Abstract: The nucleotide sequence of pC194, a small plasmid from Staphylococcus aureus which is capable of replication in Bacillus subtilis, has been determined. The genetic determinant of chloramphenicol (CAM) resistance, which includes the chloramphenicol acetyl transferase (CAT) structural gene, the putative promoter and controlling element of this determinant, have been mapped functionally by subcloning a 1,035-nucleotide fragment which specifies the resistance phenotype using plasmid pBR322 as vector. Expression of CAM resistance is autogenously regulated since the 1,035-nucleotide fragment containing the CAT gene sequence and its promoter cloned into pBR322 expresses resistance inducibly in the Escherichia coli host. A presumed controlling element of CAT expression consists of a 37-nucleotide inverted complementary repeat sequence that is located between the -10 and ribosome-loading sequences of the CAT structural gene. Whereas the composite plasmid containing the minimal CAT determinant cloned in pBR322 could not replicate in B. subtilis, ability to replicate in B. subtilis was seen if the fragment cloned included an extension consisting of an additional 300 nucleotides beyond the 59 end of the single pC194 MspI site associated with replication. This 59 extension contained a 120-nucleotide inverted complementary repeat sequence similar to that found in pE194 TaqI fragment B which contains replication sequences of that plasmid. pC194 was found to contain four opening reading frames theoretically capable of coding for proteins with maximum molecular masses, as follows: A, 27,800 daltons; B, 26,200 daltons; C, 15,000 daltons; and D, 9,600 daltons. Interruption or deletion of either frame A or D does not entail loss of ability to replicate or to express CAM resistance, whereas frame B contains the CAT structural gene and frame C contains sequences associated with plasmid replication.

Abstract: A model is proposed for the mechanism of flocculation interactions in yeasts in which flocculent cells have a recognition factor which attaches to alpha-mannan sites on other cells. This factor may be governed by the expression of the single, dominant gene FLO1. Isogenic strains of Saccharomyces cerevisiae, differing only at FLO1 and the marker genes ade1 and trp1, were developed to examine the components involved in flocculene. Electron microscopy and concanavalin Aferritin labeling of aggregated cells showed that extensive and intense interactions between cell wall mannan layers mediated cell aggregation. The components of the mannan layer essential for flocculence were Ca2+ ions, alpha-mannan carbohydrates, and proteins. By studying the divalent cation dependence at various pH values and in the presence of competing monovalent cations, flocculation was found to be Ca2+ dependent; however, Mg2+ and Mn2+ ions substituted for Ca2+ under certain conditions. Reversible inhibition of flocculation by concanavalin A and succinylated concanavalin A implicated alpha-branched mannan carbohydrates as one essential component which alone did not determine the strain specificity of flocculence, since nonflocculent strains interacted with and competed for binding sites on flocculent cells. FLO1 may govern the expression of a proteinaceous, lectin-like activity, firmly associated with the cell walls of flocculent cells, which bind to the alpha-mannan carbohydrates of adjoining cells. It was selectively and irreversibly inhibited by proteolysis and reduction of disulfide bonds. The potential of this system as a model for the genetic and biochemical control of cell-cell interactions is discussed.

Abstract: A double mutant of Cryptococcus neoformans which lacked the ability to produce melanin (Mel-) on media containing diphenols and failed to grow at 37 degrees C (temperature sensitive, Tem-) was obtained by UV irradiation and subsequent cloning The mutant showed two lesions in melanogenesis in that it lacked the active transport system for diphenolic compounds and also lacked phenoloxidase Ultrastructures of the mutant and wild-type cells grown on a medium with or without L-dopa showed that only the wild-type cells grown on L-dopa medium formed a dark cell wall layer, presumably containing melanin The mutant was crossed with a wild type, and the phenotypes of the progeny were analyzed The analysis showed no linkage between the mating type and either Mel or Tem loci, but loose linkage was seen between Mel and Tem loci The progeny, Mel+ Tem+, Mel+ Tem-, Mel- Tem+, and Mel- Tem-, were studied for their virulence in mice Only Mel+ Tem+ types killed mice with an inoculum of 5 X 10(5) cells within 50 days Images

Abstract: Pseudomonas aeruginosa is usually resistant to a wide variety of antibacterial agents, and it has been inferred, on the basis of indirect evidence, that this was due to the low permeability of its outer membrane. We determined the permeability of P. aeruginosa outer membrane directly, by measuring the rates of hydrolysis of cephacetrile, cephaloridine, and various phosphate esters by hydrolytic enzymes located in the periplasm. The permeability to these compounds was about 100-fold lower than in the outer membrane of Escherichia coli K-12. Also, we found that the apparent Km values for active transport of various carbon and energy source compounds were typically higher than 20 microM in P. aeruginosa, in contrast to E. coli in which the values are usually lower than 5 microM. These results also are consistent with the notion that the P. aeruginosa outer membrane indeed has a low permeability to most hydrophilic compounds and that this membrane acts as a rate limiting step in active transport processes with high Vmax values.

Abstract: We examined the properties of mutants of E. coli which are defective with respect to nitrate reductase activity. chlE::Mu cts and chlG::Mu cts mutants were all chlorate resistant, and the strains that we examined all synthesized nitrate reductase apoenzyme. We concluded that the chlE and chlG loci, like the chlA, chlB, and chlD loci, are involved in the synthesis of insertion of molybdenum cofactor. We identified at least four distinct phenotypic classes of chlC::Tn10 mutants, all of which were fully or partially sensitive to chlorate. Two of these classes may represent lesions in the structural genes for nitrate reductase subunits A and C. Two other classes may be altered in the regulation of the expression of nitrate reductase or other anaerobic enzymes. We propose the mnemonic nar for naming individual genes within the chlC locus.

Abstract: We cloned the DNA containing the Escherichia coli hemolysin determinant on a small, high-copy plasmid. We generated plasmids containing fragments of this DNA and used them either alone or in two-plasmid complementation systems to define the limits of the structural genes. This system also allowed us to partially characterize the function of each of the gene products in the production and transport of hemolysin. Taken with previously published data, the present experiments indicate the following. (i) At least three cistrons, hlyC, hlyA, and hlyB (these were previously designated cisC, etc. [Noegel et al., Mol. Gen. Genet. 175:343-350, 1979]), contain the specific genetic information for the hemolytic phenotype, (ii) hlyA encodes a 107,000-kilodalton protein, which seems to be an inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity inactive precursor of hemolysin. (iii) Normal amounts of hemolysin activity require only the products of hlyA and hlyC. This activity was found in the periplasm; very little hemolysin activity was found in the cytoplasm, suggesting that the hlyC product is required for transport or activation of the hlyA product or both. (iv) Active hemolysin remains in the periplasm in the absence of hlyB function, hence the hlyB product seems to be necessary for the transport of hemolysin to the exterior of the cell. We further show that overproduction of the hlyA product is lethal, probably causing lysis of the cell.

Abstract: In Klebsiella pneumoniae NCIB 418, the pathways normally responsible for aerobic growth on glycerol and sn-glycerol 3-phosphate (the glp system) are superrepressed. However, aerobic growth on glycerol can take place by the intervention of the NAD-linked glycerol dehydrogenase and the ATP-dependent dihydroxyacetone kinase of the dha system normally inducible only anaerobically by glycerol or dihydroxyacetone. Conclusive evidence that the dha system is responsible for both aerobic and anaerobic dissimilation of glycerol was provided by a Tn5 insertion mutant lacking dihydroxyacetone kinase. An enzymatically coupled assay specific for this enzyme was devised. Spontaneous reactivation of the glp system was achieved by selection for aerobic growth on sn-glycerol 3-phosphate or on limiting glycerol as the sole carbon and energy source. However, the expression of this system became constitutive. Aerobic operation of the glp system highly represses synthesis of the dha system enzymes by catabolite repression. Images

Abstract: The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12. In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques. The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined. It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values. Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized. The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis. Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed.

Abstract: Studies of trpA reversions revealed that G:C leads to A:T transitions were stimulated about 30-fold in E. coli ung mutants, whereas other base substitutions were not affected. A dUTPase (dut) mutation, which increases the incorporation of uracil into DNA in place of thymine, had no significant effect on the rate of G:C leads to A:T transitions. The results support the proposal that the glycosylase functions to reduce the mutation rate in wild-type cells by acting in the repair of DNA cytosine residues that have undergone spontaneous deamination to uracil. Further support was provided by the finding that when lambda bacteriophages were treated with bisulfite, an agent known to produce cytosine deamination, the frequency of clear-plaque mutants was increased an additional 20-fold by growth on an ung host. Bisulfite-induced mutations of the cellular chromosome, however, were about equal in ung+ and ung strains; it was found that during the treatment of ung+ cells with bisulfite, the glycosylase was inactivated.

Abstract: We found that naturally occurring tetracycline resistance in streptococci is encoded by more than one genetic determinant. Two of these distinct determinants were cloned, and the regions that are necessary and sufficient for expression of tetracycline resistance were defined by deletion analysis. These cloned determinants were further characterized by DNA-DNA hybridization experiments which also identified a third genetically unrelated tetracycline resistance determinant. Some of these genetic differences appear to represent mechanistic differences. The tetL determinant was associated with small nonconjugative plasmids and mediated resistance to tetracycline. The tetM determinant was most often "nonplasmid" associated and mediated resistance to minocycline as well as tetracycline. The tetN determinant was represented on a large conjugative plasmid and was genetically distinct from tetL and tetM, although phenotypically it resembled tetM.

Abstract: I used a chlC-lac operon fusion to study regulatory mutations which affect nitrate reductase expression in Escherichia coli. A NarL- mutant apparently lacks a nitrate-specific positive regulatory component. Furthermore, an fnr (nirR) mutation prevented enzyme induction under any conditions. These data are consistent with a two-step, positive control model for nitrate reductase regulation.

Abstract: The initial reactions in the oxidation of naphthalene by Pseudomonas sp. strain NCIB 9816 involves the enzymatic incorporation of one molecule of oxygen into the aromatic nucleus to form (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The enzyme catalyzing this reaction, naphthalene dioxygenase, was resolved into three protein components, designated A, B, and C, by DEAE-cellulose chromatography. Incubation of naphthalene with components A, B, and C in the presence of NADH resulted in the formation of (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. The ratio of oxygen and NADH utilization to product formation was 1:1:1. NADPH also served as an electron donor for naphthalene oxygenation. However, its activity was less than 50% of that observed with NADH. Component A showed NAD(P)H-cytochrome c reductase activity which was stimulated by the addition of flavin adenine dinucleotide and flavin mononucleotide. A similar stimulation was observed when these flavin nucleotides were added to the naphthalene dioxygenase assay system. These preliminary observations indicate that naphthalene dioxygenase has properties in common with both monooxygenase and dioxygenase multicomponent enzyme systems.

Abstract: Genes for indoleacetic acid production (iaaM and iaaH) are necessary for gall induction by the olive pathogen Pseudomonas savastanoi. In strain 2009 these determinants are borne on plasmid pIAA1. To map and characterize the genes, fragments of pIAA1 generated by EcoRI endonuclease treatment were cloned in Escherichia coli by using plasmid RSF1010 as vector. We isolated a recombinant plasmid encoding iaaM, the locus for tryptophan 2-monooxygenase. This plasmid, called pLUC1, was characterized by restriction endonuclease hydrolysis. It contained a 2.75-kilobase-pair segment of pIAA1. By cloning this segment in the EcoRI site of pBR328 and pBRH3B we showed that efficient expression of iaaM was dependent on the orientation with respect to the vector promoters, and thus determined the direction of transcription. To more finely map iaaM and confirm the orientation of transcription, plasmid pLUC1 was subjected to transposon Tn/mutagenesis. The promoter-distal end of iaaM was mapped between coordinates at 1.7 and 2.15 kilobase pairs of the cloned segment.

Abstract: An enzyme which liberates Pi from myo-inositol hexaphosphate (phytic acid) was shown to be present in culture filtrates of Bacillus subtilis. It was purified until it was homogeneous by ultracentrifugation, but it still showed two isozymes on polyacrylamide gel electrophoresis. The enzyme differed from other previously known phytases in its metal requirement and in its specificity for phytate. It had a specific requirement for Ca2+ for its activity. The enzyme hydrolyzed only phytate and had no action on other phosphate esters tested. This B. subtilis phytase is the only known phytate-specific phosphatase. The products of hydrolysis of phytate by this enzyme were Pi and myo-inositol monophosphate. The enzyme showed optimum activity at pH 7.5. It was inhibited by Ba2+, Sr2+, Hg2+, Cd2+, and borate. Its activity was unaffected by urea, diisopropylfluorophosphate, arsenate, fluoride, mercaptoethanol, trypsin, papain, and elastase. Images

Abstract: Thermal damage to the outer membrane of Escherichia coli W3110 was studied. When E. coli cells were heated at 55 degrees C in 50 mM Tris-hydrochloride buffer at pH 8.0, surface blebs were formed on the cell envelope, mainly at the septa of dividing cells. Membrane lipids were released from the cells during the heating period, and part of the released lipids formed vesicle-like structures from the membrane. This vesicle fraction had a lipopolysaccharide to phospholipid ratio similar to that of the outer membrane of intact cells, whereas it had a lower content of protein than the isolated outer membrane. After heating bacterial cells at 55 degrees C for 30 min, the resulting leakage from the cells of a periplasmic enzyme, alkaline phosphatase, amounted to 52% of the total activity, whereas no release of a cytoplasmic enzyme, glucose-6-phosphate dehydrogenase, was detected. The results obtained suggest that surface blebs formed by heat treatment almost completely consist of the outer membrane and that the blebs may be gradually released from the cell surface into the heating menstruum to partially form vesicles.

Abstract: Glycerol and diol dehydratases are inducible, coenzyme B12-dependent enzymes found together in Klebsiella pneumoniae ATCC 25955 during anaerobic growth on glycerol. Mutants of this strain isolated by a novel procedure were separately constitutive for either dehydratase, showing the structural genes for the two enzymes to be under independent control in vivo. Glycerol dehydratase and a trimethylene glycol dehydrogenase were implicated as members of a pleiotropic control system that includes glycerol dehydrogenase and dihydroxyacetone kinase for the anaerobic dissimilation of glycerol (the "dha system"). The dehydratase and dehydrogenases were induced by dihydroxyacetone and were jointly constitutive in mutants isolated as constitutive for either the dha system or glycerol dehydratase. These data and the stimulation of growth by Co2+ suggested that glycerol dehydratase and trimethylene glycol dehydrogenase are obligatory enzymes for anaerobic growth on glycerol as the sole carbon source. Images

Abstract: Previous studies have defined 29 genes necessary for synthesis of the Escherichia coli flagellar apparatus. This study analyzed the transcriptional control of flagellar genes, using Mu d (Apr lac) phage to generate flagellar mutants by insertion. These mutants contained operon fusions of flagellar genes to the lac genes of the Mu d phage and allowed the measurement of flagellar operon expression by detection of beta-galactosidase activity. These fusion mutants expressed the enzyme activity constitutively, and an autogenous regulation mechanism was not revealed. Lambda transducing phages carrying these chromosomal fla-lac fusions were also isolated and used to examine the effect of different fla mutations on expression of each flagellar operon. The results showed that flagellar operons are divided into six classes; (class 1) the flbB operon, which controls all of the other flagellar operons; (class 2) the flaU and flbC operons, which are controlled by the flbB operon gene products and are not required for the expression of other Fla operons; (class 3) the flbA, flaG, flaD, flaN, flaB, and flaA operons, which are under flbB operon control and are required for the expression of other fla operons; (class4) the flaZ operon, which is controlled by the gene products of the group 1 and 3 operons and is required for hag transcription; (class 5) the mocha and flaS operons, which are controlled by the gene products of the group 1 and 3 operons; and (class 6) the hag operon. These results are discussed with respect to the possible assembly sequence of the fla gene products.

Abstract: Mature, dormant spores of a marine bacillus, SG-1, bound and oxidized (precipitated) manganese on their surfaces. The binding and oxidation occurred under dormant conditions, with mature spores suspended in natural seawater. These heat-stable spores were formed in the absence of added manganese in the growth medium. The rate and amount of manganese bound by SG-1 spores was a function of spore concentration. Temperatures greater than 45 degrees C, pH values below 6.5, or the addition of EDTA or the metabolic inhibitors sodium azide, potassium cyanide, and mercuric chloride inhibited manganese binding and oxidation. However, SG-1 spores bound and oxidized manganese after treatment with glutaraldehyde, formaldehyde, ethylene oxide gas, or UV light, all of which killed the spores. Manganese oxidation never occurred in the absence of manganese binding to spores. The data suggest that Mn2+ was complexed by a spore component, perhaps an exosporium or a spore coat protein: once bound, the manganese was rapidly oxidized.

Abstract: Isolated reticulate bodies of Chlamydia psittaci were found to transport ATP and ADP by an ATP-ADP exchange mechanism. ATP uptake activity was not detected in elementary bodies. The apparent Km of transport for both ATP and ADP was approximately 5 microM, and the calculated Vmax for both was about 1 nmol of nucleotide transported per min per mg of protein. ADP competitively inhibited ATP transport with a Ki of 4.5 microM. Other nucleotides tested had no effect on the uptake of ATP. A magnesium-dependent, oligomycin-sensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was associated with reticulate bodies, and most of the transported ATP was hydrolyzed to ADP, which was exchanged for additional, extracellular nucleotide. Some ADP was hydrolyzed to AMP, which exited the cells slowly. Lysine was transported against the electrochemical gradient by reticulate bodies in the presence of ATP. Oligomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited ATP-dependent lysine transport. Lysine exited reticulate bodies when the reticulate bodies were incubated in the presence of ADP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or a reduced concentration of ATP. The results support the concept that chlamydiae are energy parasites which are capable of drawing upon the adenine nucleotides of their hosts, hydrolyzing ATP, and establishing an energized membrane.

Abstract: A new major outer membrane protein, P, was induced in Pseudomonas aeruginosa PAO1 upon growth in medium containing 0.2 mM or less inorganic phosphate. Studies with media containing different levels of phosphate and with mutants of PAO1 suggested that protein P was coregulated with alkaline phosphatase and phospholipase C. Protein P was substantially purified and shown to form sodium dodecyl sulfate-resistant oligomers on polyacrylamide gels. The incorporation of purified protein P into artificial lipid bilayers resulted in an increase of the membrane conductance by many orders of magnitude. Single-channel experiments demonstrated that protein P channels were substantially smaller than all previously studied porins from P. aeruginosa and enteric bacteria, with an average single-channel conductance in 1 M NaCl of 0.25 nS. The protein P channel was apparently not voltage induced or regulated. The results of single-channel conductance experiments, using a variety of different salts, allowed a minimum channel diameter estimate of 0.7 nm. Furthermore, from these results it was concluded that the protein P channel was highly specific for anions. Zero-current potential measurements confirmed that protein P was at least 30-fold more permeable for Cl- than for K+ ions. The possible biological role of the small, anion-specific protein P channels in phosphate uptake from the medium is discussed.

Abstract: Micrococcus sp. strain 12B was isolated by enriching for growth with dibutylphthalate as the sole carbon and energy source. A pathway for the metabolism of dibutylphthalate and phthalate by micrococcus sp. strain 12B is proposed: dibutylphthalate leads to monobutylphthalate leads to phthalate leads to 3,4-dihydro-3,4-dihydroxyphthalate leads to 3,4-dihydroxyphthalate leads to protocatechuate (3,4-dihdroxybenzoate). Protocatechuate is metabolized both by the meta-cleavage pathway through 4-carboxy-2-hydroxymuconic semialdehyde and 4-carboxy-2-hydroxymuconate to pyruvate and oxaloacetate and by the ortho-cleavage pathway to beta-ketoadipate. Dibutylphthalate- and phthalate-grown cells readily oxidized dibutylphthalate, phthalate, 3,4-dihydroxyphthalate, and protocatechuate. Extracts of cells grown with dibutylphthalate or phthalate contained the 3,4-dihydroxyphthalate decarboxylase and the enzymes of the protocatechuater 4,5-meta-cleavage pathway. Extracts of dibutylphthalate-grown cells also contained the protocatechuate ortho-cleavage pathway enzymes. The dibutylphthalate-hydrolyzing esterase and 3,4-dihydroxyphthalate decarboxylase were constitutively synthesized; phthalate-3,4-dioxygenase (and possibly the "dihydrodiol" dehydrogenase) was inducible by phthalate or a metabolite occurring before protocatechuate in the pathway; two protocatechuate oxygenases and subsequent enzymes were inducible by protocatechuate or a subsequent metabolic product. During growth at 37 degrees C, strain 12B gave clones at high frequency that had lost the ability to grow with phthalate esters. One of these nonrevertible mutants, strain 12B-Cl, lacked all of the enzymes required for the metabolism of dibutylphthalate through the protocatechuate meta-cleavage pathway. Enzymes for the metabolism of protocatechuate by the ortho-cleavage pathway were present in this strain grown with p-hydroxybenzoate or protocatechuate.

Abstract: Exogenous proline specifically stimulates the growth rate of enteric bacteria in media of inhibitory osmotic strength (J. H. B. Christian, Aust. J. Biol. Sci. 8:490-497, 1955). I observed that Salmonella typhimurium mutants which lack both of the previously known proline permeases (putP proP) are stimulated by proline in media of inhibitory osmolarity. I propose that there is a third proline permease which functions only in media of elevated osmolarity. This conclusion is based on the observations that, in media of elevated osmolarity, (i) the sensitivity of putP proP mutants to toxic proline analogs increases, (ii) proline requirements for maximal growth of proline auxotrophic putP proP mutants decreases, and (iii) the specific rate of incorporation of radioactive proline into protein of growing cells increases. I obtained a Tn10-induced mutation in a gene (proU) required for the functioning of the third proline permease and determined the map location to be at 59 map units of the chromosome, between srlA and tct, 66% linked to nalB in P22 transduction. My results suggest that the function of the third, osmotically stimulated permease might be to accumulate high intracellular proline levels during osmotic stress. Possible mechanisms by which proline might cause growth stimulation are discussed.

Abstract: Using analytical and preparative methods, we demonstrated the presence of megaplasmids with molecular weight larger than 450 x 10(6) in the two plant-associated bacteria Rhizobium meliloti and Pseudomonas solanacearum. Such giant plasmids were found in 8 of 9 P. solanacearum strains tested and in all of the 18 R. meliloti strains tested.

Abstract: The glnG gene product is both a positive regulator and a negative regulator of the expression of glnA, the structural gene for glutamine synthetase, as well as a positive regulator of the expression of a number of genes whose products are involved in the uptake and degradation of nitrogen-containing compounds. The regulation of beta-galactosidase in various strains containing a Mu d1 (lac bla) insertion within glnG leads to the following conclusions regarding the expression of this gene: (i) like the synthesis of glutamine synthetase, the synthesis of the glnG product is regulated in response to the nitrogen source; (ii) high-level expression of glnG under nitrogen-limiting growth conditions depends on transcription initiated at the glnA promoter; and (iii) there is a second, glnA-distal promoter for glnG, whose activity is negatively controlled by the glnG product. Thus, the glnG product regulates the synthesis of the glnG product at two distinct promoters (positively and negatively at the glnA promoter and negatively at the glnA-distal promoter). In addition, a high level of glnG product, corresponding to the level produced by initiation of transcription at the glnA promoter under nitrogen-limiting conditions, is necessary for activation of histidase synthesis. The lower level of glnG product originating from transcription initiated at the glnA-distal promoter is not sufficient to activate histidase synthesis, but is sufficient to activate fully and to repress glnA expression.

Abstract: A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance [Tcr]), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C. The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively. Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed. When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B. stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline. Bacillus subtilis could also be transformed with the plasmids extracted from B. stearothermophilus and vice versa. Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B. stearothermophilus and B. subtilis. The requirements for replication of pTB19 in B. subtilis and B. stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B. subtilis, whereas another deletion plasmid pTB92 could replicate solely in B. stearothermophilus. Plasmids pTB19 and pTB90 could be maintained and expressed in B. stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.

Abstract: The Marburg strain of Butyribacterium methylotrophicum did not grow on CO alone but did consume CO during growth on a variety of substrates in the presence of a 100% CO gas phase. We selected a strain (the CO strain) that grew vigorously on CO alone. The ability of the CO strain to grow on CO was stable through multiple transfers in the absence of CO. CO dehydrogenase activity was lower in the CO strain grown on CO (13.3 micromol/min per mg of protein) than in the Marburg strain grown on methanol-acetate (47.2 mumol/min per mg of protein); thus, the levels of this enzyme did not explain the growth on CO. CO was dissimilated to acetate and CO2 with the following stoichiometry: 4 CO leads to 2.17 CO2 + 0.74 acetate. We observed a growth rate of 0.05 h-1, a final optical density at 660 nm of 0.8, and a cell yield of 3.0 g of cells per mol of CO during growth of the CO strain. Growing cultures of the CO strain displayed a Ks for CO of 28 to 56 microM. The apparent thermodynamic efficiency of cell synthesis from CO was 57%. Energetic and biochemical aspects of CO metabolism are described.

Abstract: A number of strains of Candida albicans were tested for germ tube formation after induction by N-acetyl-D-glucosamine (GlcNAc) and other simple (proline, glucose plus glutamine) or complex (serum) compounds. A proportion of strains (high responders) were induced to form germ tubes evolving to true hyphae by GlcNAc alone or by proline or glucose plus glutamine mixture. The majority of strains were low responders because they could be induced only by serum or GlcNAc-serum medium. Two strains were found to be nonresponders: they grew as pseudohyphae in serum. Despite minor quantitative differences, all strains efficiently utilized GlcNAc for growth under the yeast form at 28 degrees C. They also had comparable active, inducible, and constitutive uptake systems for GlcNAc. During germ tube formation in GlcNAc, the inducible uptake system was modulated, as expected from induction and decay of GlcNAc kinase. Uranyl acetate, at a concentration of 0.01 mM, inhibited both GlcNAc uptake and germ tube formation and was reversed by phosphates. Germinating and nongerminating cells differed in the rapidity and extent of GlcNAc incorporation into acid-insoluble and alkali-acid-insoluble cell fractions. During germ tube formation induced by proline, GlcNAc was almost totally incorporated into the acid-insoluble fraction after 60 min. Moreover, hyphal development on induction by either GlcNAc or proline was characterized by an apparent "uncoupling" between protein and polysaccharide metabolism, the ratio between the two main cellular constituents falling from more than 1 to less than 0.5 after 270 min of development. The data suggest that utilization of the inducer for wall synthesis is a determinant of germ tube formation C. albicans but that the nature and extent of inducer uptake is not a key event for this phenomenon to occur.

Abstract: Streptococcus mutans JC2 produced formate, acetate, ethanol, and lactate when suspensions were incubated with an excess of galactose or mannitol under strictly anaerobic conditions. The galactose- or mannitol-grown cell suspensions produced more formate, acetate, and ethanol than the glucose-grown cells even when incubated with glucose. The levels of lactate dehydrogenase and fructose 1,6-bisphosphate were not significantly different in these cells, but the level of pyruvate formate-lyase was higher in the galactose- or mannitol-grown cells, and that of triose phosphate was lower in the galactose-grown cells. This suggests that the regulation of pyruvate formate-lyase may play a major role in the change of the fermentation patterns. The cells of S. mutans grown on glucose produced a significant amount of volatile products even in the presence of excess glucose under strictly anaerobic conditions. However, when the anaerobically grown cells were exposed to air, only lactate was produced from glucose. When cells were anaerobically grown on mannitol and then exposed to air for 2 min, only trace amounts of fermentation products were formed from mannitol under anaerobic conditions. It was found that the pyruvate formate-lyase in the cells was inactivated by exposure of the cells to air.

Abstract: Saccharomyces cerevisiae does not show a noticeable Pasteur effect (activation of sugar catabolism by anaerobiosis) when growing with an excess of sugar and nitrogen source, but it does do so after exhaustion of the nitrogen source in the medium (resting state). We have found that this different behavior of growing and resting S. cerevisiae seems due to differences in the contribution of respiration to catabolism under both states. Growing S. cerevisiae respired only 3 to 20% of the catabolized sugar, depending on the sugar present; the remainder was fermented. In contrast, resting S. cerevisiae respired as much as 25 to 100% of the catabolized sugar. These results suggest that a shift to anaerobiosis would have much greater energetic consequences in resting than in growing S. cerevisiae. In resting S. cerevisiae anaerobiosis would strongly decrease the formation of ATP; as a consequence, various regulatory mechanisms would switch on, producing the observed increase of the rate of glycolysis. The greater significance that respiration reached in resting cells was not due to an increase of the respiratory capacity itself, but to a loss of fermentation which turned respiration into the main catabolic pathway. The main mechanism involved in the loss of fermentation observed during nitrogen starvation was a progressive inactivation of the sugar transport systems that reduced the rate of fermentation to less than 10% of the value observed in growing cells. Inactivation of the sugar transports seems a consequence of the turnover of the sugar carriers whose apparent half-lives were 2 to 7 h.