Deletion of the prc (tsp) gene provides evidence for additional tail-specific proteolytic activity in Escherichia coli K-12

The Escherichia coli protease Prc (Tsp) exhibits specificity in vitro for proteins with nonpolar carboxyl termini. To determine whether Prc is responsible for the selective degradation in vivo of proteins with nonpolar carboxyl termini, we constructed a prc (tsp) deletion strain. Deletion of the prc gene did not prevent the rapid intracellular degradation of a variant of the amino-terminal domain of λ repressor with a nonpolar carboxyl terminus, even though this protein is a substrate for Prc in vitro. Our results indicate that at least one additional carboxy-terminal-specific proteolytic system must exist in E. coli.     

ThegrpD55 locus ofEscherichia coli appears to be an allele ofdnaB

Attempts to characterize thegrpD55 mutation ofEscherichia coli have led us to conclude that the gene had been assigned an incorrect map position. The mutation was found to cotransduce withmalF3089:: Tn10 (at 91.5 min) and adnaB-expressing plasmid was able to complement fully thegrpD55 defect in replication. These studies strongly suggest thatgrpD55 is an allele ofdnaB and is localized near 92 min on theE. coli linkage map.     

Protein export elements from Lactococcus lactis

Summary Broad-host-range plasmids carrying -amylase or -lactamase reporter genes lacking a signal sequence were used to select export elements from Lactococcus lactis chromosomal DNA that could function as signal sequences. Fragments containing such elements were identified by their ability to direct the export of the reporter proteins in Escherichia coli. Several of the selected export elements were also active in Bacillus subtilis and L. lactis, although the efficiencies depended strongly on the host organism and reporter gene used. The export elements AL9 and BL1 were highly efficient in L. lactis in the expression and secretion of at least two heterologous proteins (Bacillus licheniformis -amylase and E. coli TEM--lactamase). AL9 even permitted growth of this organism on starch as the sole carbon source. Nucleotide sequence analysis of five selected fragments indicated that these encode oligopeptides with the major characteristics of typical signal peptides. The putative expression signals had a limited similarity to previously described expression signals for E. coli, B. subtilis and L. lactis. Differences in both expression and export efficiency are likely to underlie the host-specific effects.     

Sensitivity to plating of Escherichia coli cells expressing the cryA gene from Bacillus thuringiensis var. israelensis

Summary The gene (cytA) coding for the 27 kDa polypeptide of the Bacillus thuringiensis var. israelensis mosquito larvicidal -endotoxin, was cloned into a plasmid containing the T7 bacteriophage promoter. The plasmid was used to transform an Escherichia coli strain containing the T7 RNA polymerase gene 1, under the control of lacP. Loss of colony-forming ability without substantial lysis, associated with immediate inhibition of DNA synthesis, was observed after induction of transformed cells. The cytA gene product may kill E. colicells by disrupting their chromosome replicating apparatus.     

Characterization of two genes encoding small heat-shock proteins in Arabidopsis thaliana

Summary Using the technique of differential hybridization screening, we have isolated the cDNAs for two low-molecular-mass heat-shock proteins and their corresponding genes, HSP17.4 and HSP18.2, from Arabidopsis thaliana. These two genes encode polypeptides that are 79.2% identical to each other with respect to amino acid sequence, and contain several overlapping sequences that are similar to the consensus sequences for the heat-shock elements (HSE) in Drosophila in the regions upstream from the promoters. The 5 region of the HSP18.2 gene has been fused, in frame, to the uidA gene from Escherichia coli which encodes -glucuronidase (GUS), and the product has been introduced into petunia by Agrobacterium-mediated transformation. We have demonstrated that the GUS activity in transformed petunia plants is enhanced by heat shock.     

Functional assignment of Erwinia herbicola Eho10 carotenoid genes expressed in Escherichia coli

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, -carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for -carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase.     

Isolation of a formamidopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12

Summary The fpg ⁺ gene of Escherichia coli coding for formamidopyrimidine-DNA glycosylase was previously cloned on a multicopy plasmid. The plasmid copy of the fpg ⁺ gene was inactivated by cloning a kanamycin resistance gene into the open reading frame, yielding the fpg-1:: Kn^r mutation. This mutation was transferred to the chromosome in the following steps: (i) linearization of the plasmid bearing the fpg-1::Kn^r mutation and transformation of competent bacteria (recB recC sbcB); (ii) selection for chromosomal integration of the fpg-1::Kn^r mutation; (iii) phage P1 mediated transduction of the fpg-1::Kn^r mutation in the AB1157 background. The resulting fpg ^- mutant exhibited no detectable Fapy-DNA glycosylase activity in crude lysates. The insertion mutation was localized by means of genetic crosses between mtl and pyrE, at 81.7 min on the E. coli linkage map. Sequence analysis confirmed this mapping and further showed that fpg is adjacent to rpmBG in the order fpg, rpmGB, pyrE. The formamidopyrimidine-DNA glycosylase defective strain does not show unusual sensitivity to the following DNA damaging treatments: (i) methylmethanesulfonate, (ii) N-methyl-N-nitro-N-nitrosoguanidine, (iii) ultraviolet light, (iv) -radiation. The fpg gene is neither part of the SOS regulon nor the adaptive response to alkylating agents.     

Co-induction of DNA relaxation and synthesis of DnaK and GroEL proteins inEscherichia coli by expression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor

We examined the influence of overexpression of LetD (CcdB) protein, an inhibitor of DNA gyrase encoded by the F factor ofEscherichia coli, on DNA supercoiling and induction of heat shock proteins. Cells were transformed with a plasmid carrying the structural gene for LetD protein under control of thetac promoter, and LetD protein was induced by adding isopropyl-d-thiogalactopyranoside (IPTG) to the culture medium. Analysis by agarose gel electrophoresis in the presence of chloroquine revealed relaxation of plasmid DNA in cells depending on the concentration of IPTG employed for induction. Protein pulse-labeling experiments with [³⁵S]methionine and cysteine revealed that synthesis of DnaK and GroEL proteins was also induced by IPTG, and concentrations necessary for DNA relaxation and induction of the heat shock proteins were much the same. Expression of mutant LetD protein lacking two amino acid residues at the C-terminus induced neither DNA relaxation nor the synthesis of DnaK and GroEL proteins. Induction of wild-type LetD protein but not mutant LetD protein markedly enhanced synthesis of ³². We interpret these results to mean that DNA relaxation in cells caused by the expression of LetD protein induces heat shock proteins via increased synthesis of ³².     

High degree of homology of the deduced protein structures of the tetracycline-resistance determinants between Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5

The amino acid sequences of the tetracycline-resistance (Tc^r) determinants of Bacillus subtilis plasmid pNS1981 and Staphylococcus aureus plasmid pTP5 have been deduced from their nucleotide sequences and compared. The deduced Tc^r proteins (TETs) of pNS1981 (458 amino acids) and pTP5 (459 amino acids) show a considerable homology (60% identical). If homologous amino acid replacement is taken into account, the homology becomes 80%. Both TET proteins are highly hydrophobic, as expected for a membrane-binding protein, and their polarities are calculated at 32–33%. The putative secondary structures of both TET proteins have been also shown to be significantly homologous, being abundant in -sheets. The predicted positions of -sheets show a nice coincidence between both TET proteins. -Helix has a tendency to be formed at nonhomologous regions of the primary structures between both TET proteins. However, the predicted positions of -helices coincide in a frequency greater than 50%. -Helix and random coil moderately occur at the hydrophilic regions in both TET proteins.     

The dominant mutation Suppressor of black indicates that de novo pyrimidine biosynthesis is involved in the Drosophila tan pigmentation pathway

A deficiency in the production of -alanine causes the black (b) phenotype of Drosophila melanogaster. This phenotype is normalized by a semi-dominant mutant gene Su(b) shown previously to be located adjacent to or within the rudimentary (r) locus. The r gene codes for three enzyme activities involved in de novo pyrimidine biosynthesis. Pyrimidines are known to give rise to -alanine. However, until recently it has been unclear whether de novo pyrimidine biosynthesis is directly coupled to -alanine synthesis during the tanning process. In this report we show that flies carrying Su(b) can exhibit an additional phenotype, resistance to toxic pyrimidine analogs (5-fluorouracil, 6-azathymine and 6-azauracil). Our interpretation of this observation is that the pyrimidine pool is elevated in the mutant flies. However, enzyme assays indicate that r enzyme activities are not increased in Su(b) flies. Genetic mapping of the Su(b) gene now places the mutation within the r gene, possibly in the carbamyl phosphate synthetase (CPSase) domain. The kinetics of CPSase activity in crude extracts has been studied in the presence of uridine triphosphate (UTP). While CPSase from wild-type flies was strongly inhibited by the end-product, UTP, CPSase from Su(b) was inhibited to a lesser extent. We propose that diminished end-product inhibition of de novo pyrimidine biosynthesis in Su(b) flies increases available pyrimidine and consequently the -alanine pool. Normalization of the black phenotype results.     

Subdomain organization ofBacillus thuringiensis entomocidal proteins'' N-terminal domains

N-Terminal domain (65 kD) of -endotoxin produced byBacillus thuringiensis ssp.alesti, as shown by limited proteolysis, consists of two subdomains of molecular mass 30 and 33 kD that correspond, respectively, to conservative and variable regions of the -endotoxin primary structure. Furthermore, proteolysis of these subdomains leads to their conversion into at least two fragments of molecular mass 10 kD stable to proteinase action. Such a pattern of molecular organization appears to be common for several structurally related -endotoxins that belong to thekurstaki group. Entomicidal protein produced by ssp.israelensis (70 kD), which differs strongly fromalesti and otherkurstaki group -endotoxins, retains a similar type of molecular organization and consists of two subdomains with molecular mass of 35 kD. Apparently, the characteristic pattern of the -endotoxins' molecular structure reflects separation of functions (e.g., host recognition and toxicityper se) between domains and subdomains of these proteins.     

Two Agrobacterium tumefaciens genes for cytokinin biosynthesis: Ti plasmid-coded isopentenyltransferases adapted for function in prokaryotic or eukaryotic cells

Summary Tzs and ipt are two Ti plasmid genes coding for proteins with isopentenyltransferase (IPT) activity in vitro. We cloned both genes for protein expression in Escherichia coli and in Agrobacterium tumefaciens, and we investigated differences between the two genes by analysing the properties of the proteins in vitro and in vivo. In vitro, extracts with tzs or ipt-coded proteins had high IPT activity, and the enzymes were identical in most properties. The most important difference was detected in vivo: the tzs-encoded protein was very active in cytokinin production, while the ipt protein required overexpression in order to obtain measurable activity in bacteria. In both cases, rans-zeatin was the major product of the gene activity. Formation of this cytokinin requires a hydroxylase function in addition to the IPT reaction. No such activity could be ascribed to tzs or ipt-encoded proteins in vitro or in vivo, but cytokinin hydroxylase activity was detected in cells and extracts of E. coli, regardless of the presence or absence of the cytokinin genes. Based on these results it is proposed that both genes code for a single enzyme activity (isopentenyltransferase), that the genes and proteins are adapted for function either in bacteria (tzs) or in transformed plant cells (ipt), and that in both prokaryotic and eukaryotic cells hydroxylation to trans-zeatin is a function contributed by host enzymes.Abbreviations DMAPP dimethylallylpyrophosphate - iP isopentenyladenine - iPA isopentenyladenosine - iPMP isopentenyladenosine 5-monophosphate - IPT isopentenyltransferase - trans-Z trans-zeatin     

Nucleotide sequence of the recF gene cluster from Staphylococcus aureus and complementation analysis in Bacillus subtilis recF mutants

We have determined the nucleotide sequence of a 3.5 kb segment in the recF region of the Staphylococcus aureus chromosome. The gene order at this locus, dnaA-dnaN-recF-gyrB is similar to that found in the replication origin region of many other bacteria. S. aureus RecF protein (predicted molecular mass 42.3 kDa), has 57% amino acid sequence identity with the Bacillus subtilis RecF protein (42.2 kDa), but only 26% with the Escherichia coli RecF protein (40.5 kDa). We have shown that the S. aureus recF gene partially complements the defect of a B. subtilis recF mutant, but does not complement an E. coli recF strain. The amino acid sequence alignment of seven available RecF proteins (five of them from bacteria of gram-negative origin) allowed us to identify eight highly conserved regions ( to ) and to predict five new conserved regions within the gram-positive group (a to f). We suggest that the basic mechanism of homologous recombination is conserved among free-living bacteria.     

ISL2, a new mobile genetic element in Lactobacillus helveticus

Spontaneous, phenotypically stable mutations at the -galactosidase locus (lacL-lacM) in Lactobacillus helveticus were identified and analyzed. We found that a significant number of mutations were caused by integration of a new IS element, ISL2, into these lac genes. ISL2 is 858 by long, flanked by 16-bp perfect inverted repeats and generates 3-bp target duplications upon insertion. It contains one open reading frame, which shows significant homology (40.1 % identity) to the putative transposase of IS702 from Cyanobacterium calothrix. ISL2 is present in 4–21 copies in the L. helveticus genome, but it is not found in other lactic acid bacteria. Its divergence in copy number and genomic locations in different L. helveticus strains makes it useful as a tool for strain identification by genetic fingerprinting.     

PWP2, a member of the WD-repeat family of proteins, is an essentialSaccharomyces cerevisiae gene involved in cell separation

WD-repeat proteins contain four to eight copies of a conserved motif that usually ends with a tryptophan-aspartate (WD) dipeptide. TheSaccharomyces cerevisiae PWP2 gene, identified by sequencing of chromosome III, is predicted to contain eight so-called WD-repeats, flanked by nonhomologous extensions. This gene is expressed as a 3.2-kb mRNA in all cell types and encodes a protein of 104 kDa. ThePWP2 gene is essential for growth because spores carrying thepwp21::HIS3 disruption germinate before arresting growth with one or two large buds. The growth defect ofpwp21::HIS3 cells was rescued by expression ofPWP2 or epitope-taggedHA-PWP2 using the galactose-inducibleGAL1 promoter. In the absence of galactose, depletion of Pwp2p resulted in multibudded cells with defects in bud site selection, cytokinesis, and hydrolysis of the septal junction between mother and daughter cells. In cell fractionation studies, HA-Pwp2p was localized in the particulate component of cell lysates, from which it would be solubulized by high salt and alkaline buffer but not by nonionic detergents or urea. Indirect immunofluorescence microscopy indicated that HA-Pwp2p was clustered at multiple points in the cytoplasm. These results suggest that Pwp2p exists in a proteinaceous complex, possibly associated with the cytoskeleton, where it functions in control of cell growth and separation.     

A novel seed protein gene from Vicia faba is developmentally regulated in transgenic tobacco and Arabidopsis plants

Summary We have isolated a novel gene, denoted USP, from Vicia faba var. minor, which corresponds to the most abundant mRNA present in cotyledons during early seed development; however, the corresponding protein does not accumulate in cotyledons. The characterized USP gene with its two introns is 1 of about 15 members of a gene family. A fragment comprising 637 bp of 5 flanking sequence and the total 5 untranslated region was shown to be sufficient to drive the mainly seed-specific expression of two reporter genes, coding for neomycin phosphotransferase 11 and -glucuronidase, in transgenic Arabidopsis thaliana and Nicotiana tabacum plants. We showed that the USP promoter becomes active in transgenic tobacco seeds in both the embryo and the endosperm, whereas its activity in Arabidopsis is detectable only in the embryo. Moreover, we demonstrated a transient activity pattern of the USP promoter in root tips of both transgenic host species.     

Levels of chromosomally encoded Umu proteins and requirements for in vivo UmuD cleavage

Summary Most of the inducible mutagenesis observed in Escherichia coli after treatment with many DNA damaging agents is dependent upon the products of the umuD,C operon. RecA-mediated proteolytic processing of UmuD yields a carboxyl-terminal fragment (UmuD) that is active for mutagenesis. Processing of UmuD is therefore a critical step in the fixation of mutations. In this paper we have analyzed the requirements for UmuD processing in vivo. Standard immuno-detection assays, coupled with a sensitive chemiluminescence detection assay, have been utilized to probe levels of chromosomally encoded Umu proteins from whole-cell E. coli extracts. We found that the derepression of additional SOS gene products, other than RecA, was not required for UmuD processing. Moreover, efficient cleavage of UmuD was observed only in the presence of elevated levels of activated RecA, suggesting that efficient processing would occur only under conditions of severe DNA damage. Detection of chromosomally encoded Umu proteins has allowed us, for the first time, to measure directly the cellular steady-state levels of these proteins under various SOS inducing conditions. UmuD was present at 180 copies per uninduced cell and was measured at 2400 copies per cell in strains that lacked a functional repressor. Induced levels of UmuC were approximately 12-fold lower than UmuD with 200 molecules per cell. These levels of cellular UmuC protein suggest that it functions through specific protein-DNA or protein-protein interactions, possibly as a lesion recognition protein or by interacting with DNA polymerase III.     

Sequencing and analysis of the cos region of the lactococcal bacteriophage c2

The cohesive termini of the DNA genome of the lactococcal bacteriophage c2 were directly sequenced and appeared to be complementary, non-symmetrical, 9-nucleotide single-stranded, 3 extended DNAs, with the following sequence: 5-GTTAGGCTT-3 3-CAATCCGAA-5. DNA located on either side of the cohesive ends was sequenced and several repeats and a region with the potential for a DNA bend were found. Previously sequenced cos regions of 13 other bacteriophages were also examined for similar sequence features. All of the bacteriophages from gram-positive hosts had 3 extended DNA termini, in contrast to the bacteriophages from gram-negative hosts, which had 5 extended DNA termini. All bacteriophages had a region of dyad symmetry close to the cohesive termini. A 7.3 kb DNA fragment of the c2 genome containing the cos sequences was cloned; transduction experiments demonstrated that these cloned sequences could act as a substrate for packaging enzymes of phage c2.