Anti-cyanobacterial fatty acids released from <Emphasis Type="Italic">Myriophyllum spicatum</Emphasis>

This study was carried out to identify unknown allelochemicals released from Myriophyllum spicatum and to investigate their anti-cyanobacterial effects. A series of analyses of culture solutions and methanol extracts of M. spicatum using gas chromatograph equipped with a mass selective detector revealed that M. spicatum released fatty acids, specifically, nonanoic, tetradecanoic, hexadecanoic, octadecanoic, and octadecenoic acids. Nonanoic, cis-6-octadecenoic, and cis-9-octadecenoic acids significantly inhibited growth of Microcystis aeruginosa, whereas tetradecanoic, hexadecanoic, and octadecanoic acids did not show any effect. When the inhibitory effect of nonanoic acid was compared with those of 4 polyphenols and eugeniin, which are anti-cyanobacterial compounds previously reported to be released by M. spicatum, nonanoic acid was found to be the most inhibitory to M. aeruginosa. These results indicate that not only polyphenols and eugeniin but also fatty acids such as nonanoic acid must be studied to reveal how M. spicatum exerts its allelopathic effect on M. aeruginosa.     

Programmed cell death in the cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> induced by allelopathic effect of submerged macrophyte <Emphasis Type="Italic">Myriophyllum spicatum</Emphasis> in co-culture system

This investigation demonstrates that programmed cell death (PCD) in a cyanobacterium, Microcystis aeruginosa, resulting from allelopathic stress induced by a submerged macrophyte, Myriophyllum spicatum, in a co-culture system. The hallmarks of PCD, caspase-3-like protease activity, DNA fragmentation, and destruction of cell ultrastructure, as well as intracellular PCD signaling radicals, reactive oxygen species (ROS), and nitric oxide (NO), were measured in M. aeruginosa cells co-cultured with M. spicatum for 7 days. The results showed a dose–response relationship between M. spicatum biomass and M. aeruginosa mortality. A caspase-3-like protease was activated and elevated from day 3. Thylakoid disintegration, cytoplasmic vacuolation, and fuzzy nuclear zone were observed by transmission electron microscopy, and distinct DNA fragmentation was detected in M. aeruginosa cells at a M. spicatum biomass of 6.0 g fresh weight (FW) L^?1 during the 7 days. Allelochemicals of total phenolic compounds (TPCs) were determined in co-culture water, and the concentration increased with increasing of M. spicatum biomass and co-culture time. Compared with the level of ROS production in the control group, a significant overproduction of ROS was detected in M. aeruginosa cells in the treatment group, and this was positively correlated with TPC concentration. Furthermore, the level of intracellular NO increased with the percent mortality of M. aeruginosa. The results indicated that a PCD pathway was induced in the cyanobacterium M. aeruginosa when co-cultured with the submerged macrophyte M. spicatum.     

Effects of Iron on Growth,Pigment Content,Photosystem II Efficiency,and Siderophores Production of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Microcystis wesenbergii</Emphasis>

Changes in growth, photosynthetic pigments, and photosystem II (PS II) photochemical efficiency as well as production of siderophores of Microcystis aeruginosa and Microcystis wesenbergii were determined in this experiment. Results showed growths of M. aeruginosa and M. wesenbergii, measured by means of optical density at 665 nm, were severely inhibited under an iron-limited condition, whereas they thrived under an iron-replete condition. The contents of chlorophyll-a, carotenoid, phycocyanin, and allophycocyanin under an iron-limited condition were lower than those under an iron-replete condition, and they all reached maximal contents on day 4 under the iron-limited condition. PS II photochemical efficiencies (maximal PS II quantum yield), saturating light levels (I _k ) and maximal electron transport rates (ETR_max) of M. aeruginosa and M. wesenbergii declined sharply under the iron-limited condition. The PS II photochemical efficiency and ETR_max of M. aeruginosa rose , whereas in the strain of M. wesenbergii, they declined gradually under the iron-replete condition. In addition, I _k of M. aeruginosa and M. wesenbergii under the iron-replete condition did not change obviously. Siderophore production of M. aeruginosa was higher than that of M. wesenbergii under the iron-limited condition. It was concluded that M. aeruginosa requires higher iron concentration for physiological and biochemical processes compared with M. wesenbergii, but its tolerance against too high a concentration of iron is weaker than M. wesenbergii.     

Phosphorus cycling between the colonial cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and attached bacteria, <Emphasis Type="Italic">Pseudomonas</Emphasis>

Phosphorus (P) transfer between Microcystis aeruginosa and the attached bacterium Pseudomonas was studied using radioactive P (³²P) and green fluorescence protein-labeled Pseudomonas. M. aeruginosa in P-starved condition took up most ³²P (70%) in water and about 50% of ³²P in ³²P-saturated bacteria in individual experiments. However, only 26% of ³²P in the ³²P-saturated M. aeruginosa was transferred to P-starved bacteria. The P-starved M. aeruginosa had an advantage to take up P over the bacteria and its growth rates and abundance were higher in combined cultures, with bacteria as the biotic P source. The rate of P transfer from bacteria to the cyanobacteria was slow. P cycles predominantly between M. aeruginosa and Pseudomonas with little variation in the water. This ability is very useful for the colony-forming M. aeruginosa, especially if phosphate concentrations in water are low during water bloom periods.     

Quantitative studies on phosphorus transference occuring between <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and its attached bacterium (<Emphasis Type="Italic">Pseudomonas</Emphasis> sp.)

Phosphorus release from Microcystis aeruginosa and attached bacterium (Pseudomonas sp.) isolated from Lake Taihu was examined using a phosphorus isotope tracer in order to investigate the phosphorus transference between the two species. Our results reveal that the amount of phosphorus released form ³²P-saturated M. aeruginosa is determined by its growth phase and most of phosphorus is assimilated by Pseudomonas finally while the amount of phosphorus released from ³²P-saturated Pseudomonas is also determined by the growth phase of M. aeruginosa and most of them are assimilated by M. aeruginosa. The results suggest that phosphorus transference occurs between M. aeruginosa and its attached Pseudomonas . This process makes microenvironment of mucilage of M. aeruginosa attached bacteria maintain relative high amounts of phosphorus. Attached bacteria may be a temporary phosphorus bank to the growth of M. aeruginosa, and assimilation of phosphorus by M. aeruginosa becomes easy when M. aeruginosa is in lag growth phase. Thus, the phosphorus exchange between M. aeruginosa and attached Pseudomonas in microenvironment may be important to microfood web and cyanobacteria bloom.     

The allelopathy and allelopathic mechanism of phenolic acids on toxic <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Cyanobacterial contamination of water has been a serious problem in recent years. Thus, the effective control of undesired cyanobacteria has become an urgent issue. We studied therefore the effects of ρ-coumaric acid and vanillic acid on toxic Microcystis aeruginosa and the allelopathic mechanisms. The results showed that the growth of toxic M. aeruginosa was significantly inhibited by ρ-coumaric acid and vanillic acid, with an EC₅₀ of 0.26 ± 0.07 and 0.34 ± 0.05 mmol L⁻¹, respectively. Our data also demonstrated that both ρ-coumaric acid and vanillic acid triggered the generation of superoxide anion radicals (O₂ ^•−). The O₂ ^•− might induce a lipid peroxidation which may change cell membrane penetrability, thereby leading to the eventual death of M. aeruginosa. Our current studies further provide evidence that some phenolic acids such as ρ-coumaric acid and vanillic acid may be a potential effective solution for aquatic management.     

Growth characteristics and growth modeling of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Planktothrix agardhii</Emphasis> under iron limitation

Although iron is a key nutrient for algal growth just as are nitrogen and phosphorus in aquatic systems, the effects of iron on algal growth are not well understood. The growth characteristics of two species of cyanobacteria, Microcystis aeruginosa and Planktothrix agardhii, in iron-limited continuous cultures were investigated. The relationships between dissolved iron concentration, cell quota of iron, and population growth rate were determined applying two equations, Monod’s and Droop’s equations. Both species produced hydroxamate-type siderophores, but neither species produced catechol-type siderophores. The cell quota of nitrogen for both M. aeruginosa and P. agardhii decreased with decreasing cell quota of iron. The cell quota of phosphorus for M. aeruginosa decreased with decreasing cell quota of iron, whereas those for P. agardhii did not decrease. Iron uptake rate was measured in ironlimited batch cultures under different degrees of iron starvation. The results of the iron uptake experiments suggest that iron uptake rates are independent of the cell quota of iron for M. aeruginosa and highly dependent on the cell quota for P. agardhii. A kinetic model under iron limitation was developed based on the growth characteristics determined in our study, and this model predicted accurately the algal population growth and iron consumption. The model simulation suggested that M. aeruginosa is a superior competitor under iron limitation. The differences in growth characteristics between the species would be important determinants of the dominance of these algal species.     

Different tolerances and responses to low temperature and darkness between waterbloom forming cyanobacterium <Emphasis Type="Italic">Microcystis</Emphasis> and a green alga <Emphasis Type="Italic">Scenedesmus</Emphasis>

The dynamics of planktonic cyanobacteria in eutrophicated freshwaters play an important role in formation of annual summer blooms, yet overwintering mechanisms of these water bloom forming cyanobacteria remain unknown. The responses to darkness and low temperature of three strains (unicellular Microcystis aeruginosa FACHB-905, colonial M. aeruginosa FACHB-938, and a green alga Scenedesmus quadricauda FACHB-45) were investigated in the present study. After a 30-day incubation under darkness and low temperature, cell morphology, cell numbers, chlorophyll a, photosynthetic activity (ETR_max and I _k), and malodialdehyde (MDA) content exhibited significant changes in Scenedesmus. In contrast, Microcystis aeruginosa cells did not change markedly in morphology, chlorophyll a, photosynthetic activity, and MDA content. The stress caused by low temperature and darkness resulted in an increase of the antioxidative enzyme-catalase (CAT) in all three strains. When the three strains re-grew under routine cultivated condition subjected to darkness and low temperature, specific growth rate of Scenedesmus was lower than that of Microcystis. Flow cytometry (FCM) examination indicated that two distinct types of metabolic response to darkness and low temperature existed in the three strains. The results from the present study reveal that the cyanobacterium Microcystis, especially colonial Microcystis, has greater endurance and adaptation ability to the stress of darkness and low temperature than the green alga Scenedesmus. Handling editor: D. Hamilton     

The tyrosine <Emphasis Type="Italic">O</Emphasis>-prenyltransferase SirD catalyzes <Emphasis Type="Italic">O</Emphasis>-, <Emphasis Type="Italic">N</Emphasis>-, and <Emphasis Type="Italic">C</Emphasis>-prenylations

Recently, the prenyltransferase SirD was found to be responsible for the O-prenylation of tyrosine in the biosynthesis of sirodesmin PL in Leptosphaeria maculans. In this study, the behavior of SirD towards phenylalanine/tyrosine and tryptophan derivatives was investigated. Product formation has been observed with 12 of 19 phenylalanine/tyrosine derivatives. It was shown that the alanine structure attached to the benzene ring and an electron donor, e.g., OH or NH₂, at its para-position are essential for the enzyme activity. Modifications were possible both at the side chain and the benzene ring. Enzyme products from seven phenylalanine/tyrosine derivatives were isolated and characterized by MS and NMR analyses including HSQC and HMBC and proven to be O- or N-prenylated derivatives at position C4 of the benzene rings. K _M values of six selected derivatives were found in the range of 0.10–0.68 mM. Catalytic efficiencies (K _cat/K _M ) were determined in the range of 430–1,110 s⁻¹·M⁻¹ with l-tyrosine as the best substrate. In addition, 7 of 14 tested tryptophan analogs were also accepted by SirD and converted to C7-prenylated derivatives, which was confirmed by comparison with products obtained from enzyme assays using a 7-dimethylallyltryptophan synthase 7-DMATS from Aspergillus fumigatus.     

Novel achiral titanocene anti-cancer drugs synthesised from bis-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylamino fulvene and lithiated heterocyclic compounds

Abstract From the carbolithiation of 6-bis-N,N-dimethylamino fulvene (3a) and different ortho-lithiated heterocycles (furan, thiophene and N-methylpyrrole), the corresponding lithium cyclopentadienide intermediate (4a–c) was formed. These three lithiated intermediates underwent a transmetallation reaction with TiCl₄ resulting in bis-N,N-dimethylamino-functionalised titanocenes 5a–c. When these titanocenes were tested against LLC-PK cells, the IC₅₀-values obtained were of 240, and 270 μM for titanocenes 5b and 5c, respectively. The most cytotoxic titanocene in this paper, 5a with an IC₅₀-value of 36 μM was found to be approximately six times less cytotoxic than its mono-N,N-dimethylamino substituted analogue Titanocene C (IC₅₀ = 5.5 μM) and almost ten times less cytotoxic than cisplatin, which showed an IC₅₀-value of 3.3 μM, when tested on the LLC-PK cell line. Graphical abstract Bis-(bis- (N,N-dimethylamino)-2-(N′-methylpyrrolyl)methylcyclopentadienyl) titanium (IV) dichloride, {η⁵-C₅H₄-CH[N(CH₃)₂]₂[C₅H₃NCH₃]}₂TiCl₂ was synthesised starting from 6-bis-(N,N-dimethylamino) fulvene and 2-N-methylpyrrolyl lithium. Herein, we present the synthesis and DFT structure of the titanocene and two further derivatives followed by MTT-based cytotoxicity tests on pig kidney epithelial (LLC-PK) cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Luminophores of the luminous fungus <Emphasis Type="Italic">Neonothopanus nambi</Emphasis>

Isolation of luminophores from the mycelium of a luminous fungus Neonothopanus nambi is reported. In addition to the emission peak with a maximum at 520–530 nm (the wavelength of visible green light) that corresponded to the maximum of light emission by the fungus in vivo, the fluorescence spectra of the raw extracts contained a peak with a maximum in the visible blue-light range. The luminophore that emitted the blue light was an individual compound with a molecular weight of 894 Da. Calculations that took the isotope composition of chemical elements into account pointed at C₅₂H₆₅N₂O₁₁, C₅₁H₆₅N₄O₁₀, C₅₃H₆₁N₆O₇, C₄₇H₆₅N₄O₁₃, and C₄₆H₆₅N₆O₁₂ as the putative chemical formulae of the luminophore. A sample that contained substances of a yellow color was obtained; these substances emitted fluorescence at the wavelengths of green visible light. The luminophores in this sample probably included riboflavin or derivatives thereof (flavin mononucleotide or flavin adenine dinucleotide).     

Effect of mixed diets (cyanobacteria and green algae) on the population growth of the cladocerans <Emphasis Type="Italic">Ceriodaphnia dubia</Emphasis> and <Emphasis Type="Italic">Moina macrocopa</Emphasis>

Microcystis aeruginosa, a cosmopolitan form, is a colonial cyanobacterium, which is also common in many freshwater bodies in Mexico. In eutrophic water bodies cyanobacteria are often the main phytoplankton that co-exist with cladocerans. We evaluated the effect of mixed diets, comprising 0, 25, 50, 75, and 100% on dry weight basis of M. aeruginosa, and the rest of one of two green algal species (Chlorella vulgaris and Scenedesmus acutus), on the population growth of the cladocerans Ceriodaphnia dubia and Moina macrocopa. Regardless of the share of M. aeruginosa in the mixed diet, C. dubia fed Chlorella had a longer initial lag phase. However, in mixed diet with S. acutus, the lag phase of C. dubia increased with increasing proportion of M. aeruginosa. When raised on 100% M. aeruginosa, the population growth of C. dubia was lowered compared with 100% S. acutus or 100% C. vulgaris. Increased proportion of M. aeruginosa in the mixed diet also resulted in decreased abundance of M. macrocopa. Irrespective of diet type, M. macrocopa had a shorter lag phase than C. dubia. Depending on the diet type, the rate of population increase (r) of C. dubia varied from 0.07 to 0.26 d⁻¹ while that of M. macrocopa was higher (0.14–0.61 d⁻¹). For both cladoceran species, the lower r values were obtained when fed Microcystis. Our study showed that the strain of M. aeruginosa was not highly toxic to cause total elimination of either C. dubia or M. macrocopa. Addition of a green algal component to the diet improved the population growth rates of both cladoceran species.     

<Emphasis Type="Italic">n</Emphasis>-Alkane assimilation and <Emphasis Type="Italic">tert</Emphasis>-butyl alcohol (TBA) oxidation capacity in <Emphasis Type="Italic">Mycobacterium austroafricanum</Emphasis> strains

Mycobacterium austroafricanum IFP 2012, which grows on methyl tert-butyl ether (MTBE) and on tert-butyl alcohol (TBA), the main intermediate of MTBE degradation, also grows on a broad range of n-alkanes (C₂ to C₁₆). A single alkB gene copy, encoding a non-heme alkane monooxygenase, was partially amplified from the genome of this bacterium. Its expression was induced after growth on n-propane, n-hexane, n-hexadecane and on TBA but not after growth on LB. The capacity of other fast-growing mycobacteria to grow on n-alkanes (C₁ to C₁₆) and to degrade TBA after growth on n-alkanes was compared to that of M. austroafricanum IFP 2012. We studied M. austroafricanum IFP 2012 and IFP 2015 able to grow on MTBE, M. austroafricanum IFP 2173 able to grow on isooctane, Mycobacterium sp. IFP 2009 able to grow on ethyl tert-butyl ether (ETBE), M. vaccae JOB5 (M. austroaafricanum ATCC 29678) able to degrade MTBE and TBA and M. smegmatis mc2 155 with no known degradation capacity towards fuel oxygenates. The M. austroafricanum strains grew on a broad range of n-alkanes and three were able to degrade TBA after growth on propane, hexane and hexadecane. An alkB gene was partially amplified from the genome of all mycobacteria and a sequence comparison demonstrated a close relationship among the M. austroafricanum strains. This is the first report suggesting the involvement of an alkane hydroxylase in TBA oxidation, a key step during MTBE metabolism.     

Anti-cyanobacterial activity of <Emphasis Type="Italic">Moringa oleifera</Emphasis> seeds

Filtrates from crushed Moringa oleifera seeds were tested for their effects on growth and Photosystem II efficiency of the common bloom-forming cyanobacterium Microcystis aeruginosa. M. aeruginosa populations exhibited good growth in controls and treatments with 4- and 8-mg crushed Moringa seeds per liter, having similar growth rates of 0.50 (±0.01) per day. In exposures of 20- to 160-mg crushed Moringa seeds L⁻¹, growth rates were negative and on average −0.23 (±0.05) .day⁻¹. Presumably, in the higher doses of 20- to 160-mg crushed seeds per liter, the cyanobacteria died, which was supported by a rapid drop in the Photosystem II efficiency (Φ_PSII), while the Φ_PSII was high and unaffected in 0, 4, and 8 mg L⁻¹. High-density populations of M. aeruginosa (chlorophyll-a concentrations of ∼270 μg L⁻¹) were reduced to very low levels within 2 weeks of exposure to ≥80-mg crushed seeds per liter. At the highest dosage of 160 mg L⁻¹, the Φ_PSII dropped to zero rapidly and remained nil during the course of the experiment (14 days). Hence, under laboratory conditions, a complete wipeout of the bloom could be achieved. This is the first study that yielded evidence for cyanobactericidal activity of filtrate from crushed Moringa seeds, suggesting that Moringa seed extracts might have a potential as an effect-oriented measure lessening cyanobacterial nuisance.     

Tetrahydrofolate-specific enzymes in <Emphasis Type="Italic">Methanosarcina barkeri</Emphasis> and growth dependence of this methanogenic archaeon on folic acid or <Emphasis Type="Italic">p</Emphasis>-aminobenzoic acid

Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H₄SPT) rather than tetrahydrofolate (H₄F) as a pterin C₁ carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN, folD, glyA and metF, that are predicted to encode for H₄F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N⁵,N¹⁰-methylene-H₄F dehydrogenase/N⁵,N¹⁰-methenyl-H₄F cyclohydrolase) and GlyA (serine:H₄F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli, purified and found to be specific for methylene-H₄F and H₄F, respectively (apparent K_m below 5 M). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri. The results thus indicate that M. barkeri should contain H₄F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p-aminobenzoic acid, a biosynthetic precursor of H₄F. From the p-aminobenzoic acid requirement, an intracellular H₄F concentration of approximately 5 M was estimated. Evidence is presented that the p-aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H₄SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H₄SPT and H₄F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C₁ pools in these methanogens.     

Biological synthesis of quercetin 3-<Emphasis Type="Italic">O</Emphasis>-<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine conjugate using engineered <Emphasis Type="Italic">Escherichia coli</Emphasis> expressing <Emphasis Type="Italic">UGT78D2</Emphasis>

Biotransformation of flavonoids using Escherichia coli harboring nucleotide sugar-dependent uridine diphosphate-dependent glycosyltransferases (UGTs) commonly results in the production of a glucose conjugate because most UGTs are specific for UDP-glucose. The Arabidopsis enzyme AtUGT78D2 prefers UDP-glucose as a sugar donor and quercetin as a sugar acceptor. However, in vitro, AtUGT78D2 could use UDP-N-acetylglucosamine as a sugar donor, and whole cell biotransformation of quercetin using E. coli harboring AtUGT78D2 produced quercetin 3-O-N-acetylglucosamine. In order to increase the production of quercetin 3-O-N-acetylglucosamine via biotransformation, two E. coli mutant strains deleted in phosphoglucomutase (pgm) or glucose-1-phosphate uridylyltransferase (galU) were created. The galU mutant produced up to threefold more quercetin 3-O-N-acetylglucosamine than wild type, resulting in the production of 380-mg/l quercetin 3-O-N-acetylglucosamine and a negligible amount of quercetin 3-O-glucoside. These results show that construction of bacterial strains for the synthesis of unnatural flavonoid glycosides is possible through rational selection of the nucleotide sugar-dependent glycosyltransferase and engineering of the nucleotide sugar metabolic pathway in the host strain.     

Synthesis and biotransformation of 2-alkyl-4(1<Emphasis Type="Italic">H</Emphasis>)-quinolones by recombinant <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

2-Alkyl-4(1H)-quinolones (AQs) and related derivatives, which exhibit a variety of biological properties, are secondary metabolites produced by, e.g., Pseudomonas and Burkholderia spp. Due to their main role as signaling molecules in the quorum sensing system of Pseudomonas aeruginosa, 2-heptyl-4(1H)-quinolone (HHQ) and its 3-hydroxy derivative, termed the “Pseudomonas quinolone signal” (PQS), have received considerable attention. Since chemical synthesis of different AQs is complex, we assessed the applicability of recombinant P. putida KT2440 strains for the biosynthetic production of AQs. In mineral salts medium supplemented with octanoate and anthranilate, batch cultures of P. putida KT2440 [pBBR-pqsABCD] produced about 45 μM HHQ, 30% and 70% of which were localized in the culture supernatant and methanolic cell extract, respectively. 2,4-Dihydroxyquinoline and minor amounts of C₃- to C₁₃-saturated and C_7:1 to C_13:1 monounsaturated AQs were formed as by-products. Mass spectrometry and nuclear magnetic resonance analyses spectroscopy indicated that unsaturated AQs having the same molecular mass are cis and trans isomers rather than position isomers, with the double bond located between the α and β carbon of the alkyl chain. Supplementing the cultures with hexanoate instead of octanoate shifted the AQ profile towards increased formation of C₅-AQ. Individual AQs can be prepared from concentrated methanolic extracts by preparative high-performance liquid chromatography (HPLC). Regioselective hydroxylation of HHQ to PQS can be achieved in >90% yield by biotransformation with P. putida KT2440 [pBBR-pqsH]. PQS can be isolated from methanolic cell extracts by HPLC, or be precipitated as Fe(III)-PQS complex. Preparation of a library of AQs will facilitate studies on the biological functions of these compounds.     

Permeabilization of <Emphasis Type="Italic">Microbacterium oxylans</Emphasis> shifts the conversion of puerarin from puerarin-7-<Emphasis Type="Italic">O</Emphasis>-glucoside to puerarin-7-<Emphasis Type="Italic">O</Emphasis>-fructoside

The main product of the conversion of puerarin by unpermeabilized cells of bacterium Microbacterium oxydans CGMCC 1788 was puerarin-7-O-glucoside (241 ± 31.9 μM). Permeabilization with 40% ethanol could not increase conversion yield, whereas it resulted in change of main product; a previous trace product became a main product (213 ± 48.0 μM) which was identified as a novel puerarin-7-O-fructoside by electrospray ionization time-of-flight MS, ¹³C NMR, ¹H NMR, and GC-MS analysis of sugar composition, and puerarin-7-O-glucoside became a trace product (14.8 ± 5.4 μM). However, the extract from cells of M. oxydans CGMCC 1788 permeabilized with ethanol converted puerarin to form 113.9 ± 27.7 μM puerarin-7-O-glucoside and 187.8 ± 29.5 μM puerarin-7-O-fructoside under the same conditions. When unpermeabilized intact cells were recovered and used repeatedly for the conversion of puerarin, with increase of reuse times, the yield of puerarin-7-O-glucoside gradually decreased, whereas the yield of puerarin-7-O-fructoside increased gradually in the conversion mixture. The main product of the conversion of puerarin by the tenth recycled unpremerbilized cells was puerarin-7-O-fructoside (288.4 ± 24.0 μM). Therefore, the change of permeability of cell membrane of bacterium M. oxydans CGMCC 1788 contributed to the change of conversion of the product’s composition.     

UDP-N-acetylglucosamine enolpyruvyl transferase from <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Multi-drug resistant Pseudomonas aeruginosa (MDRPA) are emerging as a major threat in the hospitals as they have become resistant to current antibiotics. There is an immediate requirement of drugs with novel mechanisms as the pipeline of investigational drugs against these organisms is lean. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) enzyme that catalyzes the first committed step of bacterial cell wall biosynthesis is an ideal target for the discovery of novel antibiotics against Gram negative pathogens as they have only one copy of murA gene in its genome. We have performed biochemical characterization and comparative kinetic analysis of MurA from E. coli and P. aeruginosa. Both enzymes were active at broad range of pH with temperature optima of 37°C. Metal ions did not enhance the activity of both enzymes. These enzymes had an apparent affinity constant (K _m ) for its substrate UDP-N-acetylglucosamine 36 ± 5.2 and 17.8 ± 2.5 μM and for phosphoenolpyruvate 0.84 ± 0.13 μM and 0.45 ± 0.07 μM for E. coli and P. aeruginosa enzymes respectively. Both the enzymes showed 5–7 fold shift in IC₅₀ for the known inhibitor fosfomycin upon pre-incubation with the substrate UDP-N-acetylglucosamine. This observation was used to develop a novel rapid sensitive high throughput assay for the screening of MurA inhibitors.     

Recombination,cryptic clades and neutral molecular divergence of the microcystin synthetase (<Emphasis Type="Italic">mcy</Emphasis>) genes of toxic cyanobacterium <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis>

Background  

The water-bloom-forming cyanobacterium Microcystis aeruginosa is a known producer of various kinds of toxic and bioactive chemicals. Of these, hepatotoxic cyclic heptapeptides microcystins have been studied most intensively due to increasing concerns for human health risks and environmental damage. More than 70 variants of microcystins are known, and a single microcystin synthetase (mcy) gene cluster consisting of 10 genes (mcyA to mcyJ) has been identified to be responsible for the production of all known variants of microcystins. Our previous multilocus sequence typing (MLST) analysis of the seven housekeeping genes indicated that microcystin-producing strains of M. aeruginosa are classified into two phylogenetic groups.