Derivation of High Enterotoxin B-Producing Mutants of Staphylococcus aureus from the Parent Strains

Certain pH-sensitive (membrane) mutants of Staphylococcus aureus, strains 14458 and 778, produce significantly more type-B enterotoxin (SEB) than the parent type. Some carbohydrate mutants (car) from these parent strains also are superior to the parent in SEB formation. By isolating car mutants from high-SEB-producing membrane mutants, it is possible to derive a double mutant producing from six to 50 times as much SEB as the parent type. Inversion of the sequence by isolating pH-sensitive mutants from car mutants does not yield clones with strikingly higher SEB production than the parent strain. The successful isolation sequence (pH-sensitive mutant first and car mutants derived from it) is relatively simple and virtually assures detection of a truly high-SEB-producing clone. The total number of clones whose direct assay for SEB formation is necessary for detection of a high-producing mutant is on the order of 50 to 60.     

Synthesis of Indicator Strains and Density of Ribonucleic Acid-Containing Coliphages in Sewage

Escherichia coli strains freshly isolated from natural sources are inefficient indicators of coliphages present in sewage. Four E. coli strains recently isolated from clinical specimens were mutagenized to obtain lac(-) mutants. Such mutants were infected with an F'lac(+) sex factor of E. coli K-12. Pairs of isogenic lac(-) and lac(-)/F'lac(+) strains were used as indicators of coliphages present in sewage, and it was found that such strains can be effectively used for a direct and almost selective enumeration of F-specific coliphage contents of sewage samples. Serological tests were applied to a number of F-specific phages isolated. All the isolates that were tested fell into two distinguishable antigenic classes: members of one class being related to ribonucleic acid (RNA) phage MS2 and those of the other being related to another RNA phage, namely, Qbeta. MS2-related phages have been found to be more widely distributed than the Qbeta related phages. Most habitats sampled were found to yield only one or the other kind of phage. Single-stranded deoxyribonucleic acid-containing F-specific phages were not detectable by the methods employed by us.     

Transfer of the delta (argF-lac)U169 mutation between Escherichia coli strains by selection for a closely linked Tn10 insertion

To facilitate construction of mutants harboring delta lac for use in gene fusion studies, strains were constructed that carry the transposon Tn10 next to the well defined lac deletion U169. This deletion can now be moved to other Escherichia coli strains in transductional or conjugational crosses by selecting resistance to tetracycline.     

Characterization of Marek's disease virus insertion and deletion mutants that lack US1 (ICP22 homolog), US10, and/or US2 and neighboring short-component open reading frames.

We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome. A deletion mutant (GA delta 4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli. This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorf1, Sorf2, and Sorf3. Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US2lac insertion mutant (J. L. Cantello, A. S. Anderson, A. Francesconi, and R. W. Morgan, J. Virol. 65:1584-1588, 1991). The isolation of these mutants indicates that the relevant genes are not required for growth of MDV in chicken embryo fibroblasts. The mutants had early growth kinetics indistinguishable from those of their parent viruses; however, 5 to 7 days after being plated, the US1 insertion mutant (US1lac) and the GA delta 4.8lac deletion mutant showed a 5- to 10-fold decrease in virus growth. This decrease in virus accumulation correlated with a 30 to 50% decrease in plaquing efficiency when these viruses were plated onto established versus fresh chicken embryo fibroblast monolayers compared with a 10 to 15% decrease seen for the parent viruses and for the US10lac or US2lac insertion mutants. Finally, GA delta 4.8lac could be reisolated from chickens, indicating that the deleted genes are not required for the infection of chickens following intra-abdominal inoculation of an attenuated serotype 1 MDV.     

Roles for menaquinone and the two trimethylamine oxide (TMAO) reductases in TMAO respiration in Salmonella typhimurium: Mu d(Apr lac) insertion mutations in men and tor.

Three groups of mutants defective in trimethylamine oxide (TMAO) reduction were isolated from Salmonella typhimurium LT2 subjected to transposition mutagenesis with Mu d(Apr lac). Mutants were identified by their acidic reaction on a modified MacConkey-TMAO medium. Group I consisted of pleiotropic chlorate-resistant mutants which were devoid of TMAO reductase activity. None expressed the lac operon. Group II mutants were partially defective in TMAO reductase. Electrophoretic studies revealed that they lacked the inducible TMAO reductase, but retained the constitutive activity. The genotypic designation tor was suggested for these mutants. The tor mutation in one was located between 80 and 83 U on the S. typhimurium chromosome. Expression of the lac operon in these mutants was not affected by air, TMAO, or nitrate. Group III mutants reduced little or no TMAO in vivo, but their extracts retained full capacity to reduce it with methyl viologen. These mutants also failed to produce hydrogen sulfide from thiosulfate and could not grow anaerobically on glycerol-fumarate. Two subgroups were distinguished. Vitamin K5 restored wild-type phenotype in subgroup IIIa only; vitamin K1 restored wild-type phenotype in both IIIa and IIIb isolates. The genotypic designation men (menaquinone) was suggested for group III isolates. The mutation in IIIa mutants was cotransducible with glpT, which corresponds to the menBCD site in Escherichia coli. That in IIIb mutants was cotransducible with glpK, which corresponds to the menA site in E. coli. Expression of the lac operon in IIIa, but not IIIb, mutants was repressed by air. An additional mutant group isolated on the same medium consisted of strains defective in formate hydrogenlyase.     

Anaerobiosis induces expression of ant, a new Escherichia coli locus with a role in anaerobic electron transport

Escherichia coli has a formate hydrogenlyase system which allows it to maintain an electron balance during anaerobic growth by passing electrons from formate to H+ ions, thus generating H2. The Mu d1(Ap lac) bacteriophage was used to generate mutants that were defective in passing electrons from formate to benzyl viologen, an artificial electron acceptor. A subset of these mutants was studied in which beta-galactosidase was expressed at much higher levels under anaerobic conditions than under aerobic conditions. If nitrate was present during anaerobic growth, the same levels of beta-galactosidase were seen in these fusion strains as were seen under aerobic conditions. The Mu d1(Ap lac) insertions in these mutants were genetically mapped between mutS and srl and thus define a new locus we have termed ant (anaerobic electron transport). Recombinant lambda derivatives were isolated which complemented the deficiency of the ant mutants in anaerobic electron transport and also carried a trans-acting region of DNA which reduced expression of the ant-lac fusions under anaerobic conditions; a probe to the ant region was generated from one of these recombinant lambda derivatives. Southern hybridization analysis revealed that the four independent ant::Mu d1(Ap lac) fusions we isolated spanned an approximately 5-kilobase region and that all were transcribed in the same direction, counterclockwise on the E. coli genetic map.     

UGA Nonsense Mutations in Salmonella typhimurium

Salmonella typhimurium strain LT-2 carries a weak UGA suppressor activity. This activity prevents the detection of some UGA mutants as auxotrophs and probably accounts for the rarity of his UGA mutants in this strain. A selection method is described which permits the isolation of these rare his UGA mutants. Map distribution of his UGA mutations is normal, and their polarity effects are indistinguishable from the polarity effects of amber and ochre mutations at similar locations. Isolation and properties of a prototrophic his UGA mutant are described. UGA mutants are common among lac mutants isolated from Salmonella strains carrying an F'lac episome. Apparently the suppressor activity is insufficient to prevent detection of lac UGA mutants. It is not yet clear whether the suppressor activity plays an important role in normal cell physiology.     

Fusions of the lac and trp Regions of the Escherichia coli Chromosome

Two classes of strains were studied in which the lac operon is transposed to a chromosomal site close to the tonB and trp loci. The two classes differ in the orientation of the lac region on the chromosome. In both types of strains, tonB mutants were selected in which deletions removing the tonB locus also caused a fusion of the lac and trp regions. The study of the properties of such fusion strains provides information on the control of both the lac and trp operons.     

Structural investigation of the transmembrane C domain of the mannitol permease from Escherichia coli using 5-FTrp fluorescence spectroscopy

The mannitol transporter EII(mtl) from Escherichia coli is responsible for the uptake of mannitol over the inner membrane and its concomitant phosphorylation. EII(mtl) is functional as a dimer and its membrane-embedded C domain, IIC(mtl), harbors one high affinity mannitol binding site. To characterize this domain in more detail the microenvironments of thirteen residue positions were explored by 5-fluorotryptophan (5-FTrp) fluorescence spectroscopy. Because of the simpler photophysics of 5-FTrp compared to Trp, one can distinguish between the two 5-FTrp probes present in dimeric IIC(mtl). At many labeled positions, the microenvironment of the 5-FTrps in the two protomers differs. Spectroscopic properties of three mutants labeled at positions 198, 251, and 260 show that two conserved motifs (Asn194-His195 and Gly254-Ile255-His256-Glu257) are located in well-structured parts of IIC(mtl). Mannitol binding has a large impact on the structure around position 198, while only minor changes are induced at positions 251 and 260. Phosphorylation of the cytoplasmic B domain of EII(mtl) is sensed by 5-FTrp at positions 30, 42, 251 and 260. We conclude that many parts of the IIC(mtl) structure are involved in the sugar translocation. The structure of EII(mtl), as investigated in this work, differs from the recently solved structure of a IIC protein transporting diacetylchitobiose, ChbC, and also belonging to the glucose superfamily of EII sugar transporters. In EII(mtl), the sugar binding site is more close to the periplasmic face and the structure of the 2 protomers in the dimer is different, while both protomers in the ChbC dimer are essentially the same.     

Membrane protein-ligand interactions in Escherichia coli vesicles and living cells monitored via a biosynthetically incorporated tryptophan analogue.

This paper presents a deceptively straightforward experimental approach to monitoring membrane protein-ligand interactions in vesicles and in living Escherichia coli cells. This is achieved via the biosynthetic incorporation of 7-azatryptophan, a tryptophan analogue with a red-shifted absorption spectrum, allowing collection of the emission signal of the target protein in a high tryptophan background via red-edge excitation. The approach is demonstrated for the mannitol permease of E. coli (EII(mtl)), an integral membrane protein of 637 amino acids, including four tryptophans, and single-tryptophan mutants of EII(mtl). By using a tryptophan auxotroph, a high level of 7-azatryptophan incorporation in EII(mtl) was achieved. The change in emission signal of the purified enzyme upon mannitol binding (-28%) was 4-fold larger than with EII(mtl) containing tryptophan, demonstrating the known higher sensitivity of this analogue for changes in the microenvironment [Schlesinger, R. (1968) J. Biol. Chem. 243, 3877-3883]. Changes in emission signal could also be monitored (-5%) when the enzyme was situated in vesicles, although it constituted only 10-15% of the total cytoplasmic membrane fraction. Of the five single-tryptophan mutants, the emission signal of the mutant with 7-azatryptophan at position 198 was the most sensitive for mannitol binding. Changes in emission signal not only were observed in vesicles (-18%) but also could be monitored in viable cells (-5%). The fact that only modest expression levels and no protein purification are needed makes this a useful approach for the characterization of numerous protein systems under in vitro and in vivo conditions.     

Gene expression in E. coli after treatment with streptozotocin

Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.     

LAC4 Is the Structural Gene for β-Galactosidase in KLUYVEROMYCES LACTIS

Using genetic and biochemical techniques, we have determined that β-galactosidase in the yeast Kluyveromyces lactis is coded by the LAC4 locus. The following data support this conclusion: (1) mutations in this locus result in levels of β-galactosidase activity 100-fold lower than levels in uninduced wild type and all other lac^- mutants; (2) three of five lac4 mutations are suppressible by an unlinked suppressor whose phenotype suggests that it codes for a nonsense suppressor tRNA; (3) a Lac⁺ revertant, bearing lac4–14 and this unlinked suppressor, has subnormal levels of β-galactosidase activity, and the K_m for hydrolysis of o-nitrophenyl-β, D-galactoside and the thermal stability of the enzyme are altered; (4) the level of β-galactosidase activity per cell is directly proportional to the number of copies of LAC4; (5) analysis of cell-free extracts of strains bearing mutations in LAC4 by two-dimensional acryl-amide gel electrophoresis shows that strains bearing lac4–23 and lac4–30 contain an inactive β-galactosidase whose subunit co-electrophoreses with the wild-type subunit, while no subunit or fragment of the subunit is observable in lac4–8, lac4–14 or lac4–29 mutants; (6) of all lac4 mutants, only those bearing lac4–23 or lac4–30 contain a protein that cross-reacts with anti-β-galactosidase antibody, a finding consistent with the previous result; and (7) β-galactosidase activity in several Lac⁺ revertants of strains carrying lac4–23 or lac4–30 has greatly decreased thermostability.     

Transcriptional regulation of genes required for antibiotic TA synthesis in Myxococcus xanthus

Abstract Regulatory mutants were isolated for six genes required for the production of antibiotic TA in Myxococcus xanthus , using Tn5lac as a promoter probe. Two of the regulatory mutants were closely linked to their corresponding Tn5lac insertions and three were separated by at least 40 kb from their original insertions. All of the β-galactosidase-overproducing mutants showed higher specific activities than their parent strains in three different media. The specific activity was higher throughout the growth cycle, starting earlier and finishing higher. In addition to helping to understand the mechanism of repression of TA genes, the mutants are potentially useful in the construction of antibiotic-overproducing strains.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.     

Thymidylate synthase gene from Lactococcus lactis as a genetic marker: an alternative to antibiotic resistance genes.

The potential of the thymidylate synthase thyA gene cloned from Lactococcus lactis subsp. lactis as a possible alternative selectable marker gene to antibiotic resistance markers has been examined. The thyA mutation is a recessive lethal one; thyA mutants cannot survive in environments containing low amounts of thymidine or thymine (such as Luria-Bertani medium) unless complemented by the thyA gene. The cloned thyA gene was strongly expressed in L. lactis subsp. lactis, Escherichia coli, Rhizobium meliloti, and a fluorescent Pseudomonas strain. In addition, when fused to a promoterless enteric lac operon, the thyA gene drove expression of the lac genes in a number of gram-negative bacteria. In transformation experiments with thyA mutants of E. coli and conjugation experiments with thyA mutants of R. meliloti, the lactococcal thyA gene permitted selection of transformants and transconjugants with the same efficiency as did genes for resistance to ampicillin, chloramphenicol, or tetracycline. Starting from the broad-host-range plasmid pGD500, a plasmid, designated pPR602, was constructed which is completely free of antibiotic resistance genes and has the lactococcal thyA gene fused to a promoterless lac operon. This plasmid will permit growth of thyA mutant strains in the absence of thymidine or thymine and has a number of unique restriction sites which can be used for cloning.     

Isolation of nonsense suppressor mutants in Pseudomonas.

A strain of Escherichia coli harboring the drug resistance plasmid RP1 was treated with the mutagen N-methyl-N-nitro-N-nitro-N-nitrosoguanidine, and mutants were isolated in which ampicillin resistance had been lost due to an amber mutation in the plasmid. One of these mutants was again treated, and a strain was isolated in which tetracycline resistance was also lost due to an amber mutation in the plasmid. The plasmid containing amber mutations in the genes amp and tet was named pLM2. This plasmid could be transferred to strains of Pseudomonas aeruginosa, P. phaseolicola, and P. pseudoalcaligenes. Mutants resistant to ampicillin and tetracycline could not be obtained from P. phaseolicola carrying pLM2. However, strains of E. coli, P. aeruginosa, and P. pseudoalcaligenes carrying the plasmid did produce mutants simultaneously resistant to both antibiotics. All of the mutants of E. coli had developed nonsense suppressors since they became phenotypically lac+, although harboring a lac amber mutation, and formed plaques with amber mutants of phages PRR1 and PRD1 that attack organisms carrying RP1. Approximately 20% of the resistant mutants of P. aeruginosa and P. pseudoalcaligenes were sensitive to the amber mutant of PRD1. These mutants were of variable stability and grew somewhat more slowly than their parent strains. One of the suppressor mutants of P. pseudoalcaligenes, designated ERA(pLM2)S4, was used for the isolation of nonsense mutants of bacteriophage PHA6, a virus having a segmented genome of double-stranded ribonucleic acid and an envelope of lipids and proteins.