Terrance Leighton

BACTERIAL REDUCTION OF SELENITE TO ELEMENTAL SELENIUM

Abstract It is well known that many microorganisms can reduce the highly toxic soluble selenium oxyanions (selenate and selenite) to a much less toxic insoluble form, namely elemental selenium. We are developing a bacterial treatment system to mitigate selenium-contaminated waste streams using Pseudomonas fuorescens and Bacillus subtilis , respectively, as model Gram (−) and (+) soil bacteria. Our studies demonstrate that both strains reduce selenite to elemental selenium. The selenite detoxification system is independent of nitrite and sulfite electron acceptors, but dependent on the substrate provided for growth. Elemental selenium is deposited as granules throughout the cell, or between the cell wall and the plasma membrane in P. fuorescens and B. subtilis , respectively. In B. subtilis , thioredoxin is induced in the presence of selenite.

Pesticides: Insecticides and Fungicides Are Chitin Synthesis Inhibitors

Several important groups of fungicides and insecticides are specific inhibitors of chitin synthesis in a Phycomyces enzyme system and in insect organ cultures. The recently discovered benzoylphenylurea insecticides, which prevent chitin synthesis in insect tissues, are apparently not direct-acting chitin synthetase inhibitors. These insecticides may prevent insect chitin synthesis by interfering with the proteolytic activation of the chitin synthetase zymogen.

Postexponential Regulation of sin Operon Expression in Bacillus subtilis

The expression of many gene products required during the early stages of Bacillus subtilis sporulation is regulated by sinIR operon proteins. Transcription of sinIR from the P1 promoter is induced at the end of exponential growth. In vivo transcription studies suggest that P1 induction is repressed by the transition-state regulatory protein Hpr and is induced by the phosphorylated form of Spo0A. In vitro DNase I footprinting studies confirmed that Hpr, AbrB, and Spo0A are trans-acting transcriptional factors that bind to the P1 promoter region of sinIR. We have also determined that the P1 promoter is transcribed in vitro by the major vegetative sigma factor, final sigma(A), form of RNA polymerase.

Bioavailability of selenium accumulated by selenite-reducing bacteria

The bioavailability of selenium (Se) was determined in bacterial strains that reduce selenite to red elemental Se (Seo). A laboratory strain ofBacillus subtilis and a bacterial rod isolated from soil in the vicinity of the Kesterson Reservoir, San Joaquin Valley, CA, (Microbacterium arborescens) were cultured in the presence of 1 mM sodium selenite (Na2SeO3). After harvest, the washed, lyophilizedB. subtilis andM. arborescens samples contained 2.62 and 4.23% total Se, respectively, which was shown to consist, within error, entirely of Seo. These preparations were fed to chicks as supplements to a low-Se, vitamin E-free diet. Three experiments showed that the Se in both bacteria had bioavailabilities of approx 2% that of selenite. A fourth experiment revealed that gray Seo had a bioavailability of 2% of selenite, but that the bioavailability of red Seo depended on the way it was prepared (by reduction of selenite). When glutathione was the reductant, bioavailability resembled that of gray Seo and bacterial Se; when ascorbate was the reductant, bioavailability was twice that level (3–4%). These findings suggest that aerobic bacteria such asB. subtilis andM. arborescens may be useful for the bioremediation of Se-contaminated sites, i.e., by converting selenite to a form of Se with very low bioavailability.

Erythromycin resistant mutations inBacillus subtilis cause temperature sensitive sporulation

SummaryAll of several hundred erythromycin resistant (eryR) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spots). The mutants and wild type cells grow vegetatively at essentially the same rates at both permissive (30° C) and nonpermissive (47° C) temperatures. In addition, cellular protein synthesis, cell mass increases and cell viabilities are similar in mutant and wild type strains for several hours after the end of vegetative growth (47° C). In the mutants examined, the temperature sensitive periods begin when the sporulation process is approximately 40% completed, and end when the process is 90% complete. At nonpermissive temperatures, the mutants produce serine and metal proteases at 50% of the wild type rate, accumulate serine esterase at 16% of the wild type rate, and do not demonstrate a sporulation related increase in alkaline phosphatase activity.The eryR and spots phenotypes cotransform 100%, and cotransduce 100% using phage PBS1. Revertants selected for ability to sporulate normally at 47° C (spo+), simultaneously regain parental sensitivity to erythromycin. No second site revertants are found.Ribosomes from eryR spots strains bind erythromycin at less than 1% of the wild type rate. A single 50S protein (L17) from mutant ribosomes shows an altered electrophoretic mobility. Ribosomes from spo+ revertants bind erythromycin like parental ribosomes and their proteins are electrophoretically identical to wild type. These data indicate that the L17 protein of the 50S ribosomal subunit fromBacillus subtilis may participate specifically in the sporulation process.

Intergenic suppression of spoO phenotypes by the Bacillus subtilis mutation rvtA.

SummaryA collection of intergenic suppressors of the Bacillus subtilis spoOF221 mutation has been isolated. One of these suppressors, rvtA, has been mapped between lys-1 and aroD. The rvtA suppressor restores spoOF sporulation to wild type levels and substantially improves the sporulation efficiencies of spoOB and spoOE strains. The rvtA gene does not affect the Spo phenotype of spoOH, spoOJ or spoOK mutants. The rvtA gene also prevents the induction by aliphatic alcohols of SpoO phenocopies in wild type B. subtilis cells.

AbrB and Spo0E control the proper timing of sporulation in Bacillus subtilis

We have shown previously that Spo0A∼P-dependent sinIR operon expression was substantially down-regulated in abrB null mutant backgrounds. In this report, we show that loss of function mutations in abrB also cause phosphorelay gene expression to be down regulated. abrB null mutations caused diminished vegetative growth-associated sporulation and resulted in a significant reduction in sporulation frequencies at T24. These mutants, however, sporulated at wild-type levels at T48, indicating that sporulation timing was affected. The rvtA11 mutation in spo0A, a deletion mutation in spo0E, and a null mutation in hpr (scoC) rescued sporulation and Spo0A∼P-dependent gene expression in an abrB mutant background. These data indicate that AbrB and Spo0E may comprise a checkpoint system that regulates the progression of sporulation, allowing exploration of alternate cell states prior to the irrevocable commitment to sporulation.

Intergenic suppressors of temperature-sensitive sporulation in Bacillus subtilis are allele non-specific

SummaryThe Bacillus subtilis mutant cal1 carries a non-reverting mutation in ribosomal protein L17 (r-protein L17) that causes both resistance to the antibiotic chalcomycin (Calr) and temperature-sensitive sporulation (Spots). Second-site suppressor (rev) mutations that relieve the Spots phenotype have been isolated from cal1. Three suppressor mutations—rev4, rev10, and rev11—each increase the sporulation frequency of cal1 at the nonpermissive temperature from 3% to 95% of the wild-type level. The cal1 rev strains remain resistant to chalcomycin and twodimensional gel electrophoresis analysis indicates that they contain the same altered r-protein L17 as the original cal1 strain and no additional altered r-proteins. The three rev mutations have been mapped at a single locus between narA and sacA on the B. subtilis chromosome and recombination indexes for the rev mutations indicate that they are tightly linked to one another.Antibiotic resistance Spots mutations that cause temperature-sensitive sporulation have previously been isolated in RNA polymerase, in the 30S and 50S subunits of the ribosome, and in elongation factor G. The rev4, 10, and 11 suppressor mutations are non-specific in their action in that they restore significant levels of sporulation at the non-permissive temperature in all of the Spots strains that we have tested. This result suggests that Spots mutations in components of the B. subtilis transcription and translation systems share a common molecular basis for their sporulation-defective phenotypes.

The distribution of mutagenic activity in red, rose and white wines

Using a modified Salmonella typhimurium TA98 Ames-test system, more than 150 red, white and rose wines were analyzed for direct-acting and microsomal enzyme-enhanced mutagenic activity. The following conclusions were reached from analysis of this wine mutagenicity data base. White and rose wines, as well as grape juices, exhibited little or no detectable direct-acting or microsomal enzyme-enhanced mutagenic activity. However, red wine samples contained highly variable amounts of mutagens, ranging from undetectable to levels 30-fold above the sensitivity limit of the assay system. The variations in red wine mutagenicity were unrelated to grape variety, vintage, aging methods or production region. Hence, individual winery production practices must represent the most significant contribution to the variations observed.

Suppression of defective-sporulation phenotypes by theBacillus subtilis mutationrev4

SummaryTheB. subtilis intergenic suppressor mutationrev4 suppresses defective-sporulation phenotypes caused by several mutations in RNA polymerase and in components of the ribosome. These suppressible mutations cause either temperature-sensitive sporulation (Spots) or oligosporogenous phenotypes, arresting sporulation at temporal stages 0 to IV. Double mutants containing Spots lesions in both RNA polymerase and the ribosome are also suppressible. In addition to strains altered in the transcription and translation systems, spontaneous Spots mutants and oligosporogenousspoOA andspoOK mutants respond torev4 suppression. Finally, Spots phenocopies, induced in wild-typeB. subtilis by the addition of the antibiotic cerulenin, ethanol, or phenethyl alcohol to sporulating cultures, are alleviated by therev4 mutation, suggesting thatrev4-suppressible sporulation phenotypes may be associated with defective membrane structure or function.