Isolation and characterization of OmpC porin mutants with altered pore properties.

The LamB protein is normally required for the uptake of maltodextrins. Starting with a LamB- OmpF- strain, we have isolated mutants that will grow on maltodextrins. The mutation conferring the Dex+ phenotype in the majority of these mutants has been mapped to the ompC locus. These mutants, unlike LamB- OmpF- strains, grew on maltotriose and maltotetraose, but not on maltopentaose, and showed a significantly higher rate of [14C]maltose uptake than the parent strain did. In addition, these mutants showed increased sensitivity to certain beta-lactam antibiotics and sodium dodecyl sulfate, but did not exhibit an increase in sensitivity to other antibiotics and detergents. The nucleotide sequence of these mutants has been determined. In all cases, residue 74 (arginine) of the mature OmpC protein was affected. The results suggest that this region of the OmpC protein is involved in the pore domain and that the alterations lead to an increased pore size.     

Isolation of mutants in M13 coat protein that affect its synthesis, processing, and assembly into phage

The major coat protein (gene 8 protein) of bacteriophage M13 has been studied intensively as a model of membrane assembly, protein packing, and protein-DNA interactions. Because this protein is essential for assembly of the phage, very few mutants have been isolated. We have therefore cloned the gene 8 into a plasmid under control of the araB promoter. In the presence of arabinose, the cloned gene is expressed at a rate comparable to that in an M13-infected cell. Plasmid-derived procoat is inserted across the plasma membrane and processed to coat at a normal rate. The coat can support plaque formation by a defective M13 virus (M13am8) with an amber mutation in its procoat gene. This complementation assay was used to screen the mutagenized, cloned gene 8 for mutants which fail to make fully functional coat. Mutants were obtained which fail to synthesize procoat, which do not convert procoat to mature coat protein, or in which the coat protein is incapable of assembling into infectious virions.     

Crystal structure of the potato leafroll virus coat protein and implications for viral assembly

Luteoviruses, poleroviruses, and enamoviruses are insect-transmitted, agricultural pathogens that infect a wide array of plants, including staple food crops. Previous cryo-electron microscopy studies of virus-like particles show that luteovirid viral capsids are built from a structural coat protein that organizes with T = 3 icosahedral symmetry. Here, we present the crystal structure of a truncated version of the coat protein monomer from potato leafroll virus at 1.80-Å resolution. In the crystal lattice, monomers pack into flat sheets that preserve the two-fold and three-fold axes of icosahedral symmetry and show minimal structural deviations when compared to the full-length subunits of the assembled virus-like particle. These observations have important implications in viral assembly and maturation and suggest that the CP N-terminus and its interactions with RNA play an important role in generating capsid curvature.     

Escherichia coli K12 arabinose-binding protein mutants with altered transport properties.

The arabinose-binding protein (ABP) of Escherichia coli binds L-arabinose in the periplasm and delivers it to a cytoplasmic membrane complex consisting of the AraG and AraH proteins, for uptake into the cell. To study the interaction between the soluble and membrane components of this periplasmic transport system, regions of the ABP surface containing the opening of the arabinose-binding cleft were subjected to site-directed mutagenesis. Thirty-eight ABP variants containing one to three amino acid substitutions were recovered. ABP variants were expressed with wild-type AraG and AraH from a plasmid, in a strain lacking the chromosomal araFGH operon, and the whole cell uptake parameters, Ven (maximum initial velocity of arabinose entry) and K(en) (concentration of arabinose yielding half-maximal entry) were determined. Twenty-four mutants had normal Ven values, 3 mutants had Ven and K(en) values twice wild type, and 11 mutants had Ven and K(en) values 20-50% of wild type. Binding proteins that had altered uptake properties were each expressed, processed, and localized to the periplasm at levels equivalent to wild type. The mutant binding proteins behaved the same as wild type during purification, and each had a Kd (dissociation constant for bound arabinose) comparable to that of wild-type ABP. Mutations that resulted in altered uptake identified nine amino acids surrounding the arabinose-binding cleft, all of which are charged in the wild-type protein, and all of whose side chains project outward from the cleft. The evidence suggests that this surface of the binding protein and these nine charged loci play a major role in ABP interactions with the membrane complex.     

Isolation of Vibrio cholerae mutants with altered toxin production

V. cholerae multiple-labeled mutants 569B with altered toxin production have been obtained by the method of induced mutagenesis with the use of nitrosoguonidine. These mutants can be used for the genetic mapping of tox genes on the chromosome of V. cholerae.     

Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter

A new method based on the toxicity of low intracellular pH (pHi) was developed to isolate fibroblast variants overexpressing Na+/H+ antiport activity. Chinese hamster lung fibroblasts (CCL39) were incubated for 60 min in medium containing 50 mM NH4Cl. Removal of external NH+4 induced a rapid and lethal intracellular acidification when the Na+/H+ antiporter was inhibited during the 60 min of the pHi recovery phase. The inhibition was provoked either by adding 5-(N-methyl,N-propyl)amiloride (MPA, LD50 = 0.3 microM) or by reducing external [Na+] (LD50 = 25 mM). Progressively increasing the MPA concentration during the acid-load selection led to the isolation of two stable variants: AR40 and AR300, resistant, respectively, to 40 and 300 microM MPA. In response to an acid-load, these variants display a much higher rate of pHi recovery due to an overexpression of Na+/H+ antiport activity. In addition, AR40 and AR300 have an altered Na+/H+ antiporter: in AR300 cells K0.5 of MPA for inhibiting Na+/H+ exchange is shifted from 5 X 10(-8) to 1.5 X 10(-6) M, Km (Na+) is decreased 2-fold, and Vmax is increased 4.5-fold. Alternatively reducing Na+ concentration of the pHi recovery saline medium in a stepwise manner led to the selection of another class of variants (DD8 and DD12) also characterized by an altered Na+/H+ antiporter and an increased expression level. The 10-fold increased rate of amiloride-sensitive Na+ influx of DD12 is accounted for by a 4-fold increase in Vmax and a 2.5-fold increase in affinity for Na+ or Li+ at the external site. Interestingly, the affinity for the amiloride analog MPA and for external H+ is unchanged in DD12. In conclusion, the genetic approach presented here: provides a general and specific method for selecting variants of the Na+/H+ antiporter with increased expression levels and/or with structural alterations and demonstrates that the external Na+- and amiloride-binding sites are not identical, since they can be genetically altered independently of each other.     

Isolation and characterization of 5-fluorotryptophan-resistant mutants with altered L-tryptophan transport.

Mutants of A9 mouse fibroblast, resistant to the killing effect of 0.4 mM 5-flurotryptophan (5-FT), have altered L-tryptophan transport properties. The resistant phenotype is stable for at least 90 generations of growth in MEM. A fluctuation test indicated that clones resistant to 0.4 mM 5-FT occurred spontaneously. An average mutation rate was estimated at 1.6 X 10(-6). Treatment with N-methyl-N'-nitro-N-nitrosoguanidine increased the frequency of these clones by at least 100-fold. These results indicate that the resistant clones arose as a result of a mutation. All the resistant mutant tested accumulate less 5-FT at near steady-state conditions than the wild type. Lineweaver-Burk plots of initial rates of tryptophan uptake yield a biphasic curve suggesting that tryptophan is transported by two transport systems. Kinetic constants determined by a computer program indicate that both proposed transport systems were modified in each of two 5-FT resistant mutants.     

Isolation and characterization of CHO cell mutants with altered asparagine synthetase.

Two asparagine auxotrophic mutants (N3, N4) were isolated from the Gat- strain of Chinese hamster ovary cells, using a selection procedure modified from that of Goldfarb et al. (1). The defect in these mutants is due to a deficiency in asparagine synthetase activity. N3, in particular, had no measurable enzyme activity. Complementation analysis by PEG-mediated cell fusion showed that the auxotrophic phenotype behaved as a recessive trait; complementation was obtained between N3 or N4 and the pseudoauxotroph, Asn3, which has a temperature-sensitive asparagyl-tRNA synthetase activity. Revertants obtained by plating N3 or N4 in asparagine-free medium had about normal levels of asparagine synthetase activity and were produced with a probability of about 10(-6) per cell per generation. Three particular revertants of N3 and one revertant of N4 were shown to have asparagine synthetase activities that were different in thermolability from that of the wild type. This observation is consistent with the suggestion that N3 and N4 have defective structural genes rather than defective regulatory genes for asparagine synthetase.     

Isolation and properties of Escherichia coli ATPase mutants with altered divalent metal specificity for ATP hydrolysis

A method was devised for isolation of large numbers of energy-transducing ATPase (coupling factor) mutants based on a modification of the procedure of Hong and Ames (Hong, J. and Ames, B. N. (1971) Proc. Natl. Acad. Sci. U.S. 68, 3158–3162) for localized mutagenesis of any small region of the bacterial chromosome using transducing phages. The principle of this procedure is to mutate P1-transducing phage particles carrying the ATPase genes (Unc (uncoupled) DNA) using the strong chemical mutagen hydroxylamine. By transducing ilv^? auxotrophs, a marker closely linked to Unc, to prototrophs, mutated Unc DNA can be introduced into the chromosome. We have used this method in conjunction with suitable selection procedures to isolate about 90 Unc^? strains which have been classified by physiological, genetic, and biochemical criteria into three different phenotypes (Unc A, B, D). Mutants of the Unc D phenotype which were studied in detail were found to have the following properties: (1) aerobic growth yields on glucose are considerably lower than the wild type; growth occurs on glucose under anaerobic conditions; (2) Unc D lesions map near the ilv operon; (3) O₂ uptake is comparable to the rate of wild type; (4) vesicles catalyze respiratory-dependent transhydrogenation, but show very low levels of Ca²⁺ ATP-dependent transhydrogenation; Mg²⁺ is ineffective; (5) oxidative phosphorylation is almost completely blocked irrespective of which metal ion is used; (6) the specific activity of ATPase is only about 20% of the wild type; (7) purified ATPase was found to have a marked specificity for Ca²⁺ as a divalent metal for ATP hydrolysis. A summary of properties of the new Unc mutants is discussed.     

Properties of Bacillus cereus temperature-sensitive mutants altered in spore coat formation.

Three conditional Bacillus cereus mutants altered in the assembly or formation of spore coat layers were analyzed. They all grew as well as the wild type in an enriched or minimal medium but produced lysozyme and octanol-sensitive spores at the nonpermissive temperature (35 to 38 degrees C). The spores also germinated slowly when produced at 35 degrees C. Temperature-shift experiments indicated that the defective protein or regulatory signal is expressed at the time of formation of the outer spore coat layers. Revertants regained all wild-type spore properties at frequencies consistent with initial point mutations. Spore coat defects were evident in thin sections and freeze-etch micrographs of mutant spores produced at 35 degrees C. In addition, one mutant contained an extra surface deposit, perhaps unprocessed spore coat precursor protein. A prevalent band of about 65,000 daltons (the same size as the presumptive precursor) was present in spore coat extracts of this mutant and may be incorrectly processed to mature spore coat polypeptides. Another class of mutants was defective in the late uptake of half-cystine residues into spore coats. Such a defect could lead to improper formation of the outer spore coat layers.     

Aggregation and assembly of phage P22 temperature-sensitive coat protein mutants in vitro mimic the in vivo phenotype

Aggregation is a common side reaction in the folding of proteins which is likely due to inappropriate interactions of folding intermediates. In the in vivo folding of phage P22 coat protein, amino acid substitutions that cause a temperature-sensitive-folding (tsf) phenotype lead to the localization of the mutant coat proteins to inclusion bodies. Investigated here is the aggregation of wild-type (WT) coat protein and 3 tsf mutants of coat protein. The tsf coat proteins aggregated when refolded in vitro at high temperature. If the tsf coat proteins were refolded at 4 degrees C, they were able attain an assembly active state. WT coat protein, on the other hand, did not aggregate significantly even when folded at high temperature. The refolded tsf mutants exhibited altered secondary and tertiary structures and had an increased surface hydrophobicity, which may explain the increased propensity of their folding intermediates to aggregate.     

Spore coat is altered in modB glycosylation mutants of Dictyostelium discoideum.

The modB mutation eliminates specific carbohydrate epitopes from glycoproteins which are expressed primarily in prespore and spore cells of differentiating Dictyostelium discoideum. Spores formed by the mutant show several phenotypes. Whereas mutant spores germinate efficiently after heat activation, they germinate poorly after urea activation. Following germination, at least one glycosylation-defective glycoprotein is cleaved, and the larger fragment is released in soluble form from the spore coat. However, an earlier difference in the spore coat can be traced back to the nongerminated spore coat, as detected by the elutability of protein from intact spores by chemical extraction. An altered character of the pregermination spore coat is also suggested by increased labeling by a fluorescent lectin which binds to its interior. The findings are consistent with a change in the character of certain molecular contacts leading to altered characteristics of the mutant spore coat, which are specific because they are distinctive from changes observed in another glycosylation mutant which affects a different epitope.     

Development and characterization of green fluorescent protein mutants with altered lifetimes

Multiple-probe fluorescence imaging applications demand an ever-increasing number of resolvable probes, and the use of fluorophores with resolvable fluorescence lifetimes can help meet this demand. Green fluorescent protein (GFP) and its variants have been widely used in spectrally resolved multiprobe imaging, but as yet, there has not been a systematic set of mutants generated with resolvable lifetimes. Therefore, to generate such mutants, we have utilized error-prone PCR and fluorescence lifetime imaging to screen for mutants of UV-excited green fluorescent protein (GFPuv) that exhibit altered fluorescence decay lifetimes. This has resulted in the isolation of GFPuv mutants displaying at least three distinctly different lifetimes in the range of 1.9-2.8 ns. Mutation of Y145 to either histidine or cysteine was found to shift the fluorescence lifetime of GFPuv from 3.03 +/- 0.03 to 2.78 +/- 0.05 ns for the Y145H mutant and to 2.74 +/- 0.05 ns for Y145C. Some of the shorter-lifetime mutants exhibited excitation peaks that were red-shifted relative to their maximal absorption, indicating that the mutations allowed the adoption of additional conformations relative to wtGFPuv. The utility of these mutants for applications in simultaneous imaging and quantification is shown by the ability to quantify the composition of binary mixtures in time-resolved images using a single detector channel. The application of the screening method for generating lifetime mutants of other fluorescent proteins is also discussed.     

Spectinomycin-resistant mutants of Bacillus subtilis with altered sporulation properties.

Summary Specitinomycin-resistant mutants of Bacillus subtilis show three different types of alterations in sporulation ability. Class 1 mutants can both grow and sporulate in the presence of spectinomycin. Class 2 mutants can grow in the presence of spectinomycin, but are unable to sporulate in either the presence or absence of spectinomycin. Class 3 mutants have a conditional phenotype, and are able to sporulate in the absence of spectinomycin, but not in its presence. The ability of these strains to produce alkaline phosphatase, a biochemical marker for early sporulation events, is correlated with the ability to sporulate in the presence or absence of antibiotic. All of the spectinomycin-resistance mutations could be genetically linked to the cysA marker, and a mutational alteration of a protein of the 30S ribosomal subunit has been identified in one of the Class 3 strains (Spc1–11). Fine-structure mapping of the spectinomycin resistance mutation of strain Spc 1–11 confirmed its location in the cluster of genes for ribosomal components on the B. subtilis genetic map. Genetic analysis indicated that the properties of the Class 1 and Class 2 mutants result from more than one mutation. The spectinomycin-resistance and altered sporulation properties of the two Class 3 mutants probably result from a single genetic lesion.     

Isolation and characterization of Bacillus subtilis mutants altered in competence.

We isolated and characterized four Bacillus subtilis competence-deficient mutants. The mutants were obtained by nitrosoguanidine mutagenesis and by screening for mutants unable to be transformed both on solid and in liquid medium. Most of the mutants obtained in this way were tested for their sensitivity to the DNA-damaging agents methyl methanesulfonate, mitomycin C, and UV light. Among the mutants which did not show an increased sensitivity to these agents, four were chosen for further characterization. Data were obtained which indicate that the mutants are reduced in chromosomal and plasmid transformation and in transfection, whereas they are not altered in transduction and in protoplast transformation. Transformation experiments carried out by mixing a culture of a mutant with a culture of a wild-type strain gave some complementation for competence with one of the strains. The mutants were also characterized for their capacity to bind, take up, and break down transforming DNA; furthermore, the four competence mutations were mapped, and the results indicate that they belong to four different genes.