Evolution of specialists in an experimental microcosm

The impact of adaptation on the persistence of a balanced polymorphism was explored using the lactose operon of Escherichia coli as a model system. Competition in chemostats for two substitutable resources, methylgalactoside and lactulose, generates stabilizing frequency-dependent selection when two different naturally isolated lac operons (TD2 and TD10) are used. The fate of this balanced polymorphism was tracked over evolutionary time by monitoring the frequency of fhuA-, a linked neutral genetic marker that confers resistance to the bacteriophage T5. In four out of nine chemostats the lac polymorphism persisted for 400-600 generations when the experiments were terminated. In the other five chemostats the fhuA polymorphism, and consequently the lac operon polymorphism, was lost between 86 and 219 generations. Four of 13 chemostat cultures monomorphic for the lac operon retained the neutral fhuA polymorphism for 450-550 generations until they were terminated; the remainder became monomorphic at fhuA between 63 and 303 generations. Specialists on each galactoside were isolated from chemostats that maintained the fhuA polymorphism, whether polymorphic or monomorphic at the lac operon. Strains isolated from three of four chemostats in which the lac polymorphism was preserved had switched their galactoside preference. Most of the chemostats where the fhuA polymorphism was lost also contained specialists. These results demonstrate that the initial polymorphism at lac was of little consequence to the outcome of long-term adaptive evolution. Instead, the fitnesses of evolved strains were dominated by mutations arising elsewhere in the genome, a fact confirmed by showing that operons isolated from their evolved backgrounds were alone unable to explain the presence of both specialists. Our results suggest that, once stabilized, ecological specialization prevented selective sweeps through the entire population, thereby promoting the maintenance of linked neutral polymorphisms.     

Insertion mutagenesis of the gene encoding the ferrichrome-iron receptor of Escherichia coli K-12.

The ferrichrome-iron receptor of Escherichia coli K-12 encoded by the fhuA gene is a multifunctional outer membrane receptor with an Mr of 78,000. It is required for the binding and uptake of ferrichrome and is the receptor for bacteriophages T5, T1, phi 80, and UC-1 as well as for colicin M. The fhuA gene was cloned into pBR322, and the recombinant plasmid pGC01 was mutagenized by the insertion of 6-base-pair TAB (two amino acid Barany) linkers into CfoI and HpaII restriction sites distributed throughout the coding region. A library of 18 TAB linker insertions in fhuA was generated; 8 of the mutations were at CfoI sites and 10 were at HpaII sites. All mutations inserted a hexamer that encoded a unique SacI site. A large deletion in fhuA was also isolated by TAB linker mutagenesis. Except for the deletion mutant, all of the linker insertion mutant FhuA proteins were found in the outer membrane in amounts similar to those found in the wild type. Five of the linker insertion mutants were susceptible to cleavage by endogenous proteolytic activity: a second FhuA-related band that migrated at approximately 72 kilodaltons could be detected on Coomassie blue-stained gels and on Western blots (immunoblots) by using a carboxy terminus-specific anti-peptide antibody. Receptor functions were measured with the mutated genes present in a single copy on the chromosome. Some of the receptors conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating as that of wild-type FhuA; killing by colicin M was also unaffected. Several mutants were altered in their sensitivities to the lethal agents. TAB linker insertions after amino acids 69 and 128 abolished all receptor functions. Phage T5 id not bind to these mutant FhuA proteins in detergent extracts. The deletion mutant was also defective in all FhuA functions. Sensitivity to the lethal agents of cellsl that expressed mutant FhuAs with insertions after amino acids 59 and 135 was reduced by several orders of magnitude. Insertion at other selected sites decreased some or all receptor functions only slightly. An insertion after amino acid 321 selectively eliminated ferrichrome growth promotion. Finally, a strain carrying a mutant fhuA gene on the chromosome in which the linker insertion occurred after amino acid 82 showed a tonB phenotype. These subtle perturbations that were introduced into the FhuA protein resulted in changes in its stability and in the binding and uptake of its cognate ligands.     

Second-step transfer of bacteriophage T5 DNA: purification and characterization of the T5 gene A2 protein.

Second-step transfer of bacteriophage T5 DNA requires the function of the T5 pre-early proteins A1 and A2. We have isolated and characterized the gene A2 protein as part of an effort to determine the mechanism of second-step transfer. The A2 protein was purified by DNA-cellulose column chromatography followed by gel filtration and ion-exchange column chromatography. The A2 protein's identity was confirmed by two-dimensional gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and thin-layer gel filtration in 6 M guanidine hydrochloride demonstrated a molecular weight of 15,000 for the A2 polypeptide. Migration of the A2 protein through gel filtration columns under nondenaturing conditions, in combination with sedimentation behavior, indicated dimerization of the A2 polypeptide. The existence of the A2 dimer was confirmed by protein cross-linking with dimethyl suberimidate and analysis of the cross-linked proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid composition, degree of polymerization, DNA-binding ability, and physical characteristics of the T5 gene A2 protein are consistent with a function of the A2 protein in DNA transfer.     

Polymannose O-antigens of Escherichia coli, the binding sites for the reversible adsorption of bacteriophage T5+ via the L-shaped tail fibers

A study of the adsorption kinetics of T5+ and the tail fiber-less mutant hd-2 to lipopolysaccharides of various Escherichia coli strains demonstrated T5+ binding to the O-antigen of th O8 and O9 types. Incorporation of radioactive mannose into the phosphomannose isomerase-deficient strain E. coli F860 O9 pmi allowed the derivation of the number of O-antigens per cell required to increase T5 adsorption. With more than 500 O-antigen molecules, acceleration of T5+ adsorption was observed. The highest adsorption rate was obtained when nearly all lipopolysaccharide molecules were substituted with a polymannose O-antigen. Inhibition studies with purified components of an enzymatically degraded lipopolysaccharide of the O8 type showed that among the mannosides tested the smallest unit, the trimannoside, was the strongest inhibitor of T5+ binding. We conclude that the reversible preadsorption to the O8 and O9 polymannose antigens increases the rate of infection via the cellular receptor protein encoded by the fhuA (formerly tonA) gene.     

Overproduction of the proFhuA outer membrane receptor protein of Escherichia coli K-12: isolation,properties, and immunocytochemical localization at the inner side of the cytoplasmic membrane

The fhu operon of Escherichia coli K-12 comprises four genes, termed fhuA,C,D,B, which are involved in the uptake of iron-hydroxamate compounds. The fhuA gene encodes the outer membrane receptor protein. Cells that contained three copies of the fhuACD fragment on the thermoamplifiable plasmid pHK232 accumulated at 37° C large amounts of the proFhuA protein. Most of the overproduced proFhuA protein was not translocated into the outer membrane but instead precipitated at the cytoplasmic side of the inner membrane, presumably at the sites of synthesis. Despite inhibition of export proFhuA synthesis continued.The precipitate formed was sedimented by centrifugation at 8,000xg. The proFhuA protein could be solubilized in 1% sodium dodecyl sulfate. Replacement of sodium dodecyl sulfate by Triton X-100 resulted in a proFhuA protein which exhibited 10% of the phage T5 binding activity of renatured mature FhuA protein. Binding of phage T5 was inhibited by the FhuA-specific ligands ferrichrome, albomycin and colicin M. Limited proteolysis of the isolated pro- and mature form of the FhuA protein with trypsin yielded similar oligopeptide patterns. Addition of ferrichrome affected trypsin cleavage of both proteins in the same way. The common proteolytic intermediates together with phage inactivation indicate a similar conformation of the pro- and mature form.Dedicated to Prof. G. Braunitzer on the occasion of his 60th birthday     

Internal deletions in the FhuA receptor of Escherichia coli K-12 define domains of ligand interactions.

The ferrichrome-iron receptor encoded by the fhuA gene of Escherichia coli K-12 is a multifunctional outer membrane receptor required for the binding and uptake of ferrichrome and bacteriophages T5, T1, phi 80, and UC-1 as well as colicin M. To identify domains of the protein which are important for FhuA activities, a library of 31 overlapping deletion mutants in the fhuA gene was generated. Export of FhuA deletion proteins to the outer membrane and receptor functions of the deletion proteins were analyzed. All but three of the deletion mutant FhuA proteins cofractionated with the outer membrane; no FhuA proteins were detected in outer membrane preparations or in cell extracts when the deletions spanned amino acids 418 to 440. Most deletion proteins were susceptible to cleavage by endogenous proteolytic activity; some degradation products were detected on Coomassie blue-stained gels and on Western blots (immunoblots). Receptor functions were measured with the mutated genes present on multicopy plasmids. Two deletion mutants, FhuA delta 060-069 and FhuA delta 129-168, conferred wild-type phenotypes: they demonstrated growth promotion by ferrichrome and the same efficiency of plating of bacteriophages as that of wild-type FhuA; killing by colicin M was also unaffected. For FhuA delta 021-128 and FhuA delta 406-417, reduced sensitivity to colicin M was detected; wild-type phenotypes were observed for all other FhuA functions. Deletions from amino acids 169 to 195 slightly reduced sensitivities to bacteriophages and to colicin M; ferrichrome growth promotion was unaffected. When deletions extended into the region of amino acids 196 to 405, all FhuA functions were either reduced or abolished. The results indicate that selected regions of the FhuA protein have receptor activities and demonstrate the presence of both shared and unique ligand-responsive domains.     

Ribosomes from trichomonad protozoa have prokaryotic characteristics.

1. Ribosomes from cells of the genera Trichomonas and Tritrichomonas have been isolated and characterized. The ribosomes from each organism had a sedimentation coefficient of 70S in calibrated sucrose gradients and the subunits sedimented as 50S and 30S particles under the same conditions. 2. The major ribosomal RNAs from each species were identical in size to prokaryotic ribosomal RNAs when examined by denaturing gel electrophoresis. The ribosomes contained both 5.8S and 5S RNAs. 3. The ribosomal proteins were compared by the methods of two-dimensional gel electrophoresis and reversed phase HPLC. Electrophoresis of the ribosomal proteins in two different gel systems indicated the presence of 56 proteins in T. gallinae, 40 in T. bactrachorum and 45 in the Tritrichomonas sp. The protein molecular mass range was 8.5-40 kDa. 4. The HPLC analysis confirmed the protein number established by the gel methods. 5. Both methods of analysis revealed greater similarities between the ribosomal proteins of the 2 Tritrichomonas sp. than between those of the more distantly related T. gallinae and T. bactrachorum.     

Experimental analysis of molecular events during mutational periodic selections in bacterial evolution

A fundamental feature of bacterial evolution is a succession of adaptive mutational sweeps when fitter mutants take over a population. To understand the processes involved in mutational successions, Escherichia coli continuous cultures were analyzed for changes at two loci where mutations provide strong transport advantages to fitness under steady-state glucose limitation. Three separate sweeps, observed as classic periodic selection events causing a change in the frequency of neutral mutations (in fhuA causing phage T5 resistance), were identified with changes at particular loci. Two of the sweeps were associated with a reduction in the frequency of neutral mutations and the concurrent appearance of at least 13 alleles at the mgl or mlc loci, respectively. These mgl and mlc polymorphisms were of many mutational types, so were not the result of a mutator or directed mutation event. The third sweep observed was altogether distinct and involved hitchhiking between T5 resistance and advantageous mgl mutations. Moreover, the hitchhiking event coincided with an increase in mutation rates, due to the transient appearance of a strong mutator in the population. The spectrum of mgl mutations among mutator isolates was distinct and due to mutS. The mutator-associated periodic selection also resulted in mgl and fhuA polymorphism in the sweeping population. These examples of periodic selections maintained significant genotypic diversity even in a rapidly evolving culture, with no individual "winner clone" or genotype purging the population.     

The FhuA protein is involved in microcin 25 uptake.

A chromosomal Tn5 insertion resulting in complete resistance to the peptide antibiotic microcin 25 was mapped to the min 4 region of the Escherichia coli genetic map. Additional experiments showed that the insertion disrupted the fhuA gene, which encodes the multifunctional outer membrane receptor for ferrichrome, the antibiotic albomycin, colicin M, and bacteriophages T5, T1, and phi 80. Thus, microcin 25 and all of these agents share the same receptor.     

An aspartate deletion mutation defines a binding site of the multifunctional FhuA outer membrane receptor of Escherichia coli K-12.

The FhuA protein of the outer membrane serves as a receptor for phages T5, T1, and phi 80, for colicin M, for the antibiotic albomycin, and for ferrichrome and related siderophores. To identify protein regions important for the multiple FhuA activities, fhuA genes of spontaneous chromosomal mutants which expressed wild-type amounts of the FhuA protein were sequenced. A mutant which was partially T5 sensitive but impaired in all other functions was missing aspartate residue 348 of the mature protein as a result of a three-base deletion. This aspartate residue is part of the hydrophilic sequence Asp-Asp-Glu-Lys. Replacement by site-specific mutagenesis of each of the Asp residues by Tyr, of Glu by Val, and of Lys by Met reduced FhuA activity but less than the Asp deletion did. Ferrichrome inhibited binding of phage phi 80 and of colicin M to these mutants in an allele-specific manner. A completely resistant derivative of the Asp deletion mutant contained, in addition, a leucine-to-proline substitution at position 106 and eight changed bases, converting at positions 576 to 578 an Arg-Pro-Leu sequence to Ala-Arg-Cys. The latter mutations and the Leu-to-Pro replacement alone did not alter sensitivity to the phages but reduced sensitivity to colicin M and albomycin 10- to 1,000-fold. The proline replacements probably disturb FhuA conformation and, in concert with the Asp deletion, inactivate FhuA completely. It is concluded that the Asp deletion site defines a region of FhuA which directly participates in binding of all FhuA ligands. Growth promotion studies on iron-limited media revealed that certain siderophores of the hydroxamate type, such as butylferrichrome, ferrichrysin, and ferrirubin, are taken up not only via FhuA but also via the FhuE outer membrane receptor protein.     

T-URF 13 Protein from Mitochondria of Texas Male-Sterile Maize (Zea mays L.) : Its Purification and Submitochondrial Localization, and Immunogold Labeling in Anther Tapetum during Microsporogenesis

The protein T-URF13 (URF13) is specific to mitochondria of maize (Zea mays L.) with Texas (T) male-sterile cytoplasm and has been implicated in causing male sterility and susceptibility to T-cytoplasm-specific fungal diseases. T-URF13 was purified from isolated mitochondria from maize (line B73) with T cytoplasm by gel filtration and a quasi two-dimensional polyacrylamide gel electrophoresis system. Antibodies to the purified and denatured protein were produced in rabbits. Anti-T-URF13 antiserum was used to show that T-URF13 is in the inner membrane of mitochondria and behaves as an integral membrane protein when mitochondria are fractionated with sodium carbonate or Triton X-114. The antiserum and protein A tagged with 20-nanometer-gold particles were used to localize T-URF13 in T mitochondria by electron microscopy of sections of isolated mitochondria from etiolated shoots and sections of roots and of tapetal cells at pre-and post-degeneration stages of microsporogenesis. The microscopic study confirms that T-URF13 is specifically localized in the mitochondrial membranes of all of the T mitochondria tested, notably those in the tapetum from the meiocyte stage to the late-microspore stage. No change in the amount of labeled T-URF13 protein in the mitochondria of aging tapetal cells was detected.     

Amino acid replacements in proteins S5 and S12 of two Escherichia coli revertants from streptomycin dependence to independence

Summary Ribosomes were isolated from two E. coli revertants from streptomycin dependence to independence, N660 and d1023. After separation of subunits, proteins were extracted from ribosomal 30S subunits and separated by CM-cellulose column chromatography and gel filtration. Pure S5 and S12 proteins of the two mutants were digested with trypsin and all resulting peptides were isolated by column and paper chromatography. The amino acid compositions of the peptides from the four mutant proteins were compared with the corresponding peptides of the wild type strain A19. The amino acid sequences of non-identical peptides were determined.The following amino acid replacements were found: Glycine by arginine in peptide T2 of protein S5 from mutant N660 and glycine by aspartic acid in peptide T15 of protein S12 from the same mutant. In the other mutant, d1023, arginine in peptide T2 of protein S5 was replaced by leucine and furthermore arginine by serine in peptide T10 of protein S12. Besides the single amino acid replacements mentioned above which are compatible with alterations of single nucleotides, a rather drastic difference between peptides T15 of proteins S12 isolated from strain A19 and mutant d1023 has been detected.The results presented in this paper are compared with amino acid replacements in proteins S5 and S12 from other ribosomal mutants of E. coli.Paper No. 62 on Ribosomal Proteins. Preceding paper is by Wittmann et al., Molec. gen. Genet., in press.     

Isolation and some properties of troponin T kinase from rabbit skeletal muscle.

A method for isolation of troponin T kinase (ATP-protein phosphotransferase, EC 2.7.1.37) from rabbit skeletal muscles in proposed. The method gives a 7000-10 000-fold purification and results in an enzyme with specific activity of 400-800-nmol x min-1 x mg-1 of protein. The molecular weight of tropin T kinase as determined by gel filtration exceeds 500 000. Electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate revealed that isolated preparations of the enzyme consisted of at least three distinct proteins with apparent mol.wt. of 50 000, 46 000 and 31 000. The enzyme phosphorylates isolated troponin T at a rate which exceeds the phosphorylation rates of casein, phosvitin, histones, phosphorylase b and protamine 5-30-fold. Within the whole troponin complex, only troponin T is phosphorylated by the enzyme. The enzyme phosphorylates only the N-terminal serine residue of troponin T, i.e. the site that is normally phosphorylated in the whole troponin complex isolated from rabbit skeletal muscles.     

Genome-linked protein associated with the 5' termini of bacteriophage phi29 DNA.

A DNA-protein complex was isolated from Bacillus subtilis bacteriophage phi29 by sucrose gradient sedimentation or gel filtration in the presence of agents known to break noncovalent bonds. A 28,000-dalton protein was released from this complex by subsequent hydrolysis of the DNA. The DNA-protein complex was examined for its susceptibility to enzymes which act upon the 5' and 3' termini of DNA molecules. It was susceptible to exonucleolytic degradation from the 3' termini by exonuclease III but not from the 5' termini by lambda exonuclease. Attempts to label radioactively the 5' termini by phosphorylation with T4 polynucleotide kinase were unsuccessful despite prior treatment with alkaline phosphatase or phosphatase treatment of denatured DNA. Removal of the majority of the bound protein by proteolytic digestion did not increase susceptibility. These results suggest that the linked protein is covalently attached to the 5' termini of phi29 DNA.     

Isolation of nematode inhibitor from hemolymph of the snail, Helix aspersa

Hemolymph plasma of the snail Helix aspersa which inhibits maturation and reproduction of its mantle cavity-inhabiting nematode, Rhabditis maupasi, was separated biochemically for the active proteinaceous component. Isolation of the active inhibitor was performed using ion-exchange chromatography in combination with subsequent gel filtration. The isolated peaks were bio-assayed in vitro on nematode larvae. The fractions harboring inhibitory protein suppressed larval growth and adult reproduction in vitro. The isolated fraction was purified by gel filtration and characterized on the basis of a single band on starch zone electrophoresis and positive reaction only with folin-phenol reagent.     

Phosphorylation of troponin in the heart and skeletal muscle by Ca2+-phospholipid-dependent protein kinase

The phosphorylation of the whole troponin complex and of the cardiac and skeletal troponin components by Ca2+-phospholipid-dependent protein kinase was studied. The activity of enzyme isolated from rat brain by ion-exchange chromatography on DEAE-Sephadex and by affinity chromatography on phosphatidylserine immobilized on polyacrylamide gel was shown to be completely dependent on Ca2+ and phospholipids and was equal to 0.4-0.6 mumol of phosphate/min.mg protein with histone H1 as substrate. The resulting preparation of Ca2+-phospholipid-dependent protein kinase was able to phosphorylate the isolated troponin I; the amount of phosphate transferred per mol of cardiac and skeletal troponin I was equal to 1.1 and 0.4, respectively. The maximal degree of phosphorylation of isolated troponin T by Ca2+-phospholipid-dependent protein kinase was 0.6 mol of phosphate per mol of troponin T both for skeletal and cardiac proteins. The rate and degree of phosphorylation were independent of the initial level of troponin T phosphorylation. Ca2+-phospholipid-dependent protein kinase did not phosphorylate the first serine residue of troponin T, i.e., the site which was phosphorylated in the highest degree after isolation of troponin T from skeletal muscles. The data obtained and the fact that the rate and degree of phosphorylation of troponins I and T within the whole troponin complex are 10-20 times less than those for isolated components provide little evidence for the participation of protein kinase C in troponin phosphorylation in vivo.