The DNA-binding domain in the Bacillus subtilis transition-state regulator AbrB employs significant motion for promiscuous DNA recognition.

AbrB is a Bacillus subtilis protein responsible for regulating a diverse array of unrelated genes during periods of sub-optimal growth conditions. DNA binding by AbrB is unique in that sequence recognition is specific, yet no obvious consensus sequence of bound promoter regions is apparent. The N-terminal domain is a recently characterized representative of a novel class of DNA-binding proteins that possess a looped-hinge helix DNA-binding topology. Although the structural characterization of this DNA-binding topology contributed to an understanding of the architectural basis for recognition of DNA target sequences, specific mechanisms responsible for promiscuity in DNA sequence recognition still were not apparent. Analysis of (15)N backbone relaxation parameters shows that dynamic motion of regions directly linked to DNA binding show concerted motion on the microsecond-millisecond timescale. Furthermore, dynamic motion of the hinge region suggests that the DNA-binding region is capable of conformational orientations that allow it to accommodate DNA sequence variability in the cognate binding sites.

Novel DNA binding domain and genetic regulation model of Bacillus subtilis transition state regulator abrB.

We have determined the high resolution NMR solution structure of the novel DNA binding domain of the Bacillus subtilis transition state regulator AbrB. Comparisons of the AbrB DNA binding domain with DNA binding proteins of known structure show that it is a member of a completely novel class of DNA recognition folds that employs a dimeric topology for cellular function. This new DNA binding conformation is referred to as the looped-hinge helix fold. Sequence homology investigations show that this DNA binding topology is found in other disparately related microbes. Structural analysis of the AbrB DNA binding domain together with bioanalytical and mutagenic data of full length AbrB allows us to construct a general model that describes the genetic regulation properties of AbrB.

Differential requirements of two insect cell lines for growth in serum-free medium.

The development of a serum-free medium that supports the growth of cells from a Spodoptera frugiperda and a Lymantria dispar cell line is reported. A yeast hydrolysate provided the B-vitamin complex, and a combination of a meat hydrolysate and tryptose provided most of the free amino acids required for cell growth. Supplemental cystine and methionine were required to achieve maximum cell growth. The serum or serum replacements used in earlier formulations were replaced with commercial lipid preparations and increased levels of iron salts. Although the cell growth cycle had a somewhat extended lag phase and the population doubling time of the S. frugiperda cells was longer than on serum-containing medium, the saturation densities were much higher. Spodoptera cells grown in this medium replicated the Autographa californica nuclear polyhedrosis virus well, producing 8.71 x 10(6) TCID50 extracellular virus and 4.4 x 10(6) polyhedra/ml culture. The specific activity of the polyhedra was somewhat less than that of polyhedra produced in insects.

Kinetics and motional dynamics of spin-labeled yeast iso-1-cytochrome c: 1. Stopped-flow electron paramagnetic resonance as a probe for protein folding/unfolding of the C-terminal helix spin-labeled at cysteine 102.

The kinetics of chemically induced folding and unfolding processes in spin-labeled yeast iso-1-cytochrome c were measured by stopped-flow electron paramagnetic resonance (EPR). Stopped-flow EPR, based on a new dielectric resonator structure [Sienkiewicz, A., Qu, K., & Scholes, C. P. (1994) Rev. Sci. Instrum. 65, 68-74], gives a new temporal component to probing nanosecond molecular tumbling motions that are modulated by macromolecular processes requiring time resolution of milliseconds to seconds. The stopped-flow EPR technique presented in this work is a kinetic technique that has not been previously used with such a time resolution on spin-labeled systems, and it has the potential for application to numerous spin-labeled sites in this and other proteins. The cysteine-specific spin-label, methanethiosulfonate spin-label (MTSSL), was attached to yeast iso-1-cytochrome c at the single naturally occurring cysteine102, and the emphasis for this work was on this disulfide-attached spin-labeled prototype. This probe has the advantage of reflecting the protein tertiary fold, as shown by recent, systematic site-directed spin labeling of T4 lysozyme [Mchaourab, H. S. Lietzow, M. A., Hideg, K., & Hubbell, W. L. (1996) Biochemistry 35, 7692-7704], and protein backbone dynamics, as also shown by model peptide studies [Todd, A. P., & Millhauser, G. L. (1991) Biochemistry 30, 5515-5523]. The C-terminal cytochrome c helix where the label is attached is thought to be critical in the initial steps of protein folding and unfolding. Stopped-flow EPR resolved the monoexponential, guanidinium-induced unfolding process at pH 6.5 with an approximately 20 ms time constant; this experiment required less than 150 microL of 80 microM spin-labeled protein. We observed an approximately 50-fold decrease of this unfolding time from the 1 s range to the 20 ms time range as the guanidinium denaturant concentration was increased from 0.6 to 2.0 M. The more complex refolding kinetics of our labeled cytochrome were studied by stopped-flow EPR at pH 5.0 and 6.5. The spin probe showed a fast kinetic process compatible with the time range over which hydrogen/deuterium amide protection indicates helix formation; this process was monoexponential at pH 5.0. At pH 6.5, there was evidence of an additional slower kinetic phase resolved by stopped-flow EPR and by heme-ligation-sensitive UV-Vis that indicated a slower folding where heme misligation may be involved. Since the disulfide-attached probe has reported folding and backbone dynamics in other systems, the implication is that our kinetic experiments were directly sensing events of the C-terminal helix formation and possibly the N- and C-terminal helical interaction. The cysteine-labeled protein was also studied under equilibrium conditions to characterize probe mobility and the effect of the probe on protein thermodynamics. The difference in spin probe mobility between folded and denatured protein was marked, and in the folded protein, the motion of the probe was anisotropically restricted. The motion of the attached nitroxide in the folded protein appears to be restricted about the carbon and sulfur bonds which tether it to the cysteine. The original point of cysteine sulfur attachment is approximately 11 A from the heme iron within the C-terminal helix near its interface with the N-terminal helix, but the low-temperature EPR spin probe line width showed that the probe lies more distant (> 15 A) from the heme iron. By all physical evidence, the protein labeled at cysteine102 folded, but the spin probe in this prototype system perturbed packing which lowered the thermal melting temperature, the free energy of folding, the guanidinium concentration at the midpoint of the unfolding transition, the m parameter of the denaturant, and the helical CD signature. This study prepares the way for study of protein folding/unfolding kinetics using EPR spectroscopy of spin-labels placed at specific cysteine-mutated sites within

Insect cells: adventitious agents.

The varied associations between insects and micro-organisms provides ample opportunity for the contamination of insect cell cultures. As with cell cultures from vertebrates, the addition of serum or serum products into cell culture media risks the introduction of mycoplasma and bovine viruses into cell cultures. Rarely do these organisms persist or multiply in insect cultures; however, there have been reports of the growth of Acholeplasmas introduced with fetal bovine serum. The contamination of cultures with plant or mammalian pathogens that are transmitted by insects is more common. The micro-organisms that are associated with insects, either as pathogens or commensals, frequently contaminate primary cell cultures and may also contaminate cell lines.

Branch cuts in the phase function.

It is shown that, when the scalar field associated with the propagation of a distorted wave function has nulls in its intensity pattern, the phase function that goes with that scalar field has branch points at the location of these nulls and that there are unavoidable 2pi discontinuities across the associated branch cuts in the phase function. An analytic proof of this supposition is provided. Sample computer-wave optics propagation results are presented that manifest such unavoidable discontinuities. Among other things, the numerical results are organized in a way that demonstrates that for those cases the branch points are unavoidable. It is found in the sample numerical results that the branch cuts can be positioned so that the 2pidiscontinuities are located along lines of minimum intensity. This location tends to minimize the physical significance or importance of the discontinuities, a significant consideration for deformablemirroradaptive optics, for which there is an unavoidable correction error in the vicinity of the branch cut. An algorithm is briefly described that allows the branch cuts to be located automatically and a phase function to be calculated that has discontinuities equal only to 2pidiscontinuities that are located at the branch cuts.

Lampyridae (Coleoptera): A plethora of mollicute associations.

Beetles (Coleoptera) harbor many species ofAcholeplasma andSpiroplasma (division Tenericutes, class Mollicutes). Mollicutes were isolated from guts and/or hemocoels of firefly beetles (Lampyridae) from the United States (Maryland and West Virginia), Ecuador, and Tobago. Firefly beetles were frequent hosts for the group XIV spiroplasma, isolated from Ellychnia corrusca, and the group XIX spiroplasma, isolated fromPhoturis spp. The most unusual feature of the firefly-mollicute association is the carriage of four Mycoplasma species. Recent phylogenetic studies indicate that these species are members of a clade that includes a vertebrate pathogen,Mycoplasma mycoides. The high rate of occurrence ofMycoplasma species (which are, otherwise, infrequent in insects) in lampyrid beetles suggests that the association is significant. The unusual light-producing physiology of lampyrids (which is dependent on large pools of energy) and the production of large amounts of cardenolides from cholesterol (a critical growth factor for many mollicutes) may favor colonization by mollicutes.

Large-scale propagation of insect cells.

Cultured insect cells have many uses in agriculture and medicine. They can be used in the diagnosis and isolation of a number of viruses infecting both animals and plants and for the laboratory study of these viruses. Large-volume culture of insect cells has been envisioned as a way of producing viruses for use in controlling insect pests and for the production of viral antigens for vaccine preparations. Recently they have become a potentially valuable way of producing a variety of proteins for human and veterinary medicine using the genetically engineered baculovirus expression vectors. The development of satisfactory cell lines and culture methods has proceeded at a slow, irregular pace, inhibited by the lack of knowledge of the physiology of the insect, its small size, and often by the lack of consistent, adequate support for the necessary developmental research. However, now that the basic culture systems are available, cell lines have been developed or can easily be developed for most needs. Suitable media are available and recent developments in refining existing media formations have resulted in low-cost media containing little protein to interfere with down-stream processing of cellular metabolites. Future developments are likely to further improve the media formulations and lower the cost. Technical problems relating to oxygen demand and cell fragility that inhibited the continued development of large-volume culture systems beyond the laboratory a few years ago now appear to be solved or at least are solvable. The successful culture of the Spodoptera cells in bioreactors of 40-liter capacity indicates that means of producing insect cells or their metabolic products on a commercial scale can be made economically feasible.

Effect of aluminum chloride and zinc sulfate on Autographa california nuclear polyhedrosis virus (ACNPV) replication in cell culture.

When IPL-SF-21AE III continuous insect cell line was grown and maintained in IPL-41 insect cell culture medium supplemented with 16 microM of AlCl3 or 0.24 microM of ZnSO4 . 7H2O, or both metallic salts, and then infected with Autographa california nuclear polyhedrosis virus, virus replication was increased significantly. The yield of polyhedral inclusion bodies (PIB) was enhanced up to 121%. Synthesis of cell-free nonoccluded virus was increased to 365% when infectivity was assayed by the plaque method. Newly applied electron microscopic quantitation and stereological techniques also revealed a significant increase in virus particles (VP) and in amount and size of PIB as well as number of VP per PIB.

Rifampin inhibition of the occluded virus form of a nuclear polyhedrosis virus.

Rifampin at concentrations toxic to noninfected cells but not to infected cells is a selective inhibitor of occluded virus of the group A Baculoviridae (nuclear polyhedrosis virus). However, the titer of nonoccluded virus is not affected. Rifampin blocks occlusion until late in the replication cycle (14 to 16 h), and its effects are reversible. Modes of action of polyhedral inclusion body production are unknown.

Quantitative measurement of oxygen consumption in insect cell culture infected with polyhedrosis virus.

A simple and reproducible quantitative method for measuring dissolved oxygen (DO) in uninfected and baculovirus infected cells in culture is described. To establish this method, an industrial DO measuring system for fermentation was employed. During this process the physical characteristics of the cell culture vessel were taken into account permitting a direct readout of DO in microliters per vessel. During these studies, it was experimentally documented that insect cells, particularly baculovirus infected cells, in culture from 1 to 14 days utilize an appreciable level of DO.

Physical Factors That Affect In Vitro Autographa californica Nuclear Polyhedrosis Virus Infection.

Of the physical parameters tested for in vitro baculovirus infection, multiplicity of infection was most important in governing percent cell infection. Most plaques formed within the first 5 min of incubation. Efficiency of infection, however, was low, and the virus titer did not diminish during prolonged incubation. Efficiency of infection improved markedly when cells or virus were preincubated with selected polyanions and polycations. Precise regulation of the pH, osmotic pressure, and ionic composition of the cell culture medium also promoted maximum in vivo infection.

Improved replication of Autographa californica nuclear polyhedrosis virus in roller bottles: characterization of the progeny virus.

A reproducible growth curve was established for the propagation of Autographa californica (Speyer) nuclear polyhedrosis virus (ACMNPV) In a continuous insect line from Spodoptera frugiperda (J. E. Smith) during large-volume production. A newly developed method for quantitation of polyhedra inclusion bodies (PIB) by electron microscopy (EM) during the growth cycle was compared to hemocytometer counts. The virus particles (VP) ratio to PFU and VP per PIB were established by EM methods. Optimal yields of PIB and cell-free virus with biological activities were obtained in six consecutive production lots when the growth curve conditions were followed. The DNA of the viruses produced in the 1st and 8th passages in the S. frugiperda cell line was treated with restriction endonucleases and compared with that from the E-2 cloned variant of this virus. Data confirmed that the virus was the ACMNPV and also indicated that there were no detectable changes after 8 passages in insect cell culture.

The establishment of two cell lines from the insect Spodoptera frugiperda (Lepidoptera; Noctuidae).

The history and characteristics of two cells lines developed from primary explants of pupal tissue from the insect, Spodoptera frugiperda (J.E. Smith), are described. One cell line, IPLB-SF 21, was developed with hemolymph-supplemented medium and has been maintained continuously on the medium. The second cell line, IPLB-SF-1254, was developed with a medium containing a combination of vertebrate sera plus hemolymph and was adapted to hemolyphn-free medium at the 6th passage. The IPLB-SF-21 cell line has a population doubling time of 26 to 30 hr; the doubling time of the IPLB-SF-1254 line is 36 hr. The chromosomal morphology and distribution was typical of other lepidopteran cell lines. Serological studies showed that both cell lines have at least one antigen which also is common is tissue antigens from pupae of Spodoptera frugiperda.

Characteristics of the non-occluded form of a nuclear polyhedrosis virus.

Non-occluded virions of a nuclear polyhedrosis virus of the alfalfa looper, Autographa californica, found in the medium of cell cultures of infected fall armyworm, Spodopter frugiperda, and in the hemolymph of infected S. frugiperda larvae were partially characterized by biological, chemical and physical methods. Also, the rate of appearance of the virions was studied in cell culture and the host insect to determine maximum virion production. Virions obtained from both sources were heat-sensitive, acid-labile and inactivated by several organic solvents. The non-occluded virions found in the insect cell culture fluid and in the hemolymph were identical, and both were enveloped nucleocapsids. Visualization of the fragilely enveloped nucleocapsid was accomplished only after fixation with glutaraldehyde. Differences between the non-occluded and occluded virions of nuclear polyhedrosis viruses are discussed.