The damage-inducible (din) genes of Escherichia coli are induced by various genotoxins in a different way.

The SOS response of Escherichia coli strains carrying the lacZ gene fused to the polB (dinA), dinB or dinD gene were investigated after treatment with several chemical agents and gamma-radiation. The induction levels of polB::lacZ reached levels between 4.0- and 9.0-fold 120 min after treatment with nalidixic acid, H2O2 or ethanol. Pentachlorophenol did not significantly induce any din genes. gamma-Irradiation is not an inducer of polB and ethanol failed to induce dinB::lacZ and dinD::lacZ. Following irradiation with a dose of 10 Gy the responses of dinB and dinD were induced about 2.5-3.0-fold above non-irradiated dinB and dinD. We found that the responses of din::lacZ fusion genes to these genotoxins are induced in a dose-dependent manner. The polB gene showed antagonistic responses to the simultaneous treatment of nalidixic acid and H2O2 or nalidixic acid and ethanol. In addition, dinB and dinD in the presence of both nalidixic acid and H2O2 at the same time showed no synergistic responses.     

SOS induction as an in vivo assay of enzyme-DNA interactions

We have constructed strains which are convenient and sensitive indicators of DNA damage and describe their use. These strains utilize an SOS::lac Z fusion constructed by Kenyon and Walker [Proc. Natl. Acad. Sci. USA 77 (1980) 2819-2823] and respond to DNA damage by producing beta-galactosidase. They can be used to characterize restriction systems and screen for restriction endonuclease mutants. Applications include the study of other enzymes involved in DNA metabolism, such as DNA methyltransferases, topoisomerases, recombinases, and DNA replication and repair enzymes.     

Novel Middle-Type Kenyon Cells in the Honeybee Brain Revealed by Area-Preferential Gene Expression Analysis

The mushroom bodies (a higher center) of the honeybee (Apis mellifera L) brain were considered to comprise three types of intrinsic neurons, including large- and small-type Kenyon cells that have distinct gene expression profiles. Although previous neural activity mapping using the immediate early gene kakusei suggested that small-type Kenyon cells are mainly active in forager brains, the precise Kenyon cell types that are active in the forager brain remain to be elucidated. We searched for novel gene(s) that are expressed in an area-preferential manner in the honeybee brain. By identifying and analyzing expression of a gene that we termed mKast (middle-type Kenyon cell-preferential arrestin-related protein), we discovered novel ‘middle-type Kenyon cells’ that are sandwiched between large- and small-type Kenyon cells and have a gene expression profile almost complementary to those of large– and small-type Kenyon cells. Expression analysis of kakusei revealed that both small-type Kenyon cells and some middle-type Kenyon cells are active in the forager brains, suggesting their possible involvement in information processing during the foraging flight. mKast expression began after the differentiation of small- and large-type Kenyon cells during metamorphosis, suggesting that middle-type Kenyon cells differentiate by modifying some characteristics of large– and/or small-type Kenyon cells. Interestingly, CaMKII and mKast, marker genes for large– and middle-type Kenyon cells, respectively, were preferentially expressed in a distinct set of optic lobe (a visual center) neurons. Our findings suggested that it is not simply the Kenyon cell-preferential gene expression profiles, rather, a ‘clustering’ of neurons with similar gene expression profiles as particular Kenyon cell types that characterize the honeybee mushroom body structure.     

Multicopy suppressors of the cold-sensitive phenotype of the pcsA68 (dinD68) mutation in Escherichia coli.

The Escherichia coli strain cs2-68 is a cold-sensitive (c) mutant that forms a long filamentous cell at 20 degrees C with a large nucleoid mass in its central region. We have recently shown that the pcsA68 mutation causing the cs phenotype is a single-base substitution within the dinD gene, a DNA damage-inducible gene which maps at 82 min. Since null mutants of the pcsA (dinD) gene are viable, with no discernible defect in cell growth, the cs phenotype is attributed to a toxic effect by the mutant protein. In an attempt to identify a target(s) for the toxic pcsA68 mutant protein, we screened for chromosomal fragments on multicopy plasmids that could suppress the cs phenotype. Three different BamHI fragments were found to suppress cold sensitivity, and the lexA, dinG, and dinI genes were identified to be responsible for the suppression in each fragment. DinG shares multiple motifs with many DNA helicases. The complete sequence of dinI revealed that DinI is a small protein of 81 amino acids. It is similar in size and sequence to ImpC of the Salmonella typhimurium plasmid TP110 and to a protein (ORFfs) of the retronphage phi R67, both of which are also under the control of LexA.     

The brain of the horseshoe crab (Limulus polyphemus). III. Cellular and synaptic organization of the corpora pedunculata.

The large, hemispherical mass of the Limulus corpora pedunculata consists of two highly branched lobes, each connected to the protocerebrum by a narrow stalk. About 10(4) afferent fibers enter through the stalks and make diverse, profuse, and often reciprocal contacts with several million Kenyon (intrinsic) cells and one another. The Kenyon cell axonal arborizations converge on a few hundred efferent dendrites. The afferent fiber types can be classified into five types. Type A forms the club-shaped core of glomeruli and circumglomerular annuli, and contains small flat vesicles, suggesting an inhibitory function. Type B terminates with bushy endings in glomeruli and is presynaptic to both Kenyon cells and to Type A terminals. It has clear round vesicles and is the presumptive excitatory input. Type C terminates on other afferents, in glomeruli, and rarely on Kenyon cell bodies, contains angular (neurosecretory) granules and is postulated to impart circadian rhythm. Type D terminates on Kenyon cell somata and the initial neurite segment (but not in glomeruli), and contains dense-cored vesicles. Type E terminates in peduncles on other afferents and Kenyon cell telodendria. It contains dense vesicles. The C, D, and E afferents have reciprocal synaptic connections with Kenyon cell axon terminals. Glomeruli thus receive three different inputs of presumptive inhibitory (A), excitatory (B), and neuromodulatory nature (C). Kenyon cells, increasing in number up to about 1 x 10(8) in the adult, show minor variations in their dendritic pattern and have only one rare variant cell type. Interactions between them occur primarily at their axonal boutons as they crowd around efferent fibers. The latter have large receptive fields, some of their large somata are located within the confines of the corpora pedunculata, and they receive input almost only from Kenyon cells. Numerical and directional details of the circuitry in the corpora pedunculata have been extracted by a combination of light and electron microscopy, serial sectioning, silver staining, and stereology. The corpora pedunculata appear to process primarily the voluminous chemosensory input from the appendages, an assumption that is supported by the major connections of the organ.     

The 'endo-blue method' for direct cloning of restriction endonuclease genes in E. coli.

A new E. coli strain has been constructed that contains the dinD1::LacZ+ fusion and is deficient in methylation-dependent restriction systems (McrA-, McrBC-, Mrr-). This strain has been used to clone restriction endonuclease genes directly into E. coli. When E. coli cells are not fully protected by the cognate methylase, the restriction enzyme damages the DNA in vivo and induces the SOS response. The SOS-induced cells form blue colonies on indicator plates containing X-gal. Using this method the genes coding for the thermostable restriction enzymes Taql (5'TCGA3') and Tth111l (5'GACNNNGTC3') have been successfully cloned in E. coli. The new strain will be useful to clone other genes involved in DNA metabolism.     

The pcsA gene is identical to dinD in Escherichia coli.

The pcsA68 mutant of Escherichia coli is a cold-sensitive mutant which forms long filaments with a large nucleoid in the central region at 20 degrees C. We here show that (i) the coding region for the pcsA gene is identical with orfY located upstream of pyrE and can be deleted without loss of viability; (ii) pcsA is also identical to dinD, a DNA damage-inducible gene, whose expression is regulated by the LexA-RecA system; (iii) the cold-sensitive phenotype of the pcsA68 mutation is suppressed by delta recA or lexA1 (Ind-) mutation, but not by sulA inactivation; (iv) overproduction of PcsA68 leads to inhibition of cell growth in recA+ and delta recA strains at 20 and 37 degrees C, but PcsA+ does not show such an effect at any temperature; (v) SOS response is induced in the pcsA68 mutant cells at 20 degrees C. We discuss the possible function of the pcsA gene, comparing it with the sulA or the dif-xerCD function. We also describe a new method for gene disruption with positive and negative selection.     

SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis.

We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis. One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis. In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow. Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not. The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein. In addition, cells infected with R377 formed filaments. Another deletion mutant of the minus-strand origin, M13 delta E101 (M. H. Kim, J. C. Hines, and D. S. Ray, Proc. Natl. Acad. Sci. USA 78:6784-6788, 1981), also induced the SOS response in E. coli. M13Gori101 (D. S. Ray, J. C. Hines, M. H. Kim, R. Imber, and N. Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response. These observations indicate that single-stranded DNA by itself induces the SOS response in vivo.     

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p's ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.     

ABC multidrug transporter Cdr1p of Candida albicans has divergent nucleotide-binding domains which display functional asymmetry

In order to ascertain the molecular basis of ATP-mediated drug extrusion by Cdr1p, a multidrug transporter of Candida albicans, we recently have reported that the Walker A motif of the N-terminal nucleotide biding domain (NBD) of this protein contains an uncommon cysteine residue (C193; GXXGXGCS/T) which is indispensable for ATP hydrolysis. This residue is exceptionally conserved in N-terminal NBDs of fungal ABC transporters and hence makes these transporters an evolutionarily divergent group. However, the presence of a conventional lysine residue at a similar position in the Walker A motif of the C-terminal NBD warrants the individual contribution of both the NBDs in the ATP-driven efflux function of such transporters. In this study we have investigated the contribution of this divergent Walker A motif in the context of the full Cdr1p protein under in vivo conditions by swapping these two crucial amino acids (C193K in Walker A motif of N-terminal NBD and K901C in Walker A motif of C-terminal NBD) between the two NBDs. Both the native and the mutant variants of Cdr1p were integrated at the PDR5 locus as GFP-tagged fusion proteins and were hyper-expressed. Our study shows that both C193K- and K901C-expressing cells elicit a severe impairment of Cdr1p’s ATPase function. However, both these mutations have distinct phenotypes with respect to other functional parameters such as substrate efflux and drug resistance profiles. In contrast to C193K, K901C mutant cells were substantially hypersensitive to the tested drugs (fluconazole, ansiomycin, miconazole and cycloheximide) and were unable to expel rhodamine 6G. Our results for the first time show that both NBDs influence the Cdr1p function asymmetrically, and that the positioning of the cysteine and lysine residues within the respective Walker A motifs is functionally not interchangeable.     

Error-prone repair and translesion synthesis III: the activation of UmuD (or less is more)

Following DNA damage to Escherichia coli bacteria, RecA protein is activated by binding to single stranded DNA and cleaves its own gene repressor (LexA protein). Two papers from Graham Walker's laboratory showed that several bacterial genes in addition to RecA are repressed by the LexA repressor and are inducible following DNA damage [C.J. Keyon, G.C. Walker, DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli, in: Proceedings of the National Academy of Sciences of the United States of America 77, 1980, pp. 2819--2823] and predicted that one of them (UmuD) might itself be subject to activation by a further cleavage reaction involving activated RecA protein [K.L. Perry, S.J. Elledge, B.B. Mitchell, L. Marsh, G.C. Walker, umuD,C and mucA,B operans whose products are required for UV light- and chemical-induced mutagenesis: UmuD, MucA, and LexA proteins share homology, in: Proceedings of the National Academy of Sciences of the United States of America 82, 1985, pp. 4331--4335]. The processed form of UmuD, termed UmuD', later proved to be a subunit of DNA polymerase V, a key enzyme involved in translesion synthesis.     

Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant

We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.     

An analysis of the structure of the product of the rbsA gene of Escherichia coli K12

The predicted amino acid sequence of rbsA, a gene from the high affinity ribose transport operon (rbs) of Escherichia coli K12, is homologous to the products of hisP, malK, and pstB, components of the histidine, maltose, and phosphate high affinity transport operons. The recent finding by Hobson et al. (Hobson, A. C., Weatherwax, R., and Ames, G.F.-L. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 7333-7337) that the hisP and malK products bind ATP suggests that these four gene products may be involved in coupling the energy from ATP to drive the active transport in their respective transport systems. Each gene product contains a sequence of glycine and basic residues which are characteristic of an ATP-binding site (Walker, J.E., Saraste, M., Runswick, M.J., and Gay, N.J. (1982) EMBO J. 1, 945-951). Interestingly the N- and C-terminal halves of rbsA are also homologous, suggesting that a primordial gene duplication and subsequent fusion of the products occurred.     

Self-assembly of the Agrobacterium tumefaciens VirB11 traffic ATPase

The Agrobacterium tumefaciens VirB11 ATPase is a component of a type IV transporter dedicated to T-DNA delivery to plant cells. In this study, we tested a prediction from genetic findings that VirB11 self-associates in vivo. A chimeric protein composed of VirB11 fused to the DNA binding domain of lambda cI repressor protein formed dimers, as shown by immunity of Escherichia coli to lambda superinfection. An allele encoding VirB11 fused at its C terminus to the green fluorescent protein (GFP) exerted strong negative dominance when synthesized in wild-type A. tumefaciens cells. Dominance was suppressed by overproduction of native VirB11, suggestive of titrating or competitive interactions between VirB11 and VirB11::GFP. In support of the titration model, a complex of native VirB11 and VirB11::GFP was recovered by precipitation with anti-GFP antibodies from detergent-solubilized A. tumefaciens cell extracts. VirB11 was shown by cI repressor fusion and immunoprecipitation assays to interact with VirB11 derivatives encoded by (i) 11 dominant negative alleles, (ii) recessive alleles bearing codon substitutions or deletions in the Walker A nucleotide binding motif, and (iii) alleles corresponding to the 5' and 3' halves of virB11. Further immunoprecipitation studies showed a hybrid protein composed of the N-terminal half of VirB11 fused to GFP interacted with mutant proteins exerting dominant effects and with a recessive Walker A deletion mutant (Delta GKT174-176). By contrast, a hybrid protein composed of the C-terminal half fused to GFP interacted with mutants exerting dominant effects but not the Walker A mutant protein. Together, these studies establish that VirB11 assembles as homomultimers in vivo via domains residing in each half of the protein. Furthermore, ATP binding appears to be critical for C-terminal interactions required for assembly of productive homomultimers.     

20-Hydroxyecdysone inhibits the mitotic activity of neuronal precursors in the developing mushroom bodies of the honeybee,Apis mellifera

The mushroom bodies (MBs) within the brain of the honeybee, Apis mellifera, are prominent paired neuropil structures consisting of a lateral and a median subunit. The intrinsic MB neurons (Kenyon cells) of each of these subunits are generated in four distinct proliferation centers, each associated with a calyx. Previous BrdU studies revealed that neurogenesis of Kenyon cells starts at the first larval stage (L1) by symmetrical cell division of Kenyon precursor cells, and ceases abruptly at a midpupal stage (P5). In the present work, we confirmed these results using the antiphospho histone H3 mitosis marker to label mitotically active cells in a cell culture system, in histological sections, and in whole-mount brain preparations. To elucidate whether the steroid hormone ecdysone plays a role in the termination of Kenyon cell neurogenesis, we manipulated the hormone titer by injecting 20-hydroxyecdysone (20E) into animals of those pupal stages (P0/1, P3, P4) in which neurogenesis of Kenyon cells was still extensive. The effects of 20E were evaluated by determining the number of mitotically active cells in confocal microscopic images of squash preparations of the MB proliferation centers. In all pupal stages studied, 20E caused a reduction of mitotic activity, indicating its involvement in the cessation of Kenyon cell neurogenesis.     

ChvD, a chromosomally encoded ATP-binding cassette transporter-homologous protein involved in regulation of virulence gene expression in Agrobacterium tumefaciens

A yeast two-hybrid screen searching for chromosomally encoded proteins that interact with the Agrobacterium tumefaciens VirB8 protein was carried out. This screen identified an interaction candidate homologous to the partial sequence of a gene that had previously been identified in a transposon screen as a potential regulator of virG expression, chvD. In this report, the cloning of the entire chvD gene is described and the gene is sequenced and characterized. Insertion of a promoterless lacZ gene into the chvD locus greatly attenuated virulence and vir gene expression. Compared to that of the wild-type strain, growth of the chvD mutant was reduced in rich, but not minimal, medium. Expression of chvD, as monitored by expression of beta-galactosidase activity from the chvD-lacZ fusion, occurred in both rich and minimal media as well as under conditions that induce virulence gene expression. The ChvD protein is highly homologous to a family of ATP-binding cassette transporters involved in antibiotic export from bacteria and has two complete Walker box motifs. Molecular genetic analysis demonstrated that disruption of either Walker A box, singly, does not inactivate this protein's effect on virulence but that mutations in both Walker A boxes renders it incapable of complementing a chvD mutant strain. Constitutive expression of virG in the chvD mutant strain restored virulence, supporting the hypothesis that ChvD controls virulence through effects on virG expression.     

Cloning and characterization of a gene encoding phosphoketolase in a Lactobacillus paraplantarum isolated from Kimchi

A gene coding for phosphoketolase, a key enzyme of carbohydrate catabolism in heterofermentative lactic acid bacteria (LAB), was cloned from a Lactobacillus paraplantarum C7 and expressed in Escherichia coli. The gene is 2502 bp long and codes for a 788-amino-acids polypeptide with a molecular mass of 88.7 kDa. A Shine-Dalgamo sequence (aaggag) and an inverted-repeat terminator sequence are located upstream and downstream of the phosphoketolase gene, respectively. The gene exhibits an identity of >52% with phosphoketolases of other LAB. The phosphoketolase of Lb. paraplantarum C7 (LBPK) contains several highly conserved phosphoketolase signature regions and typical thiamine pyrophosphate (TPP) binding sites, as reported for other TPP-dependent enzymes. The phosphoketolase gene was fused to a glutathione S-transferase (GST::LBPK) gene for purification. The GST::LBPK fusion protein was detected in the soluble fraction of a recombinant Escherichia coli BL21. The GST::LBPK fusion protein was purified with a yield of 4.32 mg/400 ml by GSTrap HP affinity column chromatography and analyzed by N-terminal sequencing. LBPK was obtained by factor Xa treatment of fusion protein and the final yield was 3.78 mg/400 ml. LBPK was examined for its N-terminal sequence and phosphoketolase activity. The K(M) and Vmax values for fructose-6-phosphate were 5.08 +/- 0.057 mM (mean +/- SD) and 499.21 +/- 4.33 micromol/min/mg, respectively, and the optimum temperature and pH for the production of acetyl phosphate were 45 degrees C and 7.0, respectively.     

Characterization of the ATPase activity of the N-terminal nucleotide binding domain of an ABC transporter involved in oleandomycin secretion by Streptomyces antibioticus

Abstract The ole B gene of Streptomyces antibioticus , oleandomycin producer, encodes an ABC transporter containing two putative ATP-binding domains and is involved in oleandomycin resistance and secretion in this organism. We have overexpressed in Escherichia coli the N-terminal nucleotide-binding domain of OleB (OleB') as a fusion protein to a maltose-binding protein and purified the fusion protein by affinity chromatography. The fusion protein showed ATPase activity dependent on the presence of Mg²⁺ ions. ATPase activity was resistant to specific inhibitors of P-, F-, and V-type ATPase whereas sodium azide and 7-chloro-4-nitrobenzo-2-oxa-l,3-diazole (NBD-C1) were strong inhibitors. The change of Lys71, located within the Walker A motif of the OleB' protein, to Gin or Glu caused a loss of ATPase activity, whereas changing to Gly did not impair the activity. The results suggest that the intrinsic ATPase activity of purified fusion protein can be clearly distinguished from other ATP-hydrolysing enzymes, including ion-translocating ATPases or ABC-traffic ATPases, both on the basis of inhibition by different agents and since it hydrolyzes ATP without interacting with a hydrophobic membrane component.