Cultures ofP Research Articles

Pyridine-2,6-Bis(Thiocarboxylic Acid) Produced by Pseudomonas stutzeri KC Reduces and Precipitates Selenium and Tellurium Oxyanions

The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H(2)S, forming zero-valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids.

Effects of morphology and rheology on neofructosyltransferase production byPenicillium citrinum

In this study, we investigated the relationship between the morphology and the rheological properties ofPenicillium citrinum to improve the production of neo-fructosyltransferase (neo-FTase). In a 2.5 L bioreactor culture ofP. citrinum, it was observed that agitation speed and aeration rate had significant effects on the production of neo-FTase and that maximum cell mass and neo-FTase production obtained at 500 rpm and 1.5 vvm were 8.14 g/L and 53.2×10−3 U/mL, respectively. Cell mass and neo-FTase production increased to 91.53 and 25.17%, respectively. In the morphology and rheology studies,P. citrinum showed a typical pellet morphology that was explained by a shaving mechanism; this phenomenon was significantly affected by carbon sources. The rheology of neo-FTase fermentation byP. citrinum was dependent on cell growth and fungal morphology.

Delays in Pseudomonas aeruginosa quorum-controlled gene expression are conditional

Acyl-homoserine lactone (acyl-HSL) signaling is thought to mediate quorum sensing in many species of Proteobacteria. The opportunistic human pathogen Pseudomonas aeruginosa uses acyl-HSLs to regulate hundreds of genes, including many that code for extracellular virulence factors. The idea that the P. aeruginosa acyl-HSLs serve as quorum-sensing signals has been questioned recently because microarray experiments show that the addition of signals to cultures of P. aeruginosa does not advance the onset of transcription for most acyl-HSL-dependent genes. We show that, under specific conditions, the expression of many acyl-HSL-dependent genes can be triggered at low culture density by signal addition. If complex medium is conditioned by growth of a non-acyl-HSL-producing P. aeruginosa, signals can eliminate the delay in expression of a battery of acyl-HSL-dependent genes. Furthermore, for one representative gene, lasB, there is no delay when signals are added to P. aeruginosa growing in conditioned complex medium or in minimal medium. We conclude that complex medium contains an inhibitor or inhibitors that can prevent induction of many, but not all, acyl-HSL-regulated genes and that the inhibitor is consumed by P. aeruginosa. Our results show that acyl-HSL signals can trigger expression of a large number of acyl-HSL-dependent genes regardless of growth phase. In this way, signaling in P. aeruginosa appears similar to quorum signaling in other Proteobacteria.

Efficient elicitation of ginsenoside biosynthesis in cell cultures ofPanax notoginseng by using self-chemically-synthesized jasmonates

A series of fluorine and hydroxyl containing jasmonate derivatives, which were chemically synthesized in our institute, were investigated for their effects on the biosynthesis and heterogeneity of ginsenosides in suspension cultures ofPanax notoginseng cells. Compared to the control (without addition of elicitors), 100 μM of each of the jasmonate was added on day 4 to the suspension cultures ofP. notoginseng cells. It was observed that, jasmonates greatly enhanced the ginsenoside content and the ratio of Rb group to Rg group (i.e. (Rb1+Rd)/(Rg1+Re)) in theP. notoginseng cells. Some of the synthetic jasmonates, such as pentafluoropropyl jasmonate (PFPJA), 2-hydroxyethyl jasmonate (HEJA) and 2-hydroxyethoxyethyl jasmonate (HEEJA), could promote the ginsenoside content to 2.55±0.11, 3.65±0.13 and 2.94±0.06 mg/100 mg DW, respectively, compared to that of 0.64±0.06 mg/100 mg DW for the control and 2.17±0.04 mg/100 mg DW by the commercially available methyl jasmonate (MJA); and they could change the respective Rb: Rg ratio to 1.60±0.04, 1.87±0.01 and 1.56±0.05, compared to that of 0.47±0.01 for the control and 1.42±0.06 by MJA. The results suggest that suitable esterification of MJA with fluorine or hydroxyl group could increase the elicitation activity to induce plant secondary metabolism. The information obtained from this study is useful for hyper-production of heterogeneous products by plant cell cultures.

Enhancement of growth and secondary metabolite biosynthesis: Effect of elicitors derived from plants and insects

Plant-derived natural products have been and will continue to be valuable sources. Elicitors have been employed to modify cell metabolism in order to enhance the productivity of useful metabolites in plant cell/tissue cultures. In this study, several elicitors were used to improve the productivity of useful metabolites and to reduce culture time for archiving high concentration inP. ginseng hairy root cultures. The addition of chitosan, chitosan oligosaccharide and alginate oligosaccharide to the culture ofP. ginseng hairy roots caused growth to be inhibited with the increase in elicitor concentration. The usage of the chitosan elicitor andd-glucosamine caused a slight decrease in hairy root growth, whereas total ginseng saponin accumulated slightly with the increase in elicitor concentration. When gel beads were added to the culture medium at the initial period, hairy root growth was enhanced. The maximum growth was 1.35 times higher than that of the control at 1% (w/v). Total ginseng saponin content decreased due to the addition of alginate beads. This would result in consistent diffusion of lower levels of calcium ions during the culture period that promotes biomass growth.

Modified serial analysis of gene expression method for construction of gene expression profiles of microbial eukaryotic species.

Serial analysis of gene expression (SAGE) is a powerful approach for the identification of differentially expressed genes, providing comprehensive and quantitative gene expression profiles in the form of short tag sequences. Each tag represents a unique transcript, and the relative frequencies of tags in the SAGE library are equal to the relative proportions of the transcripts they represent. One of the major obstacles in the preparation of SAGE libraries from microorganisms is the requirement for large amounts of starting material (i.e., mRNA). Here, we present a novel approach for the construction of SAGE libraries from small quantities of total RNA by using Y linkers to selectively amplify 3' cDNA fragments. To validate this method, we constructed comprehensive gene expression profiles of the toxic dinoflagellate Pfiesteria shumwayae. SAGE libraries were constructed from an actively toxic fish-fed culture of P. shumwayae and from a recently toxic alga-fed culture. P. shumwayae-specific gene transcripts were identified by comparison of tag sequences in the two libraries. Representative tags with frequencies ranging from 0.026 to 3.3% of the total number of tags in the libraries were chosen for further analysis. Expression of each transcript was confirmed in separate control cultures of toxic P. shumwayae. The modified SAGE method described here produces gene expression profiles that appear to be both comprehensive and quantitative, and it is directly applicable to the study of gene expression in other environmentally relevant microbial species.

Dimethylsulfoniopropionate metabolism by Pfiesteria-associated Roseobacter spp.

The Roseobacter clade of marine bacteria is often found associated with dinoflagellates, one of the major producers of dimethylsulfoniopropionate (DMSP). In this study, we tested the hypothesis that Roseobacter species have developed a physiological relationship with DMSP-producing dinoflagellates mediated by the metabolism of DMSP. DMSP was measured in Pfiesteria and Pfiesteria-like (Cryptoperidiniopsis) dinoflagellates, and the identities and metabolic potentials of the associated Roseobacter species to degrade DMSP were determined. Both Pfiesteria piscicida and Pfiesteria shumwayae produce DMSP with an average intracellular concentration of 3.8 microM. Cultures of P. piscicida or Cryptoperidiniopsis sp. that included both the dinoflagellates and their associated bacteria rapidly catabolized 200 microM DMSP (within 30 h), and the rate of catabolism was much higher for P. piscicida cultures than for P. shumwayae cultures. The community of bacteria from P. piscicida and Cryptoperidiniopsis cultures degraded DMSP with the production of dimethylsulfide (DMS) and acrylate, followed by 3-methylmercaptopropionate (MMPA) and methanethiol (MeSH). Four DMSP-degrading bacteria were isolated from the P. piscicida cultures and found to be taxonomically related to Roseobacter species. All four isolates produced MMPA from DMSP. Two of the strains also produced MeSH and DMS, indicating that they are capable of utilizing both the lyase and demethylation pathways. The diverse metabolism of DMSP by the dinoflagellate-associated Roseobacter spp. offers evidence consistent with a hypothesis that these bacteria benefit from association with DMSP-producing dinoflagellates.

Phenazines and other redox-active antibiotics promote microbial mineral reduction.

Natural products with important therapeutic properties are known to be produced by a variety of soil bacteria, yet the ecological function of these compounds is not well understood. Here we show that phenazines and other redox-active antibiotics can promote microbial mineral reduction. Pseudomonas chlororaphis PCL1391, a root isolate that produces phenazine-1-carboxamide (PCN), is able to reductively dissolve poorly crystalline iron and manganese oxides, whereas a strain carrying a mutation in one of the phenazine-biosynthetic genes (phzB) is not; the addition of purified PCN restores this ability to the mutant strain. The small amount of PCN produced relative to the large amount of ferric iron reduced in cultures of P. chlororaphis implies that PCN is recycled multiple times; moreover, poorly crystalline iron (hydr)oxide can be reduced abiotically by reduced PCN. This ability suggests that PCN functions as an electron shuttle rather than an iron chelator, a finding that is consistent with the observation that dissolved ferric iron is undetectable in culture fluids. Multiple phenazines and the glycopeptidic antibiotic bleomycin can also stimulate mineral reduction by the dissimilatory iron-reducing bacterium Shewanella oneidensis MR1. Because diverse bacterial strains that cannot grow on iron can reduce phenazines, and because thermodynamic calculations suggest that phenazines have lower redox potentials than those of poorly crystalline iron (hydr)oxides in a range of relevant environmental pH (5 to 9), we suggest that natural products like phenazines may promote microbial mineral reduction in the environment.

Biological aspects on the cultures of the entomophthoralean fungusPandora delphacis grown on broomcorn millets

A novel method was developed to use glutinous broomcorn millets (Panicum miliaceum L.) as solid substrate to make cultures of the entomophthoralean fungusPandora delphacis specifically pathogenic to planthoppers, leafhoppers and aphids. Steamed millets with water content of 45% were inoculated with a liquid culture ofP. delphacis at a ratio of 20% (v/w) and then incubated at 25°C and L:D 12:12. The millets cultured for 3–17 d exhibited high potential for conidial production. The 5-d-old millet culture sporulated most abundantly, discharging up to 17.12 (±1.31) × 104 conidia/ millet. The cultures incubated for 7–11 d also had a satisfactory sporulation capability, yielding 13.00–13.90 × 104 conidia/millet. Compared to 2.32 (±0.34) × 104 conidia discharged from each ofMyzus persicae adults killed byP. delphacis and a ≤60-h duration of sporulation, each of the millets cultured for 5–11 d produced 5.6–7.4 times more conidia with an over doubled duration for conidial discharge (144 h). Among 106M. persicae adults exposed to the shower of conidia discharged from the cultured millets, a total mortality of 69.8% caused byP. delphacis infection was observed within 7 d after exposure, but no death was attributed to the fungal infection in the aphids unexposed. The results indicate that the millet cultures ofP. delphacis are biologically similar to aphid cadavers killed by the same fungus. Due to the superiority of the cultured millets to the cadavers in sporulation potential and duration, the method for making cultures ofP. delphacis on the broomcorn millets is highly recommended for use in study of entomophthoralean fungi for microbial control. This is the first report on the success of the solid culture ofPandora species on cereals.

Hostile Interactions in the Middle East

Phytoplankton, microscopic plants that inhabit aquatic ecosystems, are the first link in the aquatic food chain and can influence climate globally by altering amounts of atmospheric carbon dioxide. Certain species produce toxins that can impact human health. In the Sea of Galilee, Peridinium gatunense , a dinoflagellate that forms large, dense patches, has historically dominated the phytoplankton community. Over the past decade, however, annual blooms of P. gatunense have been delayed or absent. Vardi et al. investigated interactions between P. gatunense and Microcystis , a toxic cyanobacterium, and discovered that substances released into the water by one or the other of these microorganisms influenced survival of the other through species-specific signal transduction pathways. Statistical analyses indicated a negative correlation between abundance of the two microorganisms in the wild, and long-term growth experiments showed reciprocal inhibition of growth between P. gatunense and Microcystis that depended on the initial density of P. gatunense but was independent of nutrient supply. A particular species, Microcystis MG, triggered a biphasic oxidative burst in P. gatunense and promoted cell death under some culture conditions while stimulating division of surviving cells. Pharmacological analysis with protein kinase inhibitors, together with in-gel kinase and Western blot analysis, implicated mitogen-activated protein kinase pathways in the P. gatunense response. Medium from cultures of P. gatunense triggered massive Microcystis MG lysis, but increased amounts of a protein involved in toxin biosynthesis in the remaining cells. The authors suggest that phytoplankton can influence the growth of other species through interactions with specific signal transduction pathways; such interactions raise the possibility of biological control of toxic species. A Vardi, D Schatz, K Beeri, U Motro, A Sukenik, A Levine, A Kaplan, Dinoflagellate-cyanobacterium communication may determine the composition of phytoplankton assemblage in a mesotrophic lake. Curr. Biol. 12 , 1767-1772 (2002). [Online Journal]

Characterization of Phenotypic Changes in Pseudomonas putida in Response to Surface-Associated Growth

The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface. A switch between planktonic and sessile growth is believed to result in a phenotypic change in bacteria. In this study, a global analysis of physiological changes of the plant saprophyte Pseudomonas putida following 6 h of attachment to a silicone surface was carried out by analysis of protein profiles and by mRNA expression patterns. Two-dimensional (2-D) gel electrophoresis revealed 15 proteins that were up-regulated following bacterial adhesion and 30 proteins that were down-regulated. N-terminal sequence analyses of 11 of the down-regulated proteins identified a protein with homology to the ABC transporter, PotF; an outer membrane lipoprotein, NlpD; and five proteins that were homologous to proteins involved in amino acid metabolism. cDNA subtractive hybridization revealed 40 genes that were differentially expressed following initial attachment of P. putida. Twenty-eight of these genes had known homologs. As with the 2-D gel analysis, NlpD and genes involved in amino acid metabolism were identified by subtractive hybridization and found to be down-regulated following surface-associated growth. The gene for PotB was up-regulated, suggesting differential expression of ABC transporters following attachment to this surface. Other genes that showed differential regulation were structural components of flagella and type IV pili, as well as genes involved in polysaccharide biosynthesis. Immunoblot analysis of PilA and FliC confirmed the presence of flagella in planktonic cultures but not in 12- or 24-h biofilms. In contrast, PilA was observed in 12-h biofilms but not in planktonic culture. Recent evidence suggests that quorum sensing by bacterial homoserine lactones (HSLs) may play a regulatory role in biofilm development. To determine if similar protein profiles occurred during quorum sensing and during early biofilm formation, HSLs extracted from P. putida and pure C(12)-HSL were added to 6-h planktonic cultures of P. putida, and cell extracts were analyzed by 2-D gel profiles. Differential expression of 16 proteins was observed following addition of HSLs. One protein, PotF, was found to be down-regulated by both surface-associated growth and by HSL addition. The other 15 proteins did not correspond to proteins differentially expressed by surface-associated growth. The results presented here demonstrate that P. putida undergoes a global change in gene expression following initial attachment to a surface. Quorum sensing may play a role in the initial attachment process, but other sensory processes must also be involved in these phenotypic changes.

Biochemical characterization of ochratoxin A-producing strains of the genus Penicillium.

In order to explore the biochemical scope of ochratoxin A-producing penicillia, we screened 48 Penicillium verrucosum isolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spectrometry. The following metabolites were identified: ochratoxins A and B, citrinin, verrucolones, verrucines, anacines, sclerotigenin, lumpidin, fumiquinazolines, alantrypinones, daldinin D, dipodazine, penigequinolines A and B, 2-pentanone, and 2-methyl-isoborneol. By use of average linking clustering based on binary (nonvolatile) metabolite data, the 48 isolates could be grouped into two large and clearly separated groups and a small outlying group of four non-ochratoxin-producing isolates. The largest group, containing 24 isolates, mainly originating from plant sources, included the type culture of P. verrucosum. These isolates produced ochratoxin A, verrucolones, citrinin, and verrucines and had a characteristic dark brown reverse color on yeast extract-sucrose agar medium. Almost all of a group of 20 isolates mainly originating from cheese and meat products had a pale cream reverse color on yeast extract-sucrose agar medium and produced ochratoxin A, verrucolones, anacines, and sclerotigenin. This group included the former type culture of P. nordicum. We also found that P. verrucosum isolates and three P. nordicum isolates incorporated phenylalanine into verrucine and lumpidin metabolites, a finding which could explain why those isolates produced relatively lower levels of ochratoxins than did most isolates of P. nordicum.

Reduction of ferric chelate caused by various wood-rot fungi

The reduction of ferric chelate caused by various wood-rot fungi was analyzed. Ferric chelate reductive activity was detected in cell-free extracts of seven wood-rot fungi:Phanerochaete chrysosporium, P. sordida YK-624,Ganoderma sp. YK-505,Coriolus versicolor, Bjerkandera adusta, Tyromyces palustris, andGloeophyllum trabeum. These fungi produced NADPH- or NADH-dependent ferric chelate reductive enzymes (or both) of different molecular weight. In the liquid culture ofP. sordida YK-624 andC. versicolor, a positive correlation was observed between extracellular MnP activity and intracellular NADPH-dependent ferric chelate reductive activity.

1,4-benzoquinone reductase from Phanerochaete chrysosporium: cDNA cloning and regulation of expression.

A cDNA clone encoding a quinone reductase (QR) from the white rot basidiomycete Phanerochaete chrysosporium was isolated and sequenced. The cDNA consisted of 1,007 nucleotides and a poly(A) tail and encoded a deduced protein containing 271 amino acids. The experimentally determined eight-amino-acid N-terminal sequence of the purified QR protein from P. chrysosporium matched amino acids 72 to 79 of the predicted translation product of the cDNA. The Mr of the predicted translation product, beginning with Pro-72, was essentially identical to the experimentally determined Mr of one monomer of the QR dimer, and this finding suggested that QR is synthesized as a proenzyme. The results of in vitro transcription-translation experiments suggested that QR is synthesized as a proenzyme with a 71-amino-acid leader sequence. This leader sequence contains two potential KEX2 cleavage sites and numerous potential cleavage sites for dipeptidyl aminopeptidase. The QR activity in cultures of P. chrysosporium increased following the addition of 2-dimethoxybenzoquinone, vanillic acid, or several other aromatic compounds. An immunoblot analysis indicated that induction resulted in an increase in the amount of QR protein, and a Northern blot analysis indicated that this regulation occurs at the level of the qr mRNA.

Novel interaction between laccase and cellobiose dehydrogenase during pigment synthesis in the white rot fungus Pycnoporus cinnabarinus.

When glucose is the carbon source, the white rot fungus Pycnoporus cinnabarinus produces a characteristic red pigment, cinnabarinic acid, which is formed by laccase-catalyzed oxidation of the precursor 3-hydroxyanthranilic acid. When P. cinnabarinus was grown on media containing cellobiose or cellulose as the carbon source, the amount of cinnabarinic acid that accumulated was reduced or, in the case of cellulose, no cinnabarinic acid accumulated. Cellobiose-dependent quinone reducing enzymes, the cellobiose dehydrogenases (CDHs), inhibited the redox interaction between laccase and 3-hydroxyanthranilic acid. Two distinct proteins were purified from cellulose-grown cultures of P. cinnabarinus; these proteins were designated CDH I and CDH II. CDH I and CDH II were both monomeric proteins and had apparent molecular weights of about 81,000 and 101,000, respectively, as determined by both gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pI values were approximately 5.9 for CDH I and 3.8 for CDH II. Both CDHs used several known CDH substrates as electron acceptors and specifically adsorbed to cellulose. Only CDH II could reduce cytochrome c. The optimum pH values for CDH I and CDH II were 5.5 and 4.5, respectively. In in vitro experiments, both enzymes inhibited laccase-mediated formation of cinnabarinic acid. Oxidation intermediates of 3-hydroxyanthranilic acid served as endogenous electron acceptors for the two CDHs from P. cinnabarinus. These results demonstrated that in the presence of a suitable cellulose-derived electron donor, CDHs can regenerate fungal metabolites oxidized by laccase, and they also supported the hypothesis that CDHs act as links between cellulolytic and ligninolytic pathways.

Plasmodium falciparumandPlasmodium yoelii:Effect of the Iron Chelation Prodrug Dexrazoxane onin VitroCultures

Loyevsky, M., Sacci, J. B., Jr., Boehme, P., Weglicki, W., John, C., and Gordeuk, V. R. 1999.Plasmodium falciparumandPlasmodium yoelii: Effect of the iron chelation prodrug dexrazoxane onin vitrocultures.Experimental Parasitology91, 105–114. To determine if an iron-chelating prodrug that must undergo intracellular hydrolysis to bind iron has antimalarial activity, we examined the action of dexrazoxane onPlasmodium falciparumcultured in human erythrocytes andP. yoeliicultured in mouse hepatocytes. Dexrazoxane was recently approved to protect humans from doxorubucin-induced cardiotoxicity. Using the fluorescent marker calcein, we confirmed that the iron-chelating properties of dexrazoxane are directly related to its ability to undergo hydrolysis. As a single agent, dexrazoxane inhibited synchronized cultures ofP. falciparumin human erythrocytes only at suprapharmacologic concentrations (>200 μM). In combination with desferrioxamine B, dexrazoxane in pharmacologic concentrations (100–200 μM) moderately potentiated inhibition by approximately 20%. In contrast, pharmacologic concentrations of dexrazoxane (50–200 μM) as a single agent inhibited the progression ofP. yoeliifrom sporozoites to schizonts in cultured mouse hepatocytes by 45 to 69% (P< 0.001). These results are consistent with the presence of a dexrazoxane-hydrolyzing enzyme in hepatocytes but not in erythrocytes or malaria parasites. Furthermore, these findings suggest that dexrazoxane must be hydrolyzed to an iron-chelating intermediate before it can inhibit the malaria parasite, and they raise the possibility that the iron chelator prodrug concept might be exploited to synthesize new antimalarial agents.

Characterization of Proteus mirabilis precocious swarming mutants: identification of rsbA, encoding a regulator of swarming behavior.

Proteus mirabilis swarming behavior is characterized by the development of concentric rings of growth that are formed as cyclic events of swarmer cell differentiation, swarming migration, and cellular differentiation are repeated during colony translocation across a surface. This cycle produces the bull's-eye colony often associated with cultures of P. mirabilis. How the cells communicate with one another to coordinate these perfectly synchronized rings is presently unknown. We report here the identification of a genetic locus that, when mutated, results in a precocious swarming phenotype. These mutants are defective in the temporal control of swarming migration and start swarming ca. 60 min sooner than wild-type cells. Unlike the wild type, precocious swarming mutants are also constitutive swarmer cells and swarm on minimal agar medium. The defects were found to be localized to a 5.4-kb locus on the P. mirabilis genome encoding RsbA (regulator of swarming behavior) and the P. mirabilis homologs to RcsB and RcsC. RsbA is homologous to membrane sensor histidine kinases of the two-component family of regulatory proteins, suggesting that RsbA may function as a sensor of environmental conditions required to initiate swarming migration. Introduction of a rsbA mutation back into the wild type via allelic-exchange mutagenesis reconstructed the precocious swarming phenotype, which could be complemented in trans by a plasmid-borne copy of rsbA. Overexpression of RsbA in wild-type cells resulted in precocious swarming, suggesting that RsbA may have both positive and negative functions in regulating swarming migration. A possible model to describe the role of RsbA in swarming migration is discussed.

De novo synthesis of amino acids by the ruminal bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1.

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing 15NH4Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.

Degradation and Utilization of Xylans by the Rumen AnaerobePrevotella bryantii(formerlyP. ruminicolasubsp.brevis) B14

Freshly harvested whole cells from cultures ofP. bryantiiB14 grown with oat spelt xylan (OSX) as an energy source showed less than 25% of the enzyme activity against OSX, and less than 15% of the activity against birchwood xylan (BWX) and carboxymethylcellulose, that was detectable in sonicated cell preparations. This indicates that much of this hydrolytic activity is either periplasmic, membrane-associated or intracellular and may be concerned with the processing of transported oligosaccharides.P. bryantiiB14 cultures were able to utilise up to 45% and 51% of the total pentose present in OSX and BWX, respectively, after 24 h, but could utilize 84% of a water-soluble fraction of BWX. Analysis of the xylan left undegraded after incubation withP. bryantiishowed that while xylose and arabinose were removed to a similar extent, uronic acids were utilized to a greater extent than xylose. Predigestion of xylans with two cloned xylanases from the cellulolytic rumen anaerobeRuminococcus flavefaciensgave little increase in overall pentose utilization suggesting that externalP. bryantiixylanases are as effective as the clonedR. flavefaciensenzymes in releasing products that can be utilised byP. bryantiicells. The xylanase system ofP. bryantiiis able to efficiently utilise not only xylo-oligosaccharides but also larger water-soluble xylan fragments.

High-Level Production of RecombinantGeotrichum candidumLipases in YeastPichia pastoris

We describe the heterologous high-level expression of the twoGeotrichum candidumlipase (GCL) isoenzymes from strain ATCC 34614 in the methylotrophic yeastPichia pastoris.The lipase cDNAs were placed under the control of the methanol-inducible alcohol oxidase promoter. The lipases expressed inP. pastoriswere fused to the α-factor secretion signal peptide ofSaccharomyces cerevisiaeand were secreted into the culture medium. Cultures ofP. pastorisexpressing lipase accumulated active recombinant enzyme in the supernatant to levels of ∼60 mg/L virtually free from contaminating proteins. This yield exceeds that previously reported withS. cerevisiaeby a factor of more than 60. Recombinant GCL I and GCL II had molecular masses of ∼63 and ∼66 kDa, respectively, as determined by SDS–PAGE. The result of endoglucosidase H digestion followed by Western blot analysis of the lipases suggested that the enzymes expressed inP. pastorisreceived N-linked high-mannose-type glycosylation to an extent, 6–8% (w/w), similar to that inG. candidum.The specific activities and substrate specificities of both recombinant lipases were determined and were found to agree with what has been reported for the enzymes isolated from the native source.