Delta Strain Research Articles

Resistance to Pyrethroid Insecticides in Soybean Looper (Lepidoptera: Noctuidae) in Mississippi

Recommended rates of pyrethroid insecticides failed to give adequate control of soybean looper, Pseudoplusia includens (Walker), on soybean in some areas of the alluvial plain (Delta) region of Mississippi in 1987. Permethrin provided 53.0% control 5 dafter application in an area of the Delta where permethrin control failures had been suspected in 1985 and 1986. This was lower than the 87.5% control reported in another area of the Delta where no permethrin control failures were suspected during these years. Numbers of soybean looper larvae were 69.4 and 73.4% lower in methomyl treatments and 58.5 and 56.2% lower in acephate treatments than in the untreated control in the Delta within and outside areas of suspected pyrethroid control failures, respectively. Compared with LD50’s for a laboratory strain susceptible to pyrethroids, LD50’s for permethrin applied to soybean loopers collected in the Delta were 10.4 and 21.1 times higher for two strains established from larvae collected in soybean fields treated with permethrin, 3.5 times higher for a strain from an untreated soybean field, and 7.3 times higher for a strain established from adults collected in an ultraviolet light trap. The levels of resistance of the Delta strains to cypermethrin and fenvalerate were similar to those for permethrin. LD50’s of cypermethrin and permethrin for a strain established from the northeastern (Hill) region of Mississippi were not different from the LD50, of the susceptible strain. LD50’s for methomyl applied to field strains were variable but were not different from the laboratory strain. The same was true for methyl parathion, except that a Delta strain collected from a soybean field treated with methyl parathion showed some evidence of resistance.

Cloned structural genes for the osmotically regulated binding-protein-dependent glycine betaine transport system (ProU) of Escherichia coli K-12.

The proU locus of Escherichia coli encodes a high-affinity, binding-protein-dependent transport system (ProU) for the osmoprotectant glycine betaine. We cloned this locus into both low-copy-number lambda vectors and multicopy plasmids and demonstrated that these clones restore osmotically controlled synthesis of the periplasmic glycine betaine binding protein (GBBP) and the transport of glycine betaine in a delta (proU) strain. These clones allowed us to investigate the influence of osmolarity on ProU transport activity independent of the osmotically controlled expression of proU. ProU activity was strongly stimulated by a moderate increase in osmolarity and was partially inhibited by high osmolarity. This activity profile differs from the profile of the osmotically regulated proU expression. The proU locus is organized in an operon and the position of the structural gene (proV) for GBBP is defined using a minicell system. We determined that at least three proteins (in addition to GBBP) are encoded by the proU locus. We also investigated the permeation of glycine betaine across the outer membrane. At low substrate concentration (0.7 microM), permeation of glycine betaine was entirely dependent on the OmpF and OmpC porins.

Yeast metallothionein function in metal ion detoxification.

A genetic approach was taken to test the function of yeast metallothionein in metal ion detoxification. A yeast strain was constructed in which the metallothionein locus was deleted (cup1 delta). The cup1 delta strain was complemented with normal or mutant metallothionein genes under normal or constitutive regulatory control on high copy episomal plasmids. Metal resistance of the cup1 delta strain with and without the metallothionein-expressing vectors was analyzed. The normally regulated metallothionein gene conferred resistance only to copper (1000-fold); constitutively expressed metallothionein conferred resistance to both copper (500-fold) and cadmium (1000-fold), but not to mercury, zinc, silver, cobalt, nickel, gold, platinum, lanthanum, uranium, or tin. Two mutant versions of the metallothionein gene were constructed and tested for their ability to confer metal resistance in the cup1 delta background. The first had a deletion of a highly conserved amino acid sequence (Lys-Lys-Ser-Cys-Cys-Ser). The second was a hybrid gene consisting of the sequences coding for the first 20 amino acids of the yeast protein fused to the monkey metallothionein gene. Expression of these genes under the CUP1 promoter provided significant protection from copper, but none of the other metals tested. These results demonstrate that there is significant flexibility in the structural requirements for metallothionein to function in copper detoxification and that yeast metallothionein is also capable of detoxifying cadmium under conditions of constitutive expression.

Deletion of the phosphoglucose isomerase structural gene makes growth and sporulation glucose dependent in Saccharomyces cerevisiae.

The structural gene PGI1 coding for phosphoglucose isomerase was replaced by the LEU2 gene in the genome of Saccharomyces cerevisiae. Plasmids carrying the LEU2 gene between genomic regions flanking the PGI1 gene were constructed and used to transform a PGI1/pgi1 diploid strain. Stable transformants lacking the PGI1 allele were isolated. Southern analysis of their meiotic products showed that haploid strains with a deletion of 1.6 kb within the 2.2 kb PGI1 coding region were viable. Thus, the PGI1 gene is not essential in yeasts. However, unlike pgi1 mutants with residual phosphoglucose isomerase activity, no growth was detected in the pgi1 delta haploid strains when fructose was supplied as sole carbon source. The wild-type growth rate could be restored by adding 0.1% glucose to the medium. Furthermore, pgi1 mutants with residual enzymatic activity grew very slowly on fructose-supplemented media containing up to 2% glucose. Strains carrying the deletion allele, however, failed to grow at glucose concentrations higher than 0.5%. Also the pgi1 delta strains did not grow in glucose as sole carbon source. On the other hand pgi1 delta/pgi1 delta diploid strains did not sporulate on the usual acetate medium. This defect could be alleviated by the addition of 0.05% glucose to the sporulation medium. Under these conditions the pgi1 delta mutants sporulated with an efficiency of 25% compared with the wild type. These results suggest that the phosphoglucose isomerase reaction is the only step catalysing the interconversion of glucose-6-P and fructose-6-P, glucose-6-P is essential in yeasts, and the oxidation of glucose-6-P through the glucose-6-P dehydrogenase reaction is not sufficient to support growth in yeasts.

Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12.

The fusion-generating phage lambda plac Mu1 was used to produce fusions of lacZ to fhuA, the gene encoding the ferrichrome-iron receptor (FhuA protein) in the outer membrane of Escherichia coli K-12. Fusions to the fhuA gene in a delta (lac) strain were selected by their resistance to bacteriophage phi 80 vir. Ten independent (fhuA'-'lacZ) fusions were all Lac+ and were resistant to the lethal agents which require the FhuA protein as receptor, i.e., phi 80 vir, T5, T1, UC-1, and colicin M; none could utilize ferrichrome as the sole iron source. Specialized transducing phages were obtained by illegitimate excision from the chromosome of each of the fusion-bearing strains, and EcoRI fragments which encoded the fusions were subcloned into the high-copy plasmid pMLB524. Physical mapping of the fusion-containing plasmids confirmed the presence of three restriction sites which were also located on the chromosomal DNA of sequences near the fhuA gene. The direction of transcription of the fhuA gene was deduced from the direction of transcription of the (fhuA'-'lacZ) gene fusion. Identification of the chimeric proteins was made by both radiolabeling cells and immunoprecipitating the LacZ-containing proteins with antibody to beta-galactosidase and by preparing whole cell extracts from Lac+ cells containing the cloned gene fusions. Two sizes of (FhuA'-'LacZ) proteins were detected, 121 kDa and 124 kDa. The DNA sequences at the unique fusion joints were determined. The sequence information allowed us to identify three distinct fusion joints which were grouped as follows, type I fusions, 5'-ACT GCT CAG CCA A-3'; type IIa fusions, 5'-GCG GTT GAA CCG A-3'; and type IIb fusions: 5'-ACC GCT GCA CCT G-3'. To orient these fhuA fusion joints, the complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA. A single open reading frame was found which translated into a 747-amino acid polypeptide. The signal sequence of 33 amino acids was followed by a mature protein with a molecular weight of 78,992. Alignment of the amino acid sequence of the FhuA protein with the amino acid sequences presented for two other tonB-dependent receptor proteins in the outer membrane of E. coli showed an area of local homology at the amino terminus of all three proteins.

Genetic inactivation of topoisomerase I suppresses a defect in initiation of chromosome replication in Escherichia coli.

A strain of Escherichia coli K12 harboring simultaneously the temperature-sensitive dnaA46 mutation and a deletion of the trp-topA-cysB region plates with the same full efficiency at 30 degrees C and 42 degrees C. We have analyzed the possible involvement of the gene coding for topoisomerase I, topA, in this suppression phenomenon. The Ts phenotype was retrieved upon introduction of a plasmid-borne DNA fragment including an active topA gene into this strain, but not upon introduction of the same fragment harboring a topA::Tn1000 insertion. Replication seems to remain DnaA-dependent in the delta (topA) strain, however, since we have been unable to introduce a dnaA::Tn10 allele. We propose either that the dnaA46 gene product is overproduced and compensates for its thermal inactivation, or that initiation at oriC demands less DnaA protein in the absence of topoisomerase I.

Negative complementation of recA protein by recA1 polypeptide: in vivo recombination requires a multimeric form of recA protein.

Recombination in vivo was studied in recA- heterozygous lacZ merodiploids by performing beta-galactosidase assays after infection with lambda precA+. Recombination as measured by beta-galactosidase production was a linear function of lambda pecA+ multiplicity of infection (MOI) when the strain contained a deletion of the chromosomal recA gene. However, when the strain carried a recA1 missense allele, a higher lambda precA+ MOI was required to obtain levels of recombination comparable to the delta (recA) strain, and the slope of the dose-response curve increased to approximately two. It is proposed that negative complementation occurs in mixed tetramers of wild-type and missense recA polypeptides, and that in vivo recombination is a property of a multimeric form of recA protein.

A hospital-wide outbreak of septicemia due to a few strains of Staphylococcus aureus.

During a 6-month period at Walter Reed Army Hospital the monthly attack rate of Staphylococcus aureus bacteremia increased to 3.8 +/- 0.5 (mean +/- SEM) from 2.5 +/- 0.2 cases per 1,000 dispositions for the previous 48 months (P less than 0.05). A predominant phage pattern, designated S, was found in 12 (39%) of 31 bacteremic isolates typed and another strain, delta, was associated with four catheter-related infections. Two other strains also accounted for infections. Patients with isolates of the S phage pattern had a higher mortality (59%) than patients with non-S isolates (37%). Thirty-eight per cent of S. aureus carriers among hospital personnel harbored S or delta strains. Limitation of intravascular devices, strict handwashing, and the use of gloves were associated with a significant decrease in the incidence of S. aureus bacteremia to 1.9 +/- 0.5/1,000 dispositions over the next 6 months (P less than 0.05). S and delta strains were reduced to 20% of these isolates despite their persistence in 32% of staphylococcal carriers upon reculture of personnel. We conclude that S. aureus persists as an important pathogen in the hospitals, and that phage typing S. aureus isolates remains an important tool in hospital epidemiology. The presence of multiple S. aureus strains causing this outbreak and the extent of their dissemination among patients and personnel reported here emphasizes the need to reevaluate strategies of nosocomial staphylococcal control.

The effect of urea on catabolite sensitive operons in Escherichia coli K 12.

Low concentrations of urea specifically inhibit the expression of catabolite sensitive genes (the lactose, galactose and maltose operons and the tryptophanase gene). This inhibition depends upon growth conditions, i.e. carbon source and temperature. The main effect of urea is exerted at the level of transcription initiation. However an additional inhibitory effect is observed on the decay and expression of the beta-galactosidase messenger. In a strain harboring the UV5 mutation in the lactose promoter, the effect at the level of transcription is relieved while the effect on the decay and the expression of the beta-galactosidase messenger remains the same. Just like the extreme physiological catabolite repression, the urea effect occurs even in a cya delta strain and is not antagonized by addition of adenosine 3'-5' cyclic monophosphate.

Agal region mutant that requires cAMP for growth on galactose in an adenyl cyclase negative (cyaΔ) background

Strains of Escherichia coli K12 that contain a deletion of the adenyl cyclase gen (cya delta), required for the synthesis of cyclic adenosine-3';5' monophosphate (cAMP), grow on galactose-containing minimal medium. A mutant was isolated that grows on this medium only if cAMP is added. The mutation (designated galP20) is linked to the gal operon region as determined by both generalized transduction with bacteriophage P1 and specialized transduction with bacteriophage lambda. Studies with galP20 cya delta strains as well as gal delta (deletions of the gal operon) cya delta strains indicate that synthesis of the physiologically important transport mechanism for galactose (galactose permease) requires either cAMP or a function mission from both the galdelta strains and the galP20 strain.

The Penetration and Metabolism of C14-Labeled DDT in Resistant and Susceptible Tobacco Budworm Larvae, Heliothis virescens (F.)1

The fate of C14-labeled DDT applied topically to larvae of the tobacco budworm, Heliathis virescens (F.), was determined in several DDT-resistant and susceptible strains. The DDT did not penetrate effectively the cuticle of the older 5th-instal larvae, The same phenomenon occurred in the 3rd-instar larvae of the South Delta resistaant strain. In the other resistant strain the DDT penetrated effectively the cuticle but was degraded rapidly and climinated. The resistant South Delta strain when fed a diet of colton Ieaves was susceptible to a topically applied dose of DDT which readily penetrated the cuticle. This diet, altered strain resembled one of the susceptible strains in the rate of penetration and climination of the dose as well as having a similar LD50 for DDT. The other susceptible strain showed DDT breakdown but was ineffective in reducing the DDT content interllally owing to the rapid DDT absorption in conjunction with slow excretion of the dose. In all strains where DDT breakdown occurred both DDE and DDA were found, with the DDA increasing in the excreted fraction.