Outer membrane proteins of Escherichia coli K-12: Isolation of a common receptor protein for bacteriophage T6 and colicin K

Summary tsx mutants, resistant to T6-like bacteriophages and colicin K, of Escherichia coli K-12 lack an outer membrane protein of 26,000 molecular weight. This protein is shown to have receptor activity for both bacteriophage T6 and colicin K. The protein has been purified and its amino acid composition determined. Some tsx mutants appear to have an altered receptor protein, as indicated by their ability to plate extended host-range mutants of bacteriophage T6. These mutants are also cotransducible with proC and can be arranged in an order of increasing resistance to the host range phages, which appear to have differing degrees of ability to propagate on the tsx mutants.The tsx protein was shown to be catabolite repressible both by use of varying growth conditions and cya and crp mutants.     

Recent Advances in GFP Folding Reporter and Split-GFP Solubility Reporter Technologies. Application to Improving the Folding and Solubility of Recalcitrant Proteins from Mycobacterium tuberculosis

We have improved our green fluorescent protein (GFP) folding reporter technology [Waldo et al., (1999) Nat. Biotechnol. 17, 691–695] to evolve recalcitrant proteins from Mycobacterium tuberculosis. The target protein is inserted into the scaffolding of the GFP, eliminating false-positive artifacts caused by expression of truncated protein variants from internal cryptic ribosome binding sites in the target RNA. In parallel, we have developed a new quantitative fluorescent protein tagging and detection system based on micro-domains of GFP. This split-GFP system, which works both in vivo and in vitro, is amenable to high-throughput assays of protein expression and solubility [Cabantous et al., (2005) Nat. Biotechnol. 23, 102–107]. Together, the GFP folding reporter and split-GFP technologies offer a comprehensive system for manipulating and improving protein folding and solubility.     

Selection for increased percentage phaseolin in common bean

Summary Two selection methods were compared to determine which was more efficient for increasing percentage phaseolin in the common bean (Phaseolus vulgaris L.). A base population consisting of families segregating for six seed protein alleles (Phas ^S , Phas ^C , Phas ^T , phas ^-, lec^-, and Arcl ⁺), all of which have measurable effects on percentage phaseolin, was subjected to either three cycles of S₁ family recurrent selection for increased percentage phaseolin (PPS), or one cycle of selection for combinations of the protein alleles (PAS) known to have positive effects on phaseolin accumulation. One cycle of PAS resulted in an increase in percentage phaseolin that was equivalent to three cycles of PPS. Selection under both methods produced increases in several correlated traits including percentage total protein, phaseolin as a percent of total protein, mg protein/seed, and mg phaseolin/seed. The amount of nonphaseolin protein per seed decreased, while seed yield was unaffected by either selection procedure. By selecting for favorable seed protein alleles identified by electrophoresis, it was possible to rapidly increase percentage phaseolin without the need for field evaluation.     

The product of the Drosophila melanogaster segment polarity gene armadillo is highly conserved in sequence and expression in the housefly Musca domestica

Summary Segmental pattern in Drosophila melanogaster is set up via a set of cell-cell interactions mediated by the products of the segment polarity genes. Among these is the armadillo gene, whose product seems to be required for the reception of an intercellular signal encoded by the wingless gene. As part of our effort to relate the structure of the armadillo protein to its function within the cell, we have examined the evolutionary conservation of the armadillo gene during insect evolution. We have cloned the armadillo gene from the housefly, Musca domestica, which diverged from Drosophila 100 million years ago. The Musca protein is 97.5% identical to that in Drosophila, while the noncoding sequences have diverged extensively. This remarkable degree of conservation at the protein level is mirrored in the expression pattern of the armadillo protein. Antibodies against the Drosophila protein cross-react with a Musca protein of the appropriate size. We have also used these antibodies to show that the Musca armadillo protein has a pattern of expression in larval and adult tissues similar to that of Drosophila armadillo. We discuss the implications of conservation of structure and expression for the cellular role of the armadillo protein and its mammalian homologs.Offprint requests to: M. Peifer     

The snowdrop lectin Galanthus nivalis agglutinin (GNA) and a fusion protein ButaIT/GNA have a differential affect on a pest noctuid Lacanobia oleracea and the ectoparasitoid Eulophus pennicornis

Fusion proteins have considerable potential as novel insect control agents because they enable the oral delivery of insecticidal peptides to the haemolymph of pests. Transport is achieved via fusion of the toxin to a carrier protein Galanthus nivalis agglutinin (GNA) that, after ingestion, binds to and crosses the insect gut epithelia. A fusion protein comprising a toxin from the South Indian red scorpion (Mesobuthus tamulus) that is fused to a GNA polypeptide (ButaIT/GNA) has a detrimental effect on the development of tomato moth Lacanobia oleracea (L.) (Lepidoptera: Noctuidae) larvae. The present study examines the effects of ButaIT/GNA and GNA, delivered orally or by injection, on the development of L. oleracea larvae, and the subsequent effects on the gregarious ectoparasitoid Eulophus pennicornis (Nees) (Hymenoptera: Eulophidae) developing on ButaIT/GNA‐ and GNA‐treated hosts. The fusion protein, but not GNA, reduces the growth of fifth stadium L. oleracea larvae. The development of E. pennicornis is not affected by the presence of ButaIT/GNA in hosts that ingest the protein, although it is affected when hosts are injected with the protein. This difference is considered to be a result of higher levels of fusion protein being present when the fusion protein is injected. Intact ButaIT/GNA is detected by immunoassay in the haemolymph of L. oleracea larvae after ingestion of the fusion protein. More unexpectedly, negative effects are observed for the growth of E. pennicornis larvae developing on hosts that have either ingested, or been injected with GNA.     

Subunit dissociation and metal binding by Escherichia coli apo-manganese superoxide dismutase

Metal binding by apo-manganese superoxide dismutase (apo-MnSOD) is essential for functional maturation of the enzyme. Previous studies have demonstrated that metal binding by apo-MnSOD is conformationally gated, requiring protein reorganization for the metal to bind. We have now solved the X-ray crystal structure of apo-MnSOD at 1.9 Å resolution. The organization of active site residues is independent of the presence of the metal cofactor, demonstrating that protein itself templates the unusual metal coordination geometry. Electrophoretic analysis of mixtures of apo- and (Mn₂)-MnSOD, dye-conjugated protein, or C-terminal Strep-tag II fusion protein reveals a dynamic subunit exchange process associated with cooperative metal binding by the two subunits of the dimeric protein. In contrast, (S126C) (SS) apo-MnSOD, which contains an inter-subunit covalent disulfide-crosslink, exhibits anti-cooperative metal binding. The protein concentration dependence of metal uptake kinetics implies that protein dissociation is involved in metal binding by the wild type apo-protein, although other processes may also contribute to gating metal uptake. Protein concentration dependent small-zone size exclusion chromatography is consistent with apo-MnSOD dimer dissociation at low protein concentration (K_D = 1 × 10⁻⁶ M). Studies on metal uptake by apo-MnSOD in Escherichia coli cells show that the protein exhibits similar behavior in vivo and in vitro.     

Novel class of nuclear genes involved in both mRNA splicing and protein synthesis in Saccharomyces cerevisiae mitochondria

Summary We have cloned three distinct nuclear genes, NAM1, NAM7, and NAM8, which alleviate mitochondrial intron mutations of the cytochrome b and COXI (subunit I of cytochrome oxidase) genes when present on multicopy plasmids. These nuclear genes show no sequence homology to each other and are localized on different chromosomes: NAM1 on chromosome IV, NAM7 on chromosome XIII and NAM8 on chromosome VIII. Sequence analysis of the NAM1 gene shows that it encodes a protein of 440 amino acids with a typical presequence that would target the protein to the mitochondrial matrix. Inactivation of the NAM1 gene by gene transplacement leads to a dramatic reduction of the overall synthesis of mitochondrial protein, and a complete absence of the COXI protein which is the result of a specific block in COXI pre-mRNA splicing. The possible mechanisms by which the NAM1 gene product may function are discussed.     

<Emphasis Type="Italic">Mucor rouxii</Emphasis> Rho1 protein; characterization and possible role in polarized growth

We have previously shown that protein kinase A of the medically important zygomycete Mucor rouxii participates in fungal morphology through cytoskeletal organization. As a first step towards finding the link between protein kinase A and cytoskeletal organization we here demonstrate the cloning of the Rho1 gene and the characterization of its protein product. The RHO1 protein primary sequence shows 70–85% identity with fungal RHO1 or mammalian RhoA. Two protein kinase A phosphorylation sequences in adequate context are predicted, Ser73 and Ser135. The peptide IRRNSQKFV, containing Ser135 proved to be a good substrate for M. rouxii protein kinase A catalytic subunit. The over-expressed Rho1 fully complements a Saccharomyces cerevisiae null mutant. The endogenous protein was identified by western blot against a developed antibody and by ADP-ribosylation. Localization in germlings was visualized by immunofluorescence; the protein was localized in patches in the mother cell surface and excluded from the germ tube. Measurement of Rho1 expression during germination indicates that Rho1, at both the mRNA and protein levels, correlates with differentiation and not with growth. Rho1 has been shown to be the regulatory protein of the β-1,3-glucan synthase complex in fungi in which β-1,3-glucans are major components of the cell wall. Even though glucans have not been detected in zygomycetes, caspofungin, an echinochandin known to be an inhibitor of β-1,3-glucan synthase complex, is shown here to have a negative effect on growth and to produce an alteration on morphology when added to M. rouxii growth culture medium. This result has an important impact on the possible participation of β-1,3-glucans on the regulation of morphology of zygomycetes.     

The comparison of seed protein patterns within the genusArachis by polyacrylamide gel electrophoresis

The seed protein patterns of 12Arachis species were compared by polyacrylamide gel electrophoresis (PAGE), similarities between patterns were measured by the Jaccard index. Results obtained confirm the close relationships established between members of the genus on morphological grounds and support the more recent classification schemes.A. villosa andA. correntina could well be regarded as distinct species on grounds of protein differences whileA. macedoi andA. villosulicarpa (although members of the same section, Extranervosae) show considerable differentiation of their protein patterns. Surprisingly, the formA. ×batizogaea showed less similarity in protein pattern to those of its parental species than might have been expected. The principle value of seed protein pattern data appears to be in distinguishing species within sections.     

Loss of rac locus DNA in merozygotes of Escherichia coli K12.

Summary Two genes governing ribosomal protein methylation have been located on the map of Escherichia coli by conjugation and transduction crosses between wild-type and prm (protein methylation) mutants. The Prm phenotype of recombinants was determined by an in vitro assay of methylgroups incorporation into protein.Gene prmA, governing methylation of protein L11 is situated at minute 71 on the map and is cotransduced with aroE (30%) and with rpsL (5%). Gene prmB, governing methylation of protein L3 is at minute 50, very close to aroC (98.5% co-transduction). A cold-sensitive phenotype was found associated with mutation prmB and was used to score a large number of recombinants in a three factor cross. The results of this cross suggest the order aroC-prmB-purF.The striking symmetrical clustering of aro, prm and rim (ribosome maturation) genes is discussed.     

MappingTNNC1, the Gene That Encodes Cardiac Troponin I in the Human and the Mouse

We have mapped the TNNC1 gene, whose protein product is the cardiac TnI protein. TnI is one of the proteins that makes up the troponin complex, which mediates the response of muscle to calcium ions. The human TNNC1 locus had been assigned to a large region of chromosome 19, and we have refined the mapping position to the distal end of the chromosome by amplification of DNAs from a chromosome 19 mapping panel. We have also mapped the mouse Tnnc1 locus, by following the segregation of an intron sequence through DNAs from the European Interspecific Backcross. Tnnc1 maps close to the centromere on mouse chromosome 7.     

Studies on the possible role of protein phosphorylation in the transduction of the ethylene signal

Previous work in our laboratory has demonstrated the existence of high affinity binding sites for the plant growth regulator ethylene. The ethylene binding protein (EBP), from Phaseolus cotyledons, shows many of the characteristics of a functional receptor for ethylene, has been purified on SDS-PAGE and polyclonal antibodies raised in rabbits. Current work involves the investigation of the ethylene transduction signal in a number of ethylene-responsive tissues.In peas, it has been shown that ethylene promotes the phosphorylation of specific proteins of similar molecular weight to the EBP from Phaseolus. Such ethylene-induced phosphorylation can be inhibited by the ethylene antagonist, 2,5-NBD. The antibodies raised to the EBP from Phaseolus have been shown to immunoprecipitate ³²P-labelled proteins from membrane protein preparations obtained from pea tissue. Studies on ethylene binding in pea have also shown that the binding of ethylene may be regulated by phosphorylation. Thus, under conditions which promote phosphorylation, binding is inhibited, whereas the reverse is true under conditions which enhance dephosphorylation.Further work is described which examines the effect of protein kinase, protein phosphatase and calcium channel inhibitors on ethylene-induced phosphorylation in peas, together with wild-type (WT) and ethylene insensitive (eti) mutants of Arabidopsis thaliana. The effects of these treatments can be monitored in vivo using the ethylene-induced triple response as a screen. Furthermore, the protein profiles of such treated seedlings can then be compared by labelling protein extracts with ³²P and subjecting the samples to SDS-PAGE followed by autoradiography.     

Enhanced recombinant protein production in pyruvate kinase mutant of Bacillus subtilis

Previous work demonstrated that acetate production was substantially lower in pyruvate kinase (pyk) mutant of Bacillus subtilis. The significantly lower acetate production in the pyk mutant is hypothesized to have positive effect on recombinant protein production either by lifting the inhibitory effect of acetate accumulation in the medium or redirecting the metabolic fluxes beneficial to biomass/protein synthesis. In this study, the impact of the pyk mutation on recombinant protein production was investigated. Green fluorescent protein (GFP+) was selected as a model protein and constitutively expressed in both the wild-type strain and a pyk mutant. In batch cultures, the pyk mutant produced 3-fold higher levels of recombinant protein when grown on glucose as carbon source. Experimental measurements and theoretical analysis show that the higher protein yield of the mutant is not due to removal of an acetate-associated inhibition of expression or gene dosage or protein stability but a much lower acetate production in the mutant allows for a greater fraction of carbon intake to be directed to protein synthesis.     

The AtPP gene of the Brassica napus S locus region is specifically expressed in the stigma and encodes a protein similar to a methyltransferase involved in plant defense

Self-incompatibility (SI) in Brassica is controlled by the S locus. The specificity of the SI response is controlled on the stigma side by the S receptor kinase (SRK) and on the pollen side by the SCR (S locus cysteine-rich) protein, but other proteins might be involved in the process of self-pollen rejection. In this study, we show that the AtPP gene linked to the S locus of Brassica napus is expressed in the stigmas of SI lines. AtPP has a developmental pattern of expression similar to the SRK gene. The AtPP protein has similarity with members of an Arabidopsis protein family and with an S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase, which is a plant defense-related protein of Clarkia breweri representing a new class of methyltransferases. A member of the AtPP gene family is present in the homeolog region of the S locus in Arabidopsis. Therefore, this gene might have co-evolved with S genes from an ancestral S locus of Brassicaceae. Possible functions of the AtPP protein in the self-recognition process are discussed. Received: 9 October 2000 / Revision accepted: 23 April 2001     

Networking with mitogen-activated protein kinases

Mitogen activated protein (MAP) kinases and their target ribosomal protein S6 (RSK) kinases have been recognized as shared components in the intracellular signaling pathways of many diverse cytokines. Recent studies have extended this protein kinase cascade by identifying the major activator of vertebrate MAP kinases as a serine/threonine/tyrosine-protein kinase called MEK, which is related to yeast mating factor-regulated protein kinases encoded by the STE7 and byr1 genes. MEK, in turn, may be activated following its phosphorylation on serine by either of the kinases encoded by proto-oncogenesraf1 ormos, as well as by p78 ^mekk , which is related to the yeast STE11 and byr2 gene products. Isoforms of all of these protein kinases may specifically combine to assemble distinct modules for intracellular signal transmission. However, the fundamental architecture of these protein kinase cascades has been highly conserved during eukaryotic evolution.     

Effect of phosphate on antibiotic and extracellular protein production by Myxococcus coralloides D

Summary The effect of inorganic phosphate concentrations on antibiotic and extracellular protein production by Myxococcus coralloides D have been examined. Antibiotic production by growing cells of this myxobacterium was maximal at phosphate concentrations of 10–20 mM, but was inhibited by concentrations higher than 20 mM. The total extracellular protein and the extracellular protein per cell ratio were independent of phosphate levels in the culture broth. Offprint requests to: J. M. Arias     

The ukc1 gene encodes a protein kinase involved in morphogenesis, pathogenicity and pigment formation in Ustilago maydis

The fungal phytopathogen Ustilago maydis alternates between budding and filamentous growth during its life cycle. This dimorphic transition, which is influenced by environmental factors and mating, is regulated in part by cAMP-dependent protein kinase (PKA). We have recently identified a related protein kinase, encoded by the ukc1 gene, that also plays a role in determining cell shape. The ukc1 gene is homologous to several other protein kinase-encoding genes including the cot-1 gene of Neurospora crassa, the TB3 gene of Colletotrichum trifolii, the orb6 gene of Schizosaccharomyces pombe, the warts tumor suppressor gene of Drosophila melanogaster and the myotonic dystrophy kinase gene in humans. Disruption of the ukc1 gene in U. maydis resulted in cells that were highly distorted in their morphology, incapable of generating aerial filaments during mating in culture and defective in their ability to cause disease on corn seedlings. In addition, the cells of ukc1 mutants became highly pigmented and resembled the chlamydospore-like cells that have been described for U. maydis. Overall, these results demonstrate an important role for the ukc1-encoded protein kinase in the morphogenesis, pathogenesis and pigmentation of U. maydis. Received: 6 May 1998 / Accepted: 19 November 1998     

A chimeric gene (orf256) is expressed as protein only in cytoplasmic male-sterile lines of wheat

Mitochondria derived from Triticum timopheevi have a chimeric gene, orf256, immediately upstream from coxI. Antibodies to a peptide corresponding to a part of the encoded amino acid sequence of orf256 detect a 7 kDa protein on western blots of mitochondrial proteins from cytoplasmic male-sterile (cms) wheat (T. aestivum nucleus, T. timopheevi mitochondria) but not in mitochondrial proteins from T. aestivum, T. timopheevi, or cms plants restored to fertility by introduction of nuclear genes for fertility restoration. The 7 kDa protein appears to serve as a marker for cms wheat. Its occurrence as an integral protein of the inner membrane may indicate a cms effect through an influence on mitochondrial membrane function.