Inactivation of the ecdysteroid UDP-glucosyltransferase (egt) gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV) improves its virulence towards its insect host

Some baculovirus have been genetically modified for the inactivation of their ecdysteroid glucosyltransferase (egt) gene, and these viruses were shown to kill infected larvae more rapidly when compared to wild-type virus infections. We have previously identified, cloned, and sequenced the egt gene of Anticarsia gemmatalis nucleopolyhedrovirus (AgMNPV). Here we present data regarding the construction of an egt minus (egt−) AgMNPV and its virulence towards its insect host. We have inserted an hsp70-lacZ (3.7 kb) gene cassette into the egt gene open reading frame (ORF) and purified a recombinant AgMNPV (vAgEGTΔ-lacZ). Bioassays with third-instar A. gemmatalis larvae showed that viral occlusion body (OB) production were consistently lower from infections with vAgEGTΔ-lacZ compared to the wild-type virus. A mean of 20.4×10⁸ OBs/g/larva and 40.7×10⁸ OBs/g/larva was produced from vAgEGTΔ-lacZ and AgMNPV infections, respectively. The mean lethal concentration which killed 50% of insects in a treatment group (LC₅₀) for the 10th day after virus treatment (DAT) was 3.9-fold higher for the wild-type virus compared to vAgEGTΔ-lacZ. The recombinant virus killed A. gemmatalis larvae significantly faster (ca. 1–2.8 days), than the wild-type AgMNPV. Therefore, the vAgEGTΔ-lacZ was more efficacious for the control of A. gemmatalis larvae (in bioassays) compared to wild-type AgMNPV.     

A regulatory element within a gene of a ribosomal protein operon of Escherichia coli negatively controls expression by decreasing the translational efficiency

Summary The trmD operon of Escherichia coli consists of the genes for the ribosomal protein (r-protein) S16, a 21 kDa protein (21K) of unknown function, the tRNA(m¹G37)methyltransferase (TrmD), and r-protein L19, in this order. Previously we have shown that the steady-state amount of the two r-proteins exceeds that of the 21K and TrmD proteins 12- and 40-fold, respectively, and that this differential expression is solely explained by translational regulation. Here we have constructed translational gene fusions of the trmD operon and lacZ. The expression of a lacZ fusion containing the first 18 codons of the 21K protein gene is 15-fold higher than the expression of fusions containing 49 or 72 codons of the gene. This suggests that sequences between the 18th and the 49th codon may act as a negative element controlling the expression of the 21K protein gene. Evidence is presented which demonstrates that this regulation is achieved by reducing the efficiency of translation.     

Fusion of the lac genes to the promotor for the cytidine deaminase gene of Escherichia coli K-12

Summary Phage Mu has been inserted into the structural gene for cytidine deaminase (cdd). By the use of phage (lac, Mu) the promoter for the cdd gene has been fused to lacZ. In these strains lacZ expression is regulated by the cytR repressor protein and is therefore induced by cytidine. The fusion strains were used for the isolation of cddo mutants. Plaque forming phages carrying the different cdd-lacZ fusions were isolated. Studies of the cdd-Mu strains showed that the cdd gene is transcribed clockwise with respect to the Escherichia coli map.     

Cloning and primary structure of Staphylococcus aureus DNA topoisomerase IV: a primary target of fluoroquinolones

Study of the molecular basis for Legionella pneumophila pathogenicity would be facilitated with an efficient mutagen that can not only mark genomic mutations, but can also be used to reflect gene expression during macrophage infection. A derivative of Jn903, Tn903dlllacZ, is shown to transpose with high efficiency in L. pneumophila. Tn903dlllacZ encodes resistance to kanamycin (Km^R) and carries a 5’truncated lacZ gene that can form translational fusions to L. pneumophila genes upon transposition. The cls-acting Tn903 transposase is supplied outside Tn903dlllacZ, and hence chromosomally integrated copies are stable. Km^R LacZ⁺ insertion mutants of L. pneumophila were isolated and shown by DNA hybridization to carry a single Tn903dlllacZ inserted within their chromosomes at various locations. One particular Km^R LacZ⁺ mutant, AB1156, does not produce the brown pigment (Pig) characteristic of Legionella species. Tn903dlllacZ is responsible for this phenotype since reintroduction of the transposonlinked mutation into a wild-type background results in a Pig phenotype. L. pneumophila pigment production is normally observed in stationary-phase growth of cells in culture, and β-galactosidase activity measured from the pig::lacZ fusion increased during the logarithmic-phase growth and peaked at the onset of stationary phase. Interestingly, pig::lacZ expression also increased during macrophage infection. The pigment itself, however, does not appear to be required for L. pneumophila to grow within or kill host macrophages.     

The DNA gyrase of Escherichia coli participates in the formation of a spontaneous deletion by recA-independent recombination in vivo

Summary A system for detecting a spontaneous deletion in Escherichia coli was developed and the role of DNA gyrase in deletion formation was studied. A derivative of plac5, AM36, was isolated in which whole pBR322 DNA was inserted in the lacZ gene and 227 by of the lac gene duplicated at both sides of the pBR322 DNA. E. coli lac ^–strains lysogenized by AM36 had a Lac^– phenotype and segregated Lac^– revertants. Sequence analyses showed that the revertant was formed by a deletion that eliminated the inserted pBR322 DNA and one copy of the duplicated segments. The frequency of lac ^– revertant formation was independent of recA function, was increased by oxolinic acid, an inhibitor of DNA gyrase, but was not increased in a lysogen of a nalidixic acid-resistant derivative. The reversion frequencies of temperature sensitive mutants of gyrA gene are 10 to 100 times lower than that of the wild-type strain. These results indicate that the DNA gyrase of E. coli participated in the in vivo deletion formation resulting from the direct repeats.     

Gene fusions to the ptsM/pel locus of Escherichia coli

Summary We have constructed gene fusions between ptsM/pel and lacZ. These fusions affect both phenotypes assigned to the ptsM/pel locus (at 40 min), namely, no growth on mannose or glucosamine and inhibition of the penetration of bacteriophage DNA, as well as that of other lambdoid phages such as Hy-2. Since the lacZ gene fusions are insertion mutations that abolish target gene function by disrupting the linear contiguity of the gene, it would appear that ptsM and pel are either the same gene, or two genes within the same operon. Several size classes of these ptsM/pel-lacZ fusions have been isolated and the corresponding hybrid proteins are associated with the cytoplasmic membrane of Escherichia coli. This is consistent with the proposal that ptsM/pel codes for Enzyme II of the phosphotransferase transport system (PTS) specific for mannose, glucosamine, fructose and glucose. However, we have also identified Tn10 insertion mutations that confer a Man^- phenotype but have no effect on the Pel phenotype. Complementation analysis indicates that the Tn10 insertions and the lacZ gene fusions are in different genes. Both of these genes are involved in mannose uptake. This suggests that the locus at 40 min can be subdivided into two genes whose products are required for mannose uptake and that only one of these is involved in the penetration of DNA.     

RexAB proteins of bacteriophage lambda enhance the effect of photolyase-dimer complexes on lacZ gene expression in Escherichia coli.

Summary Expression of the lacZ gene in Escherichia coli is inactivated by exposure to ultraviolet light (UV). Inactivation is exceptionally effective when cells contain amplified levels of DNA photolyase (which forms complexes with pyrimidine dimers in the absence of light for actual photoreversal) and a prophage. Without amplified photolyase, the prophage or both, inactivation rates are similar and much lower. UV-inactivation of lacZ gene expression in the presence of both amplified photolyase and is even more effective if cI857 is used in place of the wildtype prophage but is wholly unexceptional if the prophage carries defects in the genes rexA or rexB. When Rex AB proteins are provided by expression from a plasmid and the cell also contains amplified photolyase, exceptional inactivation rates again obtain; in fact inactivation is most effective under these conditions. The data are considered to reveal a role for Rex AB proteins, which mediate superinfection exclusion, in the exceptional inactivation of gene expression by photolyase bound to pyrimidine dimers in DNA. Photolyase-dimer complexes may mimic the structure of certain complexes that arise during phage development and thus influence Rex A and/or B proteins, thereby shutting down cell metabolism.     

The synthesis and role of the mechanosensitive channel of large conductance in growth and differentiation of Bacillus subtilis

A translational lacZ fusion of the Bacillus subtilis mscL gene that encodes the mechanosensitive channel of large conductance (MscL) was expressed at significant levels during log phase growth of B. subtilis, and the level of mscL–lacZ expression was increased 1.5-fold by growth in medium with high salt (1 M NaCl). However, in growth media with either low or high salt, mscL–lacZ expression fell drastically beginning in the late log phase of growth, and fell to even lower levels during sporulation, although a significant amount of β-galactosidase from mscL to lacZ was accumulated in the developing spore. Deletion of mscL had no effect on B. subtilis growth, sporulation or subsequent spore germination. The ΔmscL strain also grew as well as the wild-type parental strain in medium with 1.2 M NaCl. While log phase wild-type cells grown with 1.2 M NaCl survived a rapid 0.9 M osmotic downshift, log phase ΔmscL cells rapidly lost viability and lysed when subjected to this same osmotic downshift. However, by the early stationary phase of growth, ΔmscL cells had become resistant to a 0.9 M osmotic downshift.     

The depression of endolysin synthesis in bacteria infected with high multiplicities of phage λ

Summary The effect of multiplicity of infection was studied in Escherichia coli with phage, using phage endolysin as an example of a late gene product. A very sensitive endolysin assay method was used so that the initiation time of endolysin synthesis could be more accurately determined. It was observed that high multiplicity of infection (1) increases the rate of lysogenization, (2) progressively delays lysis time, and (3) significantly delays and reduces the synthesis of endolysin in cIII⁺-infected cells. The extent of delay and reduction in endolysin synthesis increases with increasing multiplicity. In contrast, cIII67cII68-infected cells show no delay in endolysin synthesis at high multiplicity of infection when compared with the cIII⁺ cII⁺-infected cells. The results suggest that (1) the expression of cIII and cII genes is multiplicity dependent, (2) high multiplicity of infection enhances the expression of the cIII and cII genes, and (3) the expression of the cIII and cII genes interferes with the expression of the late genes. A model to explain how the expression of the cIII and cII genes interferes with the expression of the late genes is proposed.     

Effect of an umuC mutation on phage lambda induction

Summary A possible role of the umuC gene product in the induction of the SOS responses was examined. We compared the expression of a genetic fusion, in which gene lacZ, encoding -galactosidase in Escherichia coli is under the direct control of the cI repressor from prophage , in a umuC ⁺ strain and in an otherwise isogenic umuC ^- mutant. We found that two times higher UV doses were required to obtain a similar induction in the umuC ⁺ strain as in the umuC mutant. In addition we showed that, at the same UV dose after a lag period, the specific activity of -galactosidase increased more rapidly in the umuC mutant. We suggest that the wild-type umuC gene product participates in the processing of the SOS inducing structures caused by UV irradiation or prevents formation of some of them. This is compatible with a role of the UmuC protein in DNA synthesis past a replication block.     

Inactivation of a newly transferred gene in recombination-deficient recipients of Escherichia coli

Summary The expression of a newly transferred lacZ ⁺ gene in lacZ recipients carrying various mutations in the recA and recB genes was studied by measuring the rates of induced synthesis of -galactosidase in zygotes formed after mating with either F or Hfr donors. The ability to synthesize -galactosidase decreases with time in both recA and recB zygotes when the lacZ ⁺ gene is transferred from an Hfr donor, but not when the lacZ gene is transferred from an F donor. There is no such inactivation of the newly transferred lacZ ⁺ gene in Rec⁺ zygotes. We conclude that the functioning of the transferred DNA is progressively inactivated in rec recipients unless the DNA is contained in an episome such as F.     

Identification of σs-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri

Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor σ^s. Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS ^? derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.     

Efficient RecABC-dependent, homologous recombination between coliphage lambda and plasmids requires a phage ninR region gene

Summary A phage lambda gene that gives a 100-fold increase in recombinant frequencies for RecABC pathway-mediated, phage-plasmid homologous recombination (Shen and Huang 1986) maps to ninG (orf-204) of lambda. We call this gene rap, for recombination adept with plasmid. A similar determinant exists in Charon 4A and maps in 80-derived sequences, between nin5 and the Rz homology with lambda. The absence of the Rap⁺ phenotype from certain lambda vectors explains the inefficiency of screening the resulting phage libraries using phage-plasmid homologous recombination. The mapping of rap permits the construction of lambda vectors more suitable for this screening technique.     

Transient expression of a Drosophila melanogaster hsp 70 promoter/lacZ construct injected into larvae of two species of predatory mites (Acari: Phytoseiidae)

The Drosophila melanogaster heat shock 70 promoter (hsp70) was used to regulate expression of the Escherichia coli -galactosidase gene (lacZ) in transiently-transformed predatory mite larvae. A construct containing the hsp70 promoter upstream of the D. melanogaster alcohol dehydrogenase (adh) translational start site and Escherichia coli lacZ gene fusion (adh/lacZ) was injected into larvae of Metaseiulus occidentalis and Amblyseius finlandicus. LacZ expression was compared to expression of a similar construct lacking any upstream regulatory sequence. Expression from the hsp70 promoter was strong and heat shock-dependent in both species. The Drosophila hsp70 promoter therefore appears useful for regulating expression of exogenous DNA in both phytoseiid species and may be broadly applicable in the Phytoseiidae. Furthermore, the lacZ gene is a useful gene for analysis of expression in both species. Larval microinjection provides a method of assessing transient expression and of examining native regulatory sequences in these two phytoseiids and will likely be useful in other phytoseiid mites with only minor modifications.     

Characterization and Development of Two Reporter Gene Systems for Clostridium acetobutylicum

The use of lacZ from Thermoanaerobacterium thermosulfurigenes (encoding β-galactosidase) and lucB from Photinus pyralis (encoding luciferase) as reporter genes in Clostridium acetobutylicum was analyzed with promoters of genes required for solventogenesis and acidogenesis. Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold. The luciferase assay could be performed much faster and comes close to online measurement. Resequencing of lacZ revealed a sequence error in the original database entry, which resulted in β-galactosidase with an additional 31 amino acids. Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity. The lacZ reporter data showed that bdhA (encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by sol (encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and bdhB (encoding butanol dehydrogenase B) expression. adc (encoding acetoacetate decarboxylase) was also induced early. There is about a 100-fold difference in expression between adc and bdhB (higher) and bdhA and the sol operon (lower). The lucB reporter activity could be increased 10-fold by the addition of ATP to the assay. Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data). lucB reporter measurements confirmed the expression pattern of the sol and ptb-buk (encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the lacZ reporter and showed that the expression level from the ptb promoter is 59-fold higher than that from the sol operon promoter.     

Autoinduction of RpoS Biosynthesis in the Biocontrol Strain <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. M18

The rpoS gene from Pseudomonas sp. M18, which encodes predicted protein (an alternative sigma factor s, σ^S, or σ³⁸) with 99.5% sequence identity with RpoS from Pseudomonas aeruginosa PAO1, was first cloned. In order to investigate the mechanism of rpoS expression, an rpoS null mutant, named M18S, was constructed with insertion of aacC1 cassette bearing a gentamycin resistance gene. With introduction of a plasmid containing an rpoS′–′lacZ translational fusion (pMERS) to wild-type strain M18 or M18S, it was first found that β-galactosidase activity expressed in strain M18S (pMERS) decreased to fourfold of that expressed in the strain M18 (pMERS). When strain M18S (pMERS) was introduced with another plasmid pBBS containing the wild-type rpoS gene, its β-galactosidase expression level was enhanced and almost restored to that in strain M18 (pMERS). Similarly, expression of β-galactosidase from a chromosomal fusion of the promoter of the wild-type rpoS gene with lacZ (rpoS–lacZ) was enhanced fivefold in the presence of a plasmid with the wild-type rpoS gene. With these findings, it is suggested that RpoS sigma factor may be involved in autoinducing its own gene expression in Pseudomonas sp. M18.     

Identification and characterization of stationary phase inducible genes in Escherichia coli

During transition into stationary phase a large set of proteins is induced in Escherichia coli. Only a minority of the corresponding genes has been identified so far. Using the λplacMu system and a plate screen for carbon starvation-induced fusion activity, a series of chromosomal lacZ fusions (csi::lacZ) was isolated. In complex medium these fusions were induced either during late exponential phase or during entry into stationary phase. csi::lacZ expression in minimal media in response to starvation for carbon, nitrogen and phosphate sources and the roles of global regulators such as the alternative sigma factor sigma;^S (encoded by rpoS), cAMP/CRP and the relA gene product were investigated. The results show that almost every fusion exhibits its own characteristic pattern of expression, suggesting a complex control of stationary phase-inducible genes that involves various combinations of regulatory mechanisms for different genes. All fusions were mapped to the E. coli chromosome. Using fine mapping by Southern hybridization, cloning, sequencing and/or phenotypic analysis, csi-5, csi-17, and csi-18 could be localized in osmY (encoding a periplasmic protein), glpD (aerobic glycerol-3-phosphate dehydrogenase) and glgA (glycogen synthase), respectively. The other fusions seem to specify novel genes now designated csiA through to csiF. csi-17(glpD)::lacZ was shown to produce its own glucose-starvation induction, thus illustrating the Intricacies of gene-fusion technology when applied to the study of gene regulation.     

The rep region of pR plasmid regulates the expression of SOS system

Summary By using an artificial hybrid between phage and the pR plasmid, we have shown that the rep region of the pR plasmid encodes a function which regulates the expression of the muc genes (plasmid genes that are under the negative control of lexA and responsible for an increased rate of spontaneous mutagenesis and resistance to UV and chemicals). Expression of the muc genes was monitored by a fusion between the muc promoter and the lacZ structural gene. When E. coli cells containing such a fusion are infected by the hybrid pR phasmid, -galactosidase activity is enhanced, indicating that pR encodes an antagonist of lexA. By deletion mapping we have located the gene encoding the antagonist of lexA (bat) in the rep region of the plasmid. The bat gene product can also antagonize the cI repressor as shown by the observation that pR phasmids are virulent on a homoimmune lysogen. We have exploited this latter property to carry out genetic and functional analysis of the bat region. This region is organized as a classical operon where the expression of the bat structural gene is negatively regulated by a repressor gene that encodes a proteic product.