Genetic analysis of two spored asci produced by the spo3 mutant of Saccharomyces

Summary Ascospores present in two-spored asci formed by spo3-1 diploids at a semipermissive temperature (30°C) represent random inclusion of haploid genomes into ascospores and exhibit normal viability, meiotic chromosome segregation and recombination. Genetic analysis and ultrastructural studies indicate that the function encoded by the spo3 locus is specifically required for the enclosure of the products of meiosis in prospore walls.Suppored by NSF grant GB-27688, the Wallace C. and Clara Abbott Memorial Fund from the University of Chicago, and Cancer Center Grant CCRC IIIB3.     

Erythromycin resistant mutations in Bacillus subtilis cause temperature sensitive sporulation.

Summary All of several hundred erythromycin resistant (ery^R) single site mutants ofBacillus subtilis W168 are temperature sensitive for sporulation (spo^ts). 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 ery^R and spo^ts 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 ery^R spo^ts 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.     

Stringent control of protein synthesis in E. coli

The effect of ppGpp on the rate of protein synthesis has been determined in vivo. When the stringent response is triggered and then reversed in isogenic strains carrying either spoT^? or spoT⁺, the mutants lower their elevated ppGpp only slowly, whereas wild type cells do so rapidly. Protein synthesis resumes only after a lag in spoT^? cells but almost immediately in spoT⁺ cells. In spoT^?rel^? double mutants, ppGpp does not accumulate under these conditions and protein synthesis resumes immediately. Inhibition of protein synthesis in spoT^?rel⁺ cells therefore appears to be due to elevation of ppGpp levels and not to any other effect of the spoT^? mutation.     

Aberrant nuclear behavior at meiosis and anucleate spore formation by sporulation-deficient (SPO) mutants of Saccharomyces cerevisiae

The fine structural characteristics of wild-type and sporulation-deficient mutants (spo) of yeast were examined. The results indicate that prospore wall formation, growth and closure, and nuclear budding and separation at meiosis represent parallel and normally coordinated developmental pathways of morphological change whose integration can be disrupted by gene mutation. At the restrictive temperature most cells of spo 1-1/spo 1-1 diploids terminate prior to the first spindle body duplication. In spo 2-1/spo 2-1 diploids the nucleus divides precociously both at meiosis I and at meiosis II. This aberrant behavior is followed by the formation of anucleate spores. In spo 3-1/spo 3-1 diploids development is normal until meiosis II. At this point nuclear segregation becomes retarded relative to ascospore delimitation. As a result much of the nuclear material fails to be incorporated into the ascospores.     

Single-gene regulated non-spore-forming Bacillus subtilis: Construction,transcriptome responses,and applications for producing enzymes and surfactin

Bacillus subtilis, a spore-forming industrial bacterium, is widely used for production of enzymes and valuable chemicals. The spore-formation, however, always results in remarkably reduced cell-density, thereby reducing product yield. Here, we constructed different non-spore-forming B. subtilis mutants via single-gene regulation. During the three spore-forming stages: signal sensing, transduction, and sporulation, we found that deleting only a single gene of sporulation, i.e. spo0A, spoIIIE, and spoIVB, can completely block the spore generation. Interestingly, the engineered non-sporulating mutants exhibited physiological heterogeneity and distinct synthetic capabilities. The spo0A-null spore-free mutant displayed remarkably high enzyme production capacity, such as 194% enhance amylase production. However, the spoIVB-null non-spore-forming mutant was especially efficient in producing secondary metabolites, such as surfactin; its flask titer increased significantly to 16.7 g/L, with the overexpression and Leu addition strategy. Our results offer a new strategy for re-modeling B. subtilis to further improve its fermentation efficiency and application.     

Terminal synthesis of xanthommatin in Drosophila melanogaster. I. Roles of phenol oxidase and substrate availability

Eye color mutants of Drosophila melanogaster are known which block the conversion of 3-hydroxykynurenine to xanthommatin. It has been proposed that this reaction depends on the presence of 3-hydroxykynurenine and a redox system maintained by phenol oxidase activity. The mutants st and ltd lack throughout development detectable amounts of 3-hydroxykynurenine or its metabolic derivatives. When the substrate is fed or injected, these mutants fail to form xanthommatin even though phenol oxidase activity is normal. The mutant cd accummulates excessive amounts of 3-hydroxykynurenine, has normal phenol oxidase activity, but is also deficient in xanthommatin formation. Mutants are also known which lack phenol oxidase activity but nevertheless form xanthommatin. It is concluded that the proposed relationship between 3-hydroxy-kynurenine and phenol oxidase activity is not sufficient to explain the in vivo synthesis and regulation of synthesis of xanthommatin in Drosophila. The bearing of these findings on the actual mode of synthesis is discussed.Supported by PHS 1029 and NSF GB-4539.     

Cloning and sequencing of the leu C and npr M genes and a putative spo IV gene from Bacillus megaterium DSM319

The leuC gene, encoding 3-isopropylmalate dehydrogenase, the nprM gene (neutral protease) and a sporulation gene coding for a putative spoIV protein (spoIV) from Bacillus megaterium DSM319 were cloned and the nucleotide sequences were determined. The leuC gene is 1101 bp in length, preceded by a ribosome binding site; no promoter consensus sequence could be found. The nucleotide sequence from nprM when compared to the recently published gene from B. megaterium ATCC 14581 exhibited only a 17-base pair deviation. From a sporulation mutant isolated after transposonmutagenesis with transposon Tn917 the insertion site of the transposon was cloned and adjacent chromosomal fragments were characterized. An open reading frame that encodes for a putative spo protein of 247 amino-acid residues was identified.Sequence data presented in this contribution are part of doctoral theses of the Naturwissenschaftliche Fakultät Münster, Germany nprM (KDW); leuC and spoIV (MB)     

Relaxed mutants ofSerratia marcescens SM-6. Biochemical traits and relevance of therel + allele for the formation of exoenzymes

Serratia marcescens SM-6 when starved for a required amino acid stops synthesizing protein and RNA and accumulates two nucleotides which cochromatograph with ppGpp and pppGpp. These features are characteristic of bacterial strains with stringent RNA control (rel ⁺). Two independent mutants were isolated which resemble relaxed (relA) mutants ofEscherichia coli; they continue to synthesize RNA and accumulate neither ppGpp nor pppGpp when deprived of the required amino acid. The extracellular enzyme activities (nuclease, protease, lipase) of the relaxed mutants are about the same as those of the parental stringent strain when studied under standard growth conditions. Exoenzyme-deficient (nuc; prt) and exoenzyme-hyperproducing (nuc ^su) mutants were isolated from both stringent and relaxed strains ofS. marcences SM-6 and no change of the cellular ability to form ppGpp and pppGpp could be observed. From these results it appears that the formation of exoenzymes ofS. marcescens SM-6 is independent of stringent/relaxed RNA control.Abbreviations cpd cyclic nucleotide phosphodiesterase deficient - nuc nuclease deficient - nuc ^su nuclease hyperproducing - prt protease deficient - rel relaxed control - spo ppGpp deficient (spot less) - ppGpp guanosine tetraphosphate - pppGpp guanosine pentaphosphate - TCA trichloroacetic acid - OD optical density - EU enzyme units     

Electron microscopic examination of sporulation-deficient mutants of the fission yeast Schizosaccharomyces pombe

A homothallic haploid strain of the fission yeast Schizosaccharomyces pombe initiates sexual reproduction (mating, meiosis and sporulation) in nitrogen-free sporulation medium. Cellular fine structures of eleven sporulation-deficient mutants (spo2, spo3, spo4, spo5, spo6, spo13, spo14, spo15, spo18, spo19 and spo20) of S. pombe in sporulation medium were examined by serial section-electron microscopy. The striking features of these spo mutants were: 1) the disappearance of the spindle pole bodies (SPBs) after the second meiotic division, and 2) the accumulation of unorganized structures. Based on histochemical staining, these structures were presumably unorganized spore wall precursors. In some mutants (spo3, spo5, spo6, spo19 and spo20), diploid zygotes contained four spore-like bodies which had walls similar to complete spore walls but failed to enclose any nuclei. After completion of the second meiotic division the nuclei were abnormally distributed in zygotic diploid cells. In the spo5, spo13, spo14, spo15 and spo19 mutants, the nuclei remained attached to each other. In spo5 and spo19, the inner membrane of the nuclear envelope was separated, but its outer membrane was shared by two sister nuclei. These observations suggest that the spo⁺ gene products play important roles in spatial and temporal organization of cellular structures during ascospore development.Abbreviations SPB spindle pole body - PTA-Cr phosphotungstic acid and chromic acid - PATAg periodic acid, thiocarbohydrazide and silver proteinate     

Characterization and Identification of a Novel Marine Streptomyces sp. Produced Antibacterial Substance

Strain GB-2 is a marine microbe with broad-spectrum antimicrobial activity, isolated from soil taken from the coastal city Lianyungang in the JiangSu province of China. Analysis of its morphological, physiological, and biochemical characteristics as well as chemical components of the cell wall strongly suggested that the strain GB-2 belonged to the Streptomyces sp. Analysis of the nucleotide sequence of the 16S rRNA gene of Streptomyces sp. GB-2 strain showed a strong similarity (98%) with the 16 rRNA gene of Streptomyces fradiae. Application to antibacterial substance of strain Streptomyces sp. GB-2 by various separation steps led to isolation of one active molecule having a retention time of 9.495 min, P _9.495 min, which possessed antibacterial activity against Bacillus cereus and Escherichia coli. Through analysis by liquid chromatography–mass spectrometry and mass/mass spectrometry of the peak, the molecular weight of the antibacterial substance (P _9.495 min sample) was 447.5 Da and it was determined to be sisomicin according to the analysis of ion fragments. Nuclear magnetic resonance spectrum of the peak also demonstrated that the antibacterial substance was sisomicin. This study is the first to introduce the finding of sisomicin produced from marine Streptomyces sp. This work provides a preference for the production of sisomicin in pharmaceutical industries and a probability for studying the biodiversity of marine microbe.     

Cr(III) Oxidation and Cr Toxicity in Cultures of the Manganese(II)-Oxidizing Pseudomonas putida Strain GB-1

Abstract

Mn oxides have long been considered the primary environmental oxidant of Cr(III), however, since most of the reactive Mn oxides in the environment are believed to be of biological origin, microorganisms may indirectly mediate Cr(III) oxidation and accelerate the rate over that seen in purely abiotic systems. In this study, we examined the ability of the Mn(II)-oxidizing bacterium, Pseudomonas putida strain GB-1, to oxidize Cr(III). Our results show that GB-1 cannot oxidize Cr(III) directly, but that in the presence of Mn(II), Cr(III) can be rapidly and completely oxidized. Growth studies suggest that in growth medium with few organics the resulting Cr(VI) may be less toxic to P. putida GB-1 than Cr(III), which is generally considered less hazardous. In addition, Cr(III) present during the growth of P. putida GB-1 appeared to cause iron stress as determined by the production of the fluorescent siderophore pyoverdine. When stressed by Fe limitation or Cr(III) toxicity, Mn(II) oxidation by GB-1 is inhibited.     

Effect of organic acids on inhibition of Escherichia coli O157:H7 colonization in gnotobiotic mice associated with infant intestinal microbiota

Previously, we produced two groups of gnotobiotic mice, GB-3 and GB-4, which showed different responses to Escherichia coli O157:H7 challenge. E. coli O157:H7 was eliminated from GB-3, whereas GB-4 became carriers. In this study, we analysed the mechanisms of E. coli O157:H7 elimination using GB-3 and GB-4. When GB-3 and GB-4 mice were challenged with E. coli O157:H7, the E. coli O157:H7 population was reduced in the caecum of GB-3 when compared to that in the GB-4 caecum, although the numbers of E. coli O157:H7 in the small intestine were not significantly different between these two groups of gnotobiotic mice. The lag time of E. coli O157:H7 growth in a 50% GB-3 caecal suspension increased when compared to that in a GB-4 caecal suspension. Acetate and lactate were detected in the GB-3 caecal contents, and acetate and propionate in those from GB-4. Although E. coli O157:H7 growth was not suppressed when it was cultured in anaerobic broth supplemented with these organic acids, the motility of E. coli O157:H7 was suppressed when it was cultured on semi-solid agar supplemented with the combination of acetate and lactate. These results indicate that the organic acid profile in the caecum is an important factor related to the elimination of E. coli O157:H7 from the intestine.     

The coupling effects of some thiol and other sulfur-containing compounds on the circadian rhythm of cell division in photosynthetic mutants of Euglena

Previous work has demonstrated a persisting, free-running, circadian rhythm of cell division in the P₄ZUL photosynthetic mutant of the alga Euglena gracilis Klebs (Strain Z) Pringsheim grown organotrophically in continuous light or darkness at 19° C following prior synchronization by a repetitive LD: 10,14 light cycle. A similar circadian rhythmicity has been recently discovered in the W₆ZHL heat-bleached and the Y₉ZNalL naladixic acid-induced mutants of Euglena grown under comparable conditions. Over extended timespans, however, these mutants appear to gradually lose first their ability to display persisting overt rhythms, and then even their capability of being entrained by imposed LD cycles. These properties can be restored by the addition of certain sulfur-containing compounds to the medium including cysteine, methionine, dithiothreital, sodium monosulfide, sodium sulfite, and sodium thiosulfate, as well as thioglycolic [mercaptoacetic] acid. The implications of these findings toward biological clock mechanisms are discussed: It appears that some sort of coupling process is operating as opposed to the initiation of an underlying oscillation.Non-Standard Abbreviations LL continuous illumination - DD continuous darkness - LD repetitive light-dark cycle - SS stepsize - period of biological rhythm Supported by research grants (GB-36287, GB-43543) from the National Science Foundation.Reports on portions of this work were presented at the 19th Annual Meeting of the Biophysical Society, Philadelphia, Pennsylvania, February 19–21, 1975; at the XII International Botanical Congress, Leningrad, U.S.S.R., July 3–10, 1975; and at the XII International Conference of the International Society for Chronobiology, Washington, D.C., August 10–13, 1975.     

Preferential recovery of uniparental streptomycin resistant mutants from diploid Chlamydomonas reinhardtii

Summary Analysis of the streptomycin resistant mutants recovered from control and N-methyl-N-nitro-N-nitrosoguanadine (MNNG) treated haploid cultures of C. reinhardtii reveal that approximately 60% of the mutants are of the sr-1 type known to show Mendelian inheritance while 40% are of the sr-2 and sm-3 types known to be inherited in a uniparental (UP) manner. In contrast, most if not all streptomycin mutants recovered from similarly treated diploid cultures of C. reinhardtii are of the UP variety. Failure to recover sr-1 mutants from the diploid stock is explained by our findings that diploids heterozygous for Mendelian streptomycin resistance (sr-1/sr-1 ⁺) are both stable and sensitive to streptomycin. Efficient recovery of UP streptomycin resistant mutants from diploids can be explained by the observations of Gillham (1963a, 1969) which demonstrate that diploids heterozygous for an sr-2 mutation (sr-2/sr-2 ⁺) segregate resistant and sensitive progeny during mitotic cell division.The utility of diploids for isolating new UP mutant genotypes, for establishing the cellular localization of the UP genome(s), and for characterizing the rules governing UP gene segregation is discussed.Supported by NIH postdoctoral fellowship GM 52359 to R.W.L., NIH predoctoral traineeship GM 02007 to K.P.V., and NSF grant GB-22769 to N.W.G. and J.E.B.     

spoT,a new genetic locus involved in the stringent response in E. coli

A new genetic locus, spoT, whose product is involved in the stringent response to amino acid starvation, maps very close to 72 min on the E. coli chromosome. The locus is defined by a spontaneous mutant allelle, spoT-, whose phenotypic effects are: virtually no pppGpp is produced during amino acid starvation; ppGpp is overproduced; and the stability of ppGpp upon reversal of the stringent response is greatly increased. All three phenotypic effects are recessive to wild type in heterozygotes. These three phenotypic effects are best accounted for by postulating that the spoT gene product plays a role in the phosphorylation of ppGpp to pppGpp preparatory to further metabolism. Since the stability of ppGpp has also shown to increase following a carbon and energy source downshift (Gallant, Margason, and Finch, J. Biol. Chem. 247: 6055), we suggest that the activity of the spoT gene product is regulated by some consequence of downshift.     

Gammarus desperatus,a new species from New Mexico (Crustacea : Amphipoda)

A new species of the Amphipoda (Gammarus desperatus) is described from North Spring in Roswell, Chaves County, New Mexico. Apparently this is the same species reported erroneously as G. fasciatus Say from nearby Lander Springbrook by Noel (1954).Supported in part by NSF Grants GB-2461 and GB-6477X to W. L. MinckleySupported in part by NSF Grants GB-2461 and GB-6477X to W. L. Minckley     

Genetic variation in Hawaiian Drosophila. VIII. Heterozygosity and genic changes in isolated populations of D. engyochracea

Drosophila engyochracea, an endemic Hawaiian fly found only in two, finite populations in Volcano National Park, has extensive electrophoretic heterozygosity on a par with that found in species with much wider distributions. A study of six polymorphic loci in both populations over an 18-month period revealed that the population in the more xeric environment is more dynamic genetically as well as more variable. In addition, genetic changes at one locus, Pgm, are correlated to changes in an environmental moisture parameter. These findings confirm that migration is not necessary to maintain genetic variation in isolated populations and demonstrate that D. engyochracea gene pools are susceptible to errors in Hardy-Weinberg equilibria during specific seasonal periods.Portions of this article were submitted in partial fulfillment for a Doctor of Philosophy degree in Genetics awarded to W. W. M. Steiner by the Department of Genetics, University of Hawaii, Honolulu. Research was supported by NSF Grants GB-23230, GM-27586, and GB-29288.     

Geographic variation of the lactate dehydrogenases ofRana pipiens andRana palustris

Electrophoretic, immunological, catalytic, and enzyme recombination studies were carried out on the HLDHs, and to a lesser extent on the MLDHs, of many populations of Rana pipiensand R. palustris.HLDH is far more variable from one population to the next than is MLDH, which appears to be identical in both species as far as can be determined by the methods employed. Of all the methods used, electrophoresis is capable of distinguishing the most differences between populations. However, two electrophoretically identical groups of enzymes can be differentiated immunologically. The geographic distribution of HLDH variability is generally concordant with the distributions of different mating calls, with the results of hybridization studies, and also with the old subspecies distributions. Eleven different forms of HLDH have been discovered so far in the R. pipienscomplex. This work was supported in part by research grants from the National Aeronautics and Space Administration (NSG-375), the National Science Foundation (GB-1701), and the American Cancer Society (P-77G) administered by N. O. Kaplan, and from the National Science Foundation (GB-5232 and GB-7749) administered by the author. During part of the period of this research, the author was an American Cancer Society postdoctoral fellow, PF-217.