Effects of lysine analogs on Penicillium chrysogenum.

Compounds structurally related to lysine were tested against Penicillium chrysogenum Wis. 54-1255 for inhibition of growth, sporulation, and penicillin formation. This strain is relatively resistant to lysine analogs. The compounds that were the more active inhibitors of growth and whose activities were reversed by L-lysine were diaminohexynoic acid, N-epsilon-methyllysine, N-alpha-methyllysine, and diaminopimelic acid. These four compounds also inhibited sporulation, which was more sensitive to inhibition than growth was. Analogs strongly inhibiting benzyl-penicillin formation by resting mycelia were diaminohexynoic acid and N-epsilon-methyllysine. The action of the most active analog (diaminohexynoic acid) on penicillin synthesis was reversed by DL-alpha-aminoadipic acid.     

Regulation of lysine and dipicolinic acid biosynthesis in Bacillus brevis ATCC 10068: significance of derepression of the enzymes during the change from vegetative growth to sporulation

Lysine biosynthetic pathway enzymes of Bacillus brevis ATCC 1068 were studied as a function of stage of development (growth and sporulation). The synthesis of aspartic-2-eemialdehyde dehydrogenase (ASA-dehydrogenase), dihydrodipicolinate synthase (DHDPA-synthase), DHPA-reductase and diaminopimelate decarboxylase (DAP-decarboxylase) was found not to be co-regulated, since lysine was not a co-repressor for these enzymes. Unlike the aspartokinase isoenzymes, the other enzymes of the lysine pathway were not derepressed in thiosine-resistant, lysine-excreting mutants. Thus, the aspartokinase isoenzymes were the key enzymes during growth and regulation of lysine biosynthesis through restriction of l-ASA synthesis via feedback control by lysine on the aspartokinases was therefore suggested.In contrast to other Bacillus species, the levels of the lysine biosynthetic pathway enzymes of strain ATCC 10068 were not derepressed during the change from vegetative growth to sporulation. Two control mechanisms, enabling the observed preferential channelling of carbon for the synthesis of spore-specific diaminopimelic acid (DAP) and dipicolinic acid (DPA) were a) loss of DAP-decarboxylase, b) inhibition of DHDPA-reductase by DPA. Increase in the level of the DAP pool during sporulation, as a consequence of the loss of DAP-decarboxylase, and its relevance to the non-enzymatic formation of DPA has been discussed.Abbreviations l-ASA l-aspartic-2-semialdehyde - DAP diaminopimelic acid - DPA dipicolinic acid - DHDPA dihydrodipicolinate - AGM aspargine-glycerol medium - PY peptone-yeast extract - NB+NSM nutrient broth plus nutrient sporulation medium     

Study of inhibition of outgrowth in Bacillus cereus T by ethyl picolinate.

The effects of ethyl picolinate on germination, outgrowth, and sporulation of Bacillus cereus T were studied in a synthetic medium containing glucose. Ethyl picolinate specifically inhibited at two stages, outgrowth and sporulation. The initiation of germination and cell division was not affected. The inhibition of outgrowth by ethyl picolinate could be reversed by enrichment of inoculum with aspartic acid, asparagine, lysine, phenylalanine, and tyrosine among the amino acids and by oxalacetate. Nicotinic acid and nicotinamide also possessed this ability. Ethyl picolinate failed to block outgrowth when added to cultures incubated for a short time after inoculation. Enrichment of the medium with lysine plus zinc sulfate stimulated sporulation in the presence of ethyl picolinate to a significance degree.     

Study of inhibition of outgrowth in Bacillus cereus T by ethyl picolinate.

The effects of ethyl picolinate on germination, outgrowth, and sporulation of Bacillus cereus T were studied in a synthetic medium containing glucose. Ethyl picolinate specifically inhibited at two stages, outgrowth and sporulation. The initiation of germination and cell division was not affected. The inhibition of outgrowth by ethyl picolinate could be reversed by enrichment of inoculum with aspartic acid, asparagine, lysine, phenylalanine, and tyrosine among the amino acids and by oxalacetate. Nicotinic acid and nicotinamide also possessed this ability. Ethyl picolinate failed to block outgrowth when added to cultures incubated for a short time after inoculation. Enrichment of the medium with lysine plus zinc sulfate stimulated sporulation in the presence of ethyl picolinate to a significance degree.     

Spores of microorganisms

The addition of penicillin (300–1,000 units/ml.) to a culture ofBacillus cereus during formation of the refractive prespores leads to lysis of the sporangia and to the release of spore components (calcium and dipicolinic acid) from the cells. Penicillin mildly raises the incorporation of amino acids, including diaminopimelic acid, into hot-TCA precipitate of cells, while chloramphenicol lowers it. In the later phases of penicillin inhibition, DAP-containing structures are also destroyed, including the fraction firmly bound to the envelope structures of the spore (in the control culture this fraction is not released until later, during digestion by enzymes localized in the envelope structures themselves). Penicillin inhibition of sporogenesis can be reversed by adapting the culture to penicillin or by simultaneously adding chloramphenicol. After the presporulation phase, sporogenesis is relatively resistant to chloramphenicol, but the whole process is considerably slowed down. Chloramphenicol also affects the morphology of the spores during their formation and inhibits their release from the sporangia until the late phase of sporulation.     

Differential amino acid requirements for sporulation in Bacillus subtilis

The amino acid requirements for sporulation were studied by use of auxotrophic mutants of Bacillus subtilis 168. Cells were grown to T(0) in medium containing the test amino acid and were then transferred to a minimal medium lacking that amino acid. Omission of leucine caused no reduction in sporulation. Omission of methionine, lysine, and phenylalanine appeared to cause reduced levels of sporulation, and sporulation was completely inhibited when isoleucine, tryptophan, and threonine were omitted. The amino acids in this third class showed a sequence of requirements, with tryptophan required earlier than isoleucine, which in turn was required earlier in the sporulation process than threonine. Isoleucine omission did not affect the early sporulation functions of extracellular protease formation or septum formation, but prevented the increased levels of protein synthesis and oxygen consumption that normally accompany early sporulation stages. Isoleucine did not appear to be metabolized to other compounds in significant amounts during sporulation. The role of isoleucine in the sporulation process remains unclear.     

Sporulation of auxotrophic mutants ofBacillus subtilis in suboptimal concentrations of essential amino acids

The effect of the amino acid concentration on sporulation of five mutants ofBacillus subtilis requiring histidine, isoleucine, leucine, lysine and methionine for growth was studied. Low amino acid concentrations, permitting about 10–20% of maximum growth, inhibit the ability of the bacteria to sporulate on completing vegetative growth. In medium concentrations (from 25% of maximum growth), leucine, isoleucine, histidine and methionine induce sporulation, while the concentration of the other nutrients in the medium is still adequate for further vegetative growth. The percentage of sporulation depends on the amino acid concentration and is characteristic for each amino acid. The lysine mutant strain 1399 was not capable of sporulation unless cultivated in the presence of the lysine concentration optimal for growth. The effect of amino acid deficiency depends on the cultivation method.     

Selective inhibition of Bacillus subtilis sporulation by acridine orange and promethazine.

Two structurally similar compounds were found to inhibit sporulation in Bacillus subtilis 168. A dye, acridine orange, and an antischizophrenic drug, promethazine, blocked spore formation at concentrations subinhibitory to vegetative growth, while allowing synthesis of serine protease, antibiotic, and certain catabolite-repressed enzymes. The sporulation process was sensitive to promethazine through T2, whereas acridine orange was inhibitory until T4. The drug-treated cells were able to support the replication of phages phie and phi29, although the lytic cycles were altered slightly. The selective inhibition of sporulation by these compounds may be related to the affinity of some sporulation-specific genes to intercalating compounds.     

Sporulation and regulation of homoserine dehydrogenase in Bacillus subtilis

Homoserine dehydrogenase in dialyzed cell extracts of Bacillus subtilis 168 was studied, particularly with regard to inhibition, repression, and level of activity as a function of stage of development (growth and sporulation). It was assayed in the "forward direction" using L-aspartic semialdehyde and NADPH as substrates. Of the potentials inhibitors tested, only cysteine and NADP were found to be effective. Both L- and D-cysteine were equally effective. Therefore, the physiological significance of cysteine as an inhibitor is somewhat questionable. Amino acids involved in repression of homoserine dehydrogenase included methionine, isoleucine, possibly threonine, and one or more unidentified components of Casamino acids. The specific activity of homoserine dehydrogenase was highest during the exponential phase of growth and declined steadily during the stationary phase of growth. The low specific activity during late sporulation may favor preferential funnelling of L-aspartic semialdehyde into the lysine pathway, where it is needed for synthesis of large amounts of dipicolinic acid and diaminopimelic acid.     

Catabolism of lysine in Penicillium chrysogenum leads to formation of 2-aminoadipic acid, a precursor of penicillin biosynthesis.

Penicillium chrysogenum L2, a lysine auxotroph blocked in the early steps of the lysine pathway before 2-aminoadipic acid, was able to synthesize penicillin when supplemented with lysine. The amount of penicillin produced increased as the level of lysine in the media was increased. The same results were observed in resting-cell systems. Catabolism of [U-14C]lysine by resting cells and batch cultures of P. chrysogenum L2 resulted in the formation of labeled saccharopine and 2-aminoadipic acid. Formation of [14C]saccharopine was also observed in vitro when cell extracts of P. chrysogenum L2 and Wis 54-1255 were used. Saccharopine dehydrogenase and saccharopine reductase activities were found in cell extracts of P. chrysogenum, which indicates that lysine catabolism may proceed by reversal of the two last steps of the lysine biosynthetic pathway. In addition, a high lysine:2-ketoglutarate-6-aminotransferase activity, which converts lysine into piperideine-6-carboxylic acid, was found in cell extracts of P. chrysogenum. These results suggest that lysine is catabolized to 2-aminoadipic acid in P. chrysogenum by two different pathways. The relative contribution of lysine catabolism in providing 2-aminoadipic acid for penicillin production is discussed.     

Efficient sporulation of yeast in media buffered near pH6.

Diploid cells of Saccharomyces cerevisiae underwent meiosis and sporulation when placed in 1% potassium acetate sporulation medium. In unbuffered sporulation medium the pH rose very rapidly, reaching pH 8.4 after 2 h of sporulation. Under these conditions, the uptake of radioactive adenine and lysine was extremely limited, and ascus formation was insensitive to inhibitors such as 5-fluorouracil and canavanine. By using several different buffers, we showed that an increase in the pH of sporulation media was not necessary for sporulation to occur. Spore viability and the kinetics of ascus and prototroph formation were normal for cells sporulated in several types of media buffered as low as pH 5.5. Incubation of sporulating cells below pH 6.5 did cause separation of small but viable buds from their mother cells. With sporulating cells buffered below pH 6.5, the incorporation of radioactive adenine and lysine was greatly enhanced and cells became sensitive to inhibition by 5-fluorouracil and canavanine.     

Effects of Fatty Acids on Growth and Envelope Proteins of Bacillus subtilis

Fatty acids of different chain lengths were added to cultures of Bacillus subtilis growing in nutrient sporulation medium, and the effects of these fatty acids on growth, oxygen uptake, adenosine triphosphate (ATP) concentration, and membrane protein composition were examined. All fatty acids inhibited growth, the effect being reduced in the presence of glycolytic compounds and reversed by transfer to medium without fatty acids. The inhibition of growth was correlated with a reduction in both the rate of oxygen consumption and the concentration of ATP per cell. The concentration required to obtain a certain degree of inhibition increased with decreasing molecular weight of the fatty acid. However, the reduced nicotinamide adenine dinucleotide oxidation system of cell envelope preparations (i.e., the electron transport system) was not inhibited. Submaximal growth inhibition was accompanied by the relative increase of a membrane protein band revealed by urea-acetic acid gel electrophoresis. This increase was blocked by actinomycin or chloramphenicol. All of the above changes could also be produced by 2,4-dinitrophenol. The inhibition results are best explained by assuming that the fatty acids reversibly react with the cell membrane or proteins in it; they could either alter the membrane structure or uncouple the electron transport chain from two types of proteins, those used for ATP regeneration and others needed for the transport of certain compounds into the cells.     

iTRAQ analysis reveals the effect of gabD and sucA gene knockouts on lysine metabolism and crystal protein formation in Bacillus thuringiensis

Lysine metabolism plays an important role in the formation of the insecticidal crystal proteins of Bacillus thuringiensis (Bt). The genes lam, gabD and sucA encode three key enzymes of the lysine metabolic pathway in Bt4.0718. The lam gene mainly affects the cell growth at stable period, negligibly affected sporulation and insecticidal crystal protein (ICP) production. While, the deletion mutant strains of the gabD and sucA genes showed that the growth, sporulation and crystal protein formation were inhibited, cells became slender, and insecticidal activity was significantly reduced. iTRAQ proteomics and qRT-PCR used to analyse the differentially expressed protein (DEP) between the two mutant strains and the wild type strain. The functions of DEPs were visualized and statistically classified, which affect bacterial growth and metabolism by regulating biological metabolism pathways: the major carbon metabolism pathways, amino acid metabolism, oxidative phosphorylation pathways, nucleic acid metabolism, fatty acid synthesis and peptidoglycan synthesis. The gabD and sucA genes in lysine metabolic pathway are closely related to the sporulation and crystal proteins formation. The effects of DEPs and functional genes on basic cellular metabolic pathways were studied to provide new strategies for the construction of highly virulent insecticidal strains, the targeted transformation of functional genes.     

Inhibition of staphylococcal enterotoxin B formation by cell wall blocking agents and other compounds

Enterotoxin B formation by Staphylococcus aureus S6 was inhibited by Tween 80, oleic acid, sodium deoxycholate, penicillin, d-cycloserine, or bacitracin. Toxin formation by strain 243 was sensitive to oleic acid, sodium deoxycholate, sodium lauryl sulfate, d-cycloserine, or bacitracin. The effect of d-cycloserine was reversed by d-alanine with strain 243 but not with strain S6. Neither penicillin nor bacitracin inhibited alpha-hemolysin or coagulase activity of strain S6; however, 0.118 mumoles of d-cycloserine per ml increased the alpha-hemolysin titer more than eightfold. Pigmentation of strain 243 was reduced by oleic acid, sodium deoxycholate, or methicillin, and was completely inhibited by d-cycloserine or bacitracin. Glucose was required for the inhibition by spermine of (14)C-valine incorporation into cellular protein of strain S6. These data indicate that the cell surface may contain sites important to the synthesis of enterotoxin B.     

Inhibition of Growth of Lactobacillus bulgaricus by Purine Deoxyribonucleotides.

Morris, George K. (University of Georgia, Athens), and William L. Williams. Inhibition of growth of Lactobacillus bulgaricus by purine deoxyribonucleotides. J. Bacteriol. 90:715-719. 1965.-An inhibition of growth of Lactobacillus bulgaricus GS was observed with deoxyadenylic acid and deoxyguanylic acid. Deoxynucleotides of cytosine, thymine, and uracil, and the deoxynucleosides of adenine, guanine, cytosine, and thymine were inactive as inhibitors. The inhibition was reversed by liver extract (a crude source of two unidentified growth factors for this organism). With suboptimal concentrations of liver extract, the inhibition was reversed by nucleotides of adenine, guanine, uracil, cytosine, and thymine. When the medium contained partially purified sources of the two growth factors rather than crude liver extract, fewer compounds reversed the inhibition. Adenylic acid and guanylic acid reversed the action of either inhibitor. Inosinic acid reversed inhibition caused by deoxyguanylic acid, but not that caused by deoxyadenylic acid. Thymidylic acid reversed inhibition caused by deoxyadenylic acid better than that caused by deoxyguanylic acid. Uridylic acid and cytidylic acid were no longer effective in reversing the inhibitions. This organism preferentially responded to monophosphorylated compounds as inhibitors and as reversers of inhibitions. Studies on the acid-soluble nucleotide pool revealed an accumulation of adenosine triphosphate, guanosine triphosphate, and an unidentified compound which resembled a nucleotide in its physical properties. These data cannot be explained by known metabolic pathways of nucleic acid biosynthesis. This organism responds differently from other related organisms to nucleic acid derivatives; therefore, it may be a new useful tool for studying nucleic acid metabolism and biosynthesis.     

Tannins as gibberellin antagonists

Fourteen chemically defined hydrolyzable tannins and six impure mixtures of either condensed or hydrolyzable tannins were found to inhibit the gibberellin-induced growth of light-grown dwarf pea seedlings. The highest ratio of tannins to gibberellic acid tested (1000: 1 by weight) inhibited from 80 to 95% of the induced growth for all tannins tested except for two monogalloyl glucose tannins which inhibited only 50% of the induced growth. The lowest ratio tested (10: 1) inhibited the induced growth by less than 25% except for the case of terchebin where 50% inhibition was found. The inhibition of gibberellin-induced growth was found to be completely reversed by increasing the amount of gibberellin in three cases tested. Tannins alone did not inhibit endogenous growth of either dwarf or nondwarf pea seedlings. Eight compounds related to tannins, including coumarin, trans-cinnamic acid, and a number of phenolic compounds were also tested as gibberellin antagonists. Most of these compounds showed some inhibition of gibberellin-induced growth, but less than that of the tannins. At the highest ratio (1000: 1) the greatest inhibition was 55%; at the lowest ratio (10: 1) no more than 17% was observed. These compounds did not inhibit endogenous growth, and the inhibition of gibberellin-induced growth could be reversed by increasing the amount of gibberellin in two cases tested.     

Inhibition and repression of homocitrate synthase by lysine in Penicillium chrysogenum

Homocitrate synthase in the first enzyme of the lysine biosynthetic pathway. It is feedback regulated by L-lysine. Lysine decreases the biosynthesis of penicillin (determined by the incorporation of [14C]valine into penicillin) by inhibiting and repressing homocitrate synthase, thereby depriving the cell of alpha-aminoadipic acid, a precursor of penicillin. Lysine feedback inhibited in vivo the biosynthesis and excretion of homocitrate by a lysine auxotroph, L2, blocked in the lysine pathway after homocitrate. Neither penicillin nor 6-aminopenicillanic acid exerted any effect at the homocitrate synthase level. The molecular mechanism of lysine feedback regulation in Penicillium chrysogenum involved both inhibition of homocitrate synthase activity and repression of its synthesis. In vitro studies indicated that L-lysine feedback inhibits and represses homocitrate synthase both in low- and high-penicillin-producing strains. Inhibition of homocitrate synthase activity by lysine was observed in cells in which protein synthesis was arrested with cycloheximide. Maximum homocitrate synthase activity in cultures of P. chrysogenum AS-P-78 was found at 48 h, coinciding with the phase of high rate of penicillin biosynthesis.     

Regulation of alpha-aminoadipate reductase from Penicillium chrysogenum in relation to the flux from alpha-aminoadipate into penicillin biosynthesis.

The activity and regulation of alpha-aminoadipate reductase in three Penicillium chrysogenum strains (Q176, D6/1014/A, and P2), producing different amounts of penicillin, were studied. The enzyme exhibited decreasing affinity for alpha-aminoadipate with increasing capacity of the respective strain to produce penicillin. The enzyme from all three strains was inhibited by L-lysine, and the enzyme from the lowest producer, Q176, was least sensitive. Between pH 7.5 and 6.5, inhibition of alpha-aminoadipate reductase by L-lysine was pH dependent, being more pronounced at lower pH. The highest producer strain, P2, displayed the lowest alpha-aminoadipate reductase activity at pH 7.0. In Q176, the addition of 0.5-1 mM of exogenous lysine stimulated penicillin formation, whereas the same concentration was ineffective or inhibitory with strains D6/1014/A and P2. The addition of higher (up to 5 mM) lysine concentrations inhibited penicillin production in all three strains. In mutants of P. chrysogenum D6/1014/A, selected for resistance to 20 mM alpha-aminoadipate, highest penicillin production was observed in those strains whose alpha-aminoadipate reductase was most strongly inhibited by L-lysine. The results support the conclusion that the in vivo activity of alpha-aminoadipate reductase from superior penicillin producer strains of P. chrysogenum is more strongly inhibited by lysine, and that this is related to their ability to accumulate increased amounts of alpha-aminoadipate, and hence penicillin.     

Glucose represses formation of delta-(L-alpha-aminoadipyl)-L-cysteinyl-D-valine and isopenicillin N synthase but not penicillin acyltransferase in Penicillium chrysogenum.

The content of alpha-aminoadipyl-cysteinyl-valine, the first intermediate of the penicillin biosynthetic pathway, decreased when Penicillium chrysogenum was grown in a high concentration of glucose. Glucose repressed the incorporation of [14C]valine into alpha-aminoadipyl-cysteinyl-[14C]valine in vivo. The pool of alpha-aminoadipic acid increased sevenfold in control (lactose-grown) penicillin-producing cultures, coinciding with the phase of rapid penicillin biosynthesis, but this increase was very small in glucose-grown cultures. Glucose stimulated homocitrate synthase and saccharopine dehydrogenase activities in vivo and increased the incorporation of lysine into proteins. These results suggest that glucose stimulates the flux through the lysine biosynthetic pathway, thus preventing alpha-aminoadipic acid accumulation. The repression of alpha-aminoadipyl-cysteinyl-valine synthesis by glucose was not reversed by the addition of alpha-aminoadipic acid, cysteine, or valine. Glucose also repressed isopenicillin N synthase, which converts alpha-aminoadipyl-cysteinyl-valine into isopenicillin N, but did not affect penicillin acyltransferase, the last enzyme of the penicillin biosynthetic pathway.     

Induction of sporulation by inhibitory purines and related compounds.

Sporulation of Bacillus subtilis can be induced, in the presence of excess ammonia, glucose and phosphate, by many purine derivatives under conditions of partial growth inhibition. Some of the compounds are known inhibitors of purine nucleotide synthesis. For most compounds the effect is counteracted by adenine and guanine. Partial growth inhibition by amethopterin (methotrexate) causes sporulation in the absence of purines but not in their presence. Unable to induce sporulation at any concentration are inhibitors of DNA, RNA, and protein synthesis as well as base or amino acid analogs that are incorporated into these polymers.