Genotypic exclusion: a novel relationship between the ribitol-arabitol and galactitol genes of E. coli

Summary Genetic studies indicate that the E. coli C chromosomal genes which are responsible for catabolism of the pentitol sugars, ribitol and D-arabitol, are not present in the closely related E. coli K12 strains (Reiner 1975). Molecular studies of these tightly linked genes reveal that they are surrounded by 1.4 kilobase inverted repeats of imperfect homology (Link and Reiner 1982). Here we report that E. coli C lacks genes for catabolism of the hexitol sugar galactitol, genes which are present in E. coli K12. Furthermore, the ribitol-arabitol and galactitol genes, which show no mutual homology, are mutually exclusive when exchanged (by homologous recombination) between E. coli C and K12. Physical characterization of specialized transducing phages carrying the ribitol-arabitol or galactitol genes demonstrates that this exclusion results because these genes have identical locations in their respective chromosomes. This novel type of allelic relationship between nonhomologous genes has not been previously described in prokaryotes. Analysis of the catabolic capabilities of a collection of natural E. coli strains suggests that this exclusion relationship extends to strains in the natural E. coli population. We suggest an insertion/deletion model to account for the origins of this unusual gene arrangement.     

Occurrence and expression of β-galactosidase in dextran-producing Leuconostoc species

The occurrence and expression of -galactosidase among various dextran-producing Leuconostoc strains was determined. -Galactosidase was detected from four of twelve Leuconostoc strains tested. -Galactosidase in L. mesenteroides was induced by lactose and was repressed by glucose. Growth curves of L. mesenteroides on lactose indicated extended lag and late growth phases that were shortened when the inoculum was preexposed to lactose.     

σs-dependent overexpression offtsZ in anEscherichia coli K-12 rpoB mutant that is resistant to the division inhibitors DicB and DicF RNA

TheEscherichia coli genesdicF anddicB encode division inhibitors, which prevent the synthesis and activity, respectively, of the essential division protein FtsZ. A mutation at the C-terminal end of the RNA polymerase subunit renders cells resistant to both inhibitors. In the mutant strain the level of theftsZ gene product is higher than in the wild type. Disruption ofrpoS, which encodes the stationary phase sigma factor ^S, lowers FtsZ protein levels in the mutant, and partially restores sensitivity to the inhibitors.     

Fusion of two F-prime factors inEscherichia coli studied by electron microscope heteroduplex analysis

Summary A fused F prime factor was obtained from a mating of arecA donor carrying an F' factor containing the genesmetBJF, ppc andargECBH (KLF5) with arecA recipient carrying an F' factor containingatt80, trp andlac (F155). Lysogenization of this fused F-prime factor with cI⁸⁵⁷ h80 phage followed by thermoinduction produced the transducing phages 80dmetBJF and 80dppcargECBH. This kind of fusion provides a general procedure for the construction of transducing phages carrying genes from different regions of theE. coli genome. To understand the mechanism of this fusion, the parental F prime factors (F155 and KLF5) were analyzed by the electron microscope heteroduplex technique.F155 has a length of 176±3 kilobases including two substitutions. The F sequence 0 F-2.8 F has been substituted by 53 kb of chromosomal DNA including thelac operon and the F sequences 8.5 F-16.3 F has been substituted by 27 kb of a chromosomal sequence includingatt80 and thetrp operon.KLF5 contains 221±4 kilobases of DNA (molecular weight, 148 megadaltons). It contains complete F and the segment of theE. coli chromosome frompolA torif. The F sequence 2.8 F-8.5 F known to be involved in F specific recombination inrecA ⁺ andRecA backgrounds occurs twice on KLF5, once at each of the junctions of F DNA with chromosomal DNA. The population of closed circular plasmid molecules extracted from KLF5-containing strains is heterogeneous. It is proposed that this heterogeneity is due to intramolecular recombination events occurring in KLF5 between the duplicated 2.8 F-8.5 F sequences. Such recombination can account for the genetic instability of KLF5 observed in bothrecA ⁺ andrecA hosts. The F sequence 2.8 F-8.5 F (also called ) is one of the characterized integration sequences on F.A model for the fusion of the parental F prime factors is proposed in which recombination between sequences bringsatt80 close to themetBJF genes. This is followed by a deletion of an F'lac factor. The resulting fused F' factor still carries two sequences and is therefore expected to be unstable. The closed circular molecules isolated from the fused F' containing strains show two different sizes of molecules. Genetic and physical analyses of these molecules are in agreement with the predicted instability of the fused F' factor and the existance of the sequence in the 80dmet phages isolated from fused F' and previously analyzed by the electron microscope heteroduplex technique.     

The β-tubulin gene family evolution in theDrosophila montium subgroup of themelanogaster species group

The 1-, 2-, and 3-tubulin genes have been mapped by in situ hybridization on the polytene chromosomes of 11 selected species (15 strains) belonging to theDrosophila montium subgroup. Although the hybridization pattern among the strains of the same species does not differ, this pattern is significantly different among the species. The -tubulin genes in themontium subgroup seem to be organized in a cluster, or in a semi-cluster, or are completely dispersed. The clustered arrangement is found in the North-Oriental sibling speciesD. auraria, D. triauraria, andD. quadraria. The semi-clustered arrangement, wherein the 1 and 2 genes are located at the same locus while 3 is at a different one, appears in the South-Oriental speciesD. bicomuta, D. serrata, andD. birchii, as well as in the Afrotropical speciesD. diplacantha andD. seguyi. The complete separation of the genes is observed in the Indian speciesD. kikkawai andD. jambulina and in the Afrotropical speciesD. vulcana. Based on the above results, a possible mode of evolution of the -tubulin genes in the montium subgroup is attempted. In addition, phylogenetic relationships among themontium species are discussed. Correspondence to: Z.G. Scouras     

CYP175A1 from<Emphasis Type="Italic"> Thermus thermophilus</Emphasis> HB27, the first β-carotene hydroxylase of the P450 superfamily

The biological function of thermostable P450 monooxygenase CYP175A1 from Thermus thermophilus HB27 was studied by functional complementation in Escherichia coli. The gene product of CYP175A1 added hydroxyl groups to both rings of -carotene to form zeaxanthin (,-carotene-3,3-diol) in E. coli, which produces -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. In addition, spectroscopic methods revealed that E. coli carrying CYP175A1 and the cDNA of the Haematococcus pluvialis carotene ketolase was able to synthesise hydroxyechinenone. The predicted amino acid sequence of the enzyme from T. thermophilus does not show substantial similarity with other known -carotene hydroxylases, but 41% with the cytochrome P450 monooxygenase from Bacillus megaterium (CYP102A1, P450 BM3). It is concluded that CYP175 A1 represents a new type of -carotene hydroxylase of the P450 superfamily.     

Genetics of Ia-like alloantigens in chickens and linkage withB major histocompatibility complex

Two sets of backcross matings were performed to test for linkage between genes coding for the Ia-like antigens (Ia) and the B erythrocyte antigens (Ea-B) of the chicken. Evidence is presented which indicates that the la antigens are determined by a single codominant locus and that theEa-B and Ia loci are on the same chromosome. Failure to detect a single recombinant between theEa-B and Ia loci out of 208 progeny suggests close linkage of the two genes with a map distance of up to about 2 centimorgans. The Ia genes are thus included in theB major histocompatibility complex of the chicken.     

Elektronenmikroskopische Untersuchungen über Bildung und Struktur der Zygotenwand beiMicrasterias papillifera (Desmidiaceae)

Zusammenfassung Die Feinstruktur von Mesospor und Endospor reifer Zygoten vonMicrasterias papillifera wurde untersucht. Das für die Resistenz der Zygoten gegenüber ungünstigen Umweltbedingungen wichtige Mesospor besteht aus vier Schichten von unterschiedlicher Elektronendichte. Es ist insgesamt 460–500 nm dick. Die Schichten Mes a und Mes c bestehen aus akkrustierten, dicht gepackten globulären Elementen eines Stoffes, der dem Sporopollenin ähnlich ist. Die Schicht Mes b zeigt ein fibrilläres Grundgerüst, wahrscheinlich aus Zellulose, in das Stoffe inkrustiert sind, die nicht mit denen der Schichten Mes a und Mes c identisch, aber gegen Abbauversuche ähnlich résistent sind.Die Schicht Mes d ist eine Übergangsschicht zum Endospor. Zwischen die Zellulose-Mikrofibrillen in Streutextur sind isolierte Partikel des Materials der Schicht Mes c eingestreut. Das etwa 650 nm dicke Endospor ist eine Zelluloseschicht mit Streutextur. Es wird als Wand der sogenannten Keimblase bei der Zygotenkeimung nach Sprengung von Exospor und Mesospor stark gedehnt.

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Electron microscopical investigations on the structure and formation of the zygote wall inMicrasterias papillifera (Desmidiaceae) II. The structure of the mesospore and the endospore

Summary The mesospore (460–500 nm thick) which is responsible for the resistance of the zygote against desiccation during its resting period, consists of four layers of different electron density. The layers Mes a and Mes c are composed of densely packed amorphous globular elements of a substance resembling sporopollenine. The layer Mes b has a fibrillar, probably cellulosic frame. It is incrusted by a substance which is not identical with that of Mes a or Mes c but which shows a comparable resistance against degradation.The layer Mes d contains isolated particles such as in Mes c and cellulose microfibrils of the endospore. The endospore (650 nm thick) has foliate texture. This layer surrounds the protoplast after it has escaped from the ruptured exospore and mesospore during zygospore germination.

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Der Deutschen Forchungsgemeinschaft dank ich f:ur Sachbeihilfe.     

Comparison of the in situ survival and activity ofKlebsiella pneumoniae andEscherichia coli in tropical marine environments

A near-shore coastal mangrove island receiving untreated sewage and a coastal cove receiving rum distillery effluent in Puerto Rico were examined for their ability to support survival and activity ofKlebsiella pneumoniae andEscherichia coli. Pure cultures of both bacteria were monitored for 96 hours in situ at both locations using membrane diffusion chambers.K. pneumoniae survived at all sites as measured by AODC and Coulter Counter direct counts. However, at the mangrove island less than 20% of theK. pneumoniae population was active (AODC) after the first 3 hours and less than 10% of this population was respiring (INT). In contrast, the coastal area which was receiving rum distillery effluent was able to maintain 40% of theK. pneumoniae population in an active state with 90% respiring. TheE. coli population declined by two orders of magnitude at the mangrove island, but remained unchanged at the rum distillery outfall. TheE. coli population had a higher proportion of active cells and respiring cells thanK. pneumoniae at all sites. At the rum distillery site, theE. coli population was remarkable in that 95% remained active and 99% were respiring. This study suggests that, when sufficient organic loading exists,E. coli, a nonsurvivor, can overcome the bactericidal effects of tropical marine waters.K. pneumoniae, a survivor, could survive under all conditions but could not maintain the activity or respiration that theE. coli population could, even when high organic loads were present. Morphological changes related to nutrient stress in the tropical marine environment were apparent inE. coli, but not inK. pneumoniae. Based on physiological activityE. coli is just as much a survivor asK. pneumoniae in tropical marine waters.     

Genetic mapping of the component chains of Ia antigens

The genes encoding the two polypeptide chains ( and) that comprise the murine Ia antigens were localized within distinct regions of the major histocompatibility complex (MHC). This was accomplished by correlating allelic forms of the and chains with the MHC congenic strains of mice from which they were isolated. Allelic forms of and chains were distinguished by their unique structural markers, such as isoelectric points, amino acid sequences or peptide maps. The results indicate that the structural genes for both the and chains of I-A subregion antigens are located within the K to I-A genetic interval. In contrast, the gene encoding the chain of I-E subregion antigens is located outside of theI-E subregion and within the K to I-B genetic interval. These findings may have important implications for analysis of observations that complementation by twoI-region genes is sometimes required for development of immune responses.     

Effect of<Emphasis Type="Italic"> p</Emphasis>CO<Subscript>2</Subscript> and temperature on the boron isotopic composition of the zooxanthellate coral<Emphasis Type="Italic"> Acropora</Emphasis> sp.

Culture experiments were carried out with Acropora sp. (a branching scleractinian coral) in seawater at two pCO₂ conditions (438 and 725 µatm) and two temperatures (25 and 28 °C) in order to establish the pH and temperature dependence of the boron isotopic composition of the skeleton. A clear pCO₂ effect, but no temperature effect, on the coral boron isotope composition is seen. For corals cultured at normal pCO₂ (438 µatm), the ¹¹B of the skeleton was 24.0±0.2 at 25 °C, and 23.9±0.3 at 28 °C. The values of ¹¹B measured for corals cultured at higher pCO₂ (725 µatm) were lower: 22.5±0.1, and 22.8±0.1 at 25 and 28 °C, respectively. The ¹¹B of corals cultivated at both high and normal pCO₂ conditions are consistent with a dominant pH control, and are very close to that calculated from theoretical considerations. Thus, the corals do not seem to significantly alter ambient seawater for calcification with respect to pH. Co-variation between boron and carbon isotope values is explored.Communicated by: Guest Editor A. Grottoli     

Recombinational hotspot specific to female meiosis in the mouse major histocompatibility complex

Thewm7 haplotype of the major histocompatibility complex (MHC), derived from the Japanese wild mouseMus musculus molossinus, enhances recombination specific to female meiosis in theK/A interval of the MHC. We have mapped crossover points of fifteen independent recombinants from genetic crosses of thewm7 and laboratory haplotypes. Most of them were confined to a short segment of approximately 1 kilobase (kb) of DNA between theA 3 andA 2 genes, indicating the presence of a female-specific recombinational hotspot. Its location overlaps with a sex-independent hotspot previously identified in theMus musculus castaneus CAS3 haplotype. We have cloned and sequenced DNA fragments surrounding the hotspot from thewm7 haplotype and the corresponding regions from the hotspot-negative B10.A and C57BL/10 strains. There is no significant difference between the sequences of these three strains, or between these and the published sequences of the CAS3 and C57BL/6 strains. However, a comparison of this A3/A2 hotspot with a previously characterized hotspot in theE gene revealed that they have a very similar molecular organization. Each hotspot consists of two elements, the consensus sequence of the mouse middle repetitive MT family and the tetrameric repeated sequences, which are separated by 1 kb of DNA.The nucleotide sequence data reported in this paper have been submitted to the DNA Data Bank of Japan nucleotide sequence database and have been assigned the accession numbers d90007-9. Offprint requests to: T. Shiroishi.     

Expression inEscherichia coli K12 strains of two synthetic human calcitonin genes differing in codon composition

Two genes coding for a Val⁸-variant of the human calcitonin (hCT) are synthesized on two different codon biases: the native codons for the hCT gene and the codons preferential forEscherichia coli. Both genes are fused to a synthetic human interferon-gamma (IF) gene [6] and expressed in various strains ofE. coli K12. It is found that, in all host strains used, the level of expression of both genes is similar and much lower (1/50–1/100) than that of the IF gene alone.     

Isolation of a new cellulase gene from a thermophilic anaerobe and its expression inEscherichia coli

Summary A new cellulase gene was cloned and expressed inEscherichia coli from a thermophilic anaerobe, strain NA10. A 7.4 kbEcoRI fragment of NA10 DNA encoded the cellulase which hydrolyzed carboxymethyl cellulose, lichenan, andp-nitrophenyl--d-cellobioside, but could not digest laminarin andp-nitrophenyl--d-glucoside. The cloned enzyme could digest cellooligosaccharides and release cellobiose as a main product from cellotetraose but could not digest cellobiose. It was distinct from the endoglucanase which was cloned by us previously from NA10 strain in terms ofp-nitrophenyl--d-cellobioside degradation activity and the location of restriction enzyme sites. The enzyme produced byE. coli transformant was extremely heat-stable and the optimum temperature for the enzymatic reaction was 80°C. Fifty three percent of the cloned enzyme was detected in the periplasm and the remaining activity existed in the cellular fraction in theE. coli transformant.     

Tidal Influence on Spatial Dynamics of Leopard Sharks, Triakis semifasciata, in Tomales Bay, California

We used ultrasonic telemetry to determine the movement directions and movement rates of leopard sharks, Triakis semifasciata, in Tomales Bay, California. To analyze tide and time of day effects, we surgically implanted transmitters in the peritoneal cavities of one male and five female leopard sharks, which we located during summer for three to five sampling sessions lasting 12 to 24h each. All leopard sharks showed strong movement direction patterns with tide. During incoming tides, sharks moved significantly (p<0.0001) towards the inner bay, apparently to exploit the extensive inner bay muddy littoral zones' food resources. On outgoing tides, sharks showed significant (p<0.0001) movements towards the outer bay. During high tide, there was no discernible pattern to their movements (p=0.092). Shark movement rates were significantly (p<0.0001) greater during dark periods (mean±SE: 10.5±1.0m min^–1), compared with fully lighted ones (6.7±0.5m min^–1). Movement rates of longer sharks tended to be greater than those of shorter ones (range means±SE: 5.8±0.6m min^–1 for the 91cm shark, to 12.8±1.6m min^–1 for the 119cm shark), but the leopard sharks' overall mean movement rate (8.1±0.5m min^–1) was slower than other (more pelagic) sharks.     

Isolation of large linear plasmids from β-lactam producing actinomycete strains

Summary Sixteen -lactam producing actinomycete strains were screened for the presence of large linear plasmids. Among these, four strains contained linear plasmids of 12–450 kb in size. Southern blot analysis using a synthetic oligonucleotide for the isopenicillin N synthase gene and plasmid pBROC137 containing thecefD andcefE genes in the cephalosporin biosynthesis, respectively, showed no hybridization. This result suggests that these linear plasmids are not involved in the biosynthesis of -lactam antibiotics.     

Isolation and functional identification of a novel cDNA for astaxanthin biosynthesis from Haematococcus pluvialis,and astaxanthin synthesis in Escherichia coli

We succeeded in isolating a novel cDNA involved in astaxanthin biosynthesis from the green alga Haematococcus pluvialis, by an expression cloning method using an Escherichia coli transformant as a host that synthesizes -carotene due to the Erwinia uredovora carotenoid biosynthesis genes. The cloned cDNA was shown to encode a novel enzyme, -carotene ketolase (-carotene oxygenase), which converted -carotene to canthaxanthin via echinenone, through chromatographic and spectroscopic analysis of the pigments accumulated in an E. coli transformant. This indicates that the encoded enzyme is responsible for the direct conversion of methylene to keto groups, a mechanism that usually requires two different enzymatic reactions proceeding via a hydroxy intermediate. Northern blot analysis showed that the mRNA was synthesized only in the cyst cells of H. pluvialis. E. coli carrying the H. pluvialis cDNA and the E. uredovora genes required for zeaxanthin biosynthesis was also found to synthesize astaxanthin (3S, 3S), which was identified after purification by a variety of spectroscopic methods.     

Molecular cloning,expression, and characterization of a new endoglucanase gene fromFibrobacter succinogenes S85

A DNA fragment coding for a carboxymethylcellulase (CMCase) ofFibrobacter succinogenes S85 was isolated from a pUC18 gene library inEscherichia coli JM109. The CMCase gene was present as a single copy in theF. succinogenes S85 genome and was found in all the otherF. succinogenes strains tested. The gene was expressed from an endogenous promoter inE. coli and was not subject to glucose repression. Most of the CMCase activity was located in the membrane ofE. coli. Zymogram analysis and³⁵S labeling of the proteins encoded by the CMCase gene-containing plasmid indicated that the enzyme has a molecular mass of 58,000. The optimal pH and temperature of activity on CMC were respectively 6.4 and 30°C. The enzyme was active on CMC, barley -glucan, and lichenan but would not hydrolyze laminarin and exhibited no exoglucanase-type activity, suggesting that it is an endo-(1,4)--d-glucanase.     

Operon fusions of Mu d1(Ap,lac) to thel-proline biosynthetic genes ofescherichia coli K-12

Operon fusions to the promoter of either theproA,proB, orproC genes of the proline biosynthetic pathway were obtained by the use of the Mu d1(Ap,lac) bacteriophage. These fusions were further stabilized by transformation with plasmid pGW600 containing the wildtype Mu repressor gene or by transduction with phage pSG1. The level of -galactosidase in the fusion strains was not affected by the presence of exogenously addedl-proline or high concentrations of NaCl in the growth medium. A Tn5 insertion nearproBA increased -galactosidase expression 140- to 200-fold in strains carrying theproA-lac andproB-lac fusions, but the level of this enzyme was unaltered in strains carrying theproC-lac fusion. The Tn5 insertion increased intracellular proline concentrations 8- to 10-fold, suggesting that mechanisms other than allosteric inhibition may regulate proline biosynthesis, but did not confer osmotolerance to cells growing in a medium with a high concentration of salt.     

Effects of mutations altering SOS regulation on a nalidixic acid-inducible system for the production of heterologous proteins inEscherichia coli

Summary The major leftward early promoter of phage p _L, has frequently been used to drive expression of heterologous genes inEscherichia coli.p _L is typically maintained fully repressed by the lambda cl protein. When induction of heterologous protein synthesis is desired, one of several potential mechanisms of destroying cl function is employed and the expression of the foreign gene commences. One method of derepressingp _L involves exposing cells to nalidixic acid, which results in the activation of RecA protein and the subsequent RecA-mediated proteolytic cleavage of cl. Activated RecA also mediates the cleavage of theE. coli LexA protein, resulting in induction of the SOS regulon (at least 15E. coli genes, includingrec A). We have examined the effect of two chromosomal mutations on the productivity of nalidixic acid inductions. One of the tested mutations (recA o) increased the intracellular concentration of RecA prior to induction; the other (lexAind^–) resulted in a mutated lexA protein insensitive to RecA-mediated cleavage. These mutations were introduced into a strain carrying acl⁺ defective lysogen. Synthesis of two heterologous proteins, human 1-antitrypsin and a fusion protein partially derived from thePlasmodium falciparum circumsporozooite surface antigen, was examined in the wild-type and mutant strains. The maximum -1 antitrypsin concentration achieved was improved by 50% when therecA o strain was used rather than the wild type; however; only smaller changes (20% or less) in the maximum concentration of the malaria fusion protein wer observed. Use of thelexAind^– strain resulted in a decrease in the maximum concentration attained for both heterologous products.