoisomerization of their chromophores. Squid metarhodopsin is a pH indicator, red (λ_max 500 mµ) near neutrality, yellow (λ_max 380 mµ) in alkaline solution. The two forms—acid and alkaline metarhodopsin—are interconverted according to the equation, Alkaline metarhodopsin + H⁺ acid metarhodopsin, with pK 7.7. In both forms, retinene is attached to opsin at the same site as in rhodopsin. However, metarhodopsin decomposes more readily than rhodopsin into retinene and opsin. The opsins apparently fit the shape of the neo-b chromophore. When light isomerizes the chromophore to the all-trans configuration, squid opsin accepts the all-trans chromophore, while vertebrate opsins do not and hence release all-trans retinene. Light triggers vision by affecting directly the shape of the retinene chromophore. This changes its relationship with opsin, so initiating a train of chemical reactions.     

Membrane Fusion Mediated by Baculovirus gp64 Involves Assembly of Stable gp64 Trimers into Multiprotein Aggregates

The baculovirus fusogenic activity depends on the low pH conformation of virally-encoded trimeric glycoprotein, gp64. We used two experimental approaches to investigate whether monomers, trimers, and/or higher order oligomers are functionally involved in gp64 fusion machine. First, dithiothreitol (DTT)- based reduction of intersubunit disulfides was found to reversibly inhibit fusion, as assayed by fluorescent probe redistribution between gp64-expressing and target cells (i.e., erythrocytes or Sf9 cells). This inhibition correlates with disappearance of gp64 trimers and appearance of dimers and monomers in SDS-PAGE. Thus, stable (i.e., with intact intersubunit disulfides) gp64 trimers, rather than independent monomers, drive fusion. Second, we established that merger of membranes is preceded by formation of large (greater than 2 MDa), short-lived gp64 complexes. These complexes were stabilized by cell–surface cross-linking and characterized by glycerol density gradient ultracentrifugation. The basic structural unit of the complexes is stable gp64 trimer. Although DTT-destabilized trimers were still capable of assuming the low pH conformation, they failed to form multimeric complexes. The fact that formation of these complexes correlated with fusion in timing, and was dependent on (a) low pH application, (b) stable gp64 trimers, and (c) cell–cell contacts, suggests that such multimeric complexes represent a fusion machine.