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Background: Our previous comparative metabolomics research revealed that betaine (N,N,Ntrimethylglycine, a typically essential methyl-group donor for vitamin B12 biosynthesis) had powerful promoting effect on the generation of vitamin B12 precursors and intermediates in vitamin B12-producing Pseudomonas denitrificans. However, the integral effect of betaine on the vitamin B12 biosynthetic pathway is still unclear. Objective: Considering the vitamin B12 biosynthetic pathway of P. denitrificans as a whole, this work aimed to reveal the biological function of betaine on the vitamin B12 biosynthetic pathway in P. denitrificans, which would sharpen and expand understanding of betaine as the methyl-group donor for vitamin B12 biosynthesis. Materials and Methods: By using a proteomics method based on the iTRAQ technique, the present study compared and analyzed the differential expression of proteins involved in vitamin B12 biosynthetic pathway under 10 g/L betaine in addition to P. denitrificans fermentation medium. Results: The results showed that betaine could significantly up-regulate the expression of proteins related to the vitamin B12 biosynthetic pathway, which was mainly reflected in the following three aspects: 1) the δ-aminolevulinic acid (ALA) synthase and porphobilinogen synthase that were responsible for the formation of the committed precursors for tetrapyrrole-derived macrocycle in vitamin B12 molecule; 2) the C-methylation-related enzymes (such as precorrin-4 C(11)-methyltransferase, precorrin-2 C(20)- methyltransferase, precorrin-8X methylmutase, and precorrin-6Y C5,15-methyltransferase) and methionine synthase that were crucial to the C-methylation reactions for vitamin B12 biosynthesis; 3) the latestage key enzymes (Cobaltochelatase, and Cob(I)yrinic acid a,c-diamide adenosyltransferase) that were related to cobalt chelation of vitamin B12 molecule. Conclusions: The present study demonstrated clearly that betaine could significantly promote the expression of the integral enzymes involved in the vitamin B12 biosynthetic pathway of P. denitrificans, thus promoting vitamin B12 biosynthesis.

Here, we characterized a gene expression system induced by 3-hydroxypropionic acid (3-HP) in Pseudomonas denitrificans. The system consists of a putative LysR-type transcriptional regulator (LTTR) encoded by hpdR and a LTTR-responsive promoter that positively controls the expression of hpdH, encoding 3-HP dehydrogenase in the presence of 3-HP. In the hpdH-responsive promoter region, two operators exhibiting dyad symmetry and designated O1 and O2 and centered at the −73 and −30 positions, respectively, were located upstream of the hpdH transcription start site. When either O1, O2, or both regions were mutated, the inducibility by the HpdR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcriptional activation. The HpdR protein and its operator sites on hpdH were highly specific for each other, and did not engage in cross-talk with another similar 3-HP-inducible system that is also present in P. denitrificans. This 3-HP-inducible promoter system should be useful in developing biosensors for 3-HP, and/or dynamic gene expression systems for metabolic engineering purposes.

Autotoxins play important roles in problems with peach replanting. Benzoic acid (BA), one of the major autotoxins, is identified in peach root exudates and rhizosphere soil, leading to stunted seedling growth or even death. In this research, we aimed to screen the most efficient BA-degrading strain (BDS) from peach rhizosphere soil and assess its potential as a biodegradation strain that could relieve peach replant stress. In the work, three BDSs (WH-B1, WH-B2 and WH-B3) were isolated and identified as Pseudomonas denitrificans, Acinetobacter calcoaceticus and P. putida, respectively. Through bacterial growth and BA degradation tests, the strain WH-B3 was found to show the highest growth rate and the greatest degradation capacity and was selected for soil inoculation. Meanwhile, the phytotoxicity analysis revealed that the BA degradation products of the three BDSs were almost completely nontoxic to seedlings. Moreover, the pot experiments indicated that a high concentration of soil BA (≥ 40 mg kg−1) significantly restrained an increase in peach seedling height, stem diameter, and biomass, while the WH-B3 inoculum distinctly decreased the soil BA content and fully counteracted the BA-mediated inhibition of seedling growth. Furthermore, to investigate the performance of the potent strain under authentic replanted soil (RS) conditions, WH-B3 was inoculated into the RS for peach seedling growth. The results demonstrated that the WH-B3 inoculum degraded approximately 65% of the BA in RS and significantly improved replant-stressed seedling growth. Altogether, the characteristics of P. putida WH-B3 show that it is a potential strain for BA biodegradation and has promise for alleviating problems in peach replanting.

An alternative to reduce the effect of chemical fertilization on soils and favor the growth of sweet potato crops is the incorporation of microorganisms that promote plant growth in fertilization. In the Colombian Caribbean, sweet potato is currently one of the emerging crops due to its high nutritional properties in the human diet. The objective of this study was to evaluate the effect of two bacterial strains, Pseudomonas denitrificans IBVS2 and Azotobacter vinelandii IBVS13, on the yield of sweet potato clone Tainung 66 in field conditions, in the Valle del Sinú, Córdoba, Colombia. The research was carried out during second semester of 2017, at the Turipaná-Agrosavia Research Center, using apical cuttings from the Tainung 66 clone. A randomized complete block design with a 3x2+2 factorial arrangement was implemented, physiological component, foliar and edaphic chemical analysis, yield and bromatological composition variables were evaluated. The results showed that yield and dry root mass showed significant differences with respect to the chemical control when fertilization consisted of IBVS2 strain plus 50% of chemical fertilizer, obtaining a yield of 11.32 t.ha-1 and 5,78 t.ha-1 respectively. These results showed that inoculation of PGPRs constitutes an alternative for sweet potatoes cultivation with positive effects when compared to complete chemical fertilization.

Many microbial producers of coenzyme B12 family cofactors together with their metabolically interdependent pathways are comprehensively studied and successfully used both in natural ecosystems dominated by auxotrophs, including bacteria and mammals, and in the safe industrial production of vitamin B12. Metabolic reconstruction for genomic and metagenomic data and functional genomics continue to mine the microbial and genetic resources for biosynthesis of the vital vitamin B12. Availability of metabolic engineering techniques and usage of affordable and renewable sources allowed improving bioprocess of vitamins, providing a positive impact on both economics and environment. The commercial production of vitamin B12 is mainly achieved through the use of the two major industrial strains, Propionobacterium shermanii and Pseudomonas denitrificans, that involves about 30 enzymatic steps in the biosynthesis of cobalamin and completely replaces chemical synthesis. However, there are still unresolved issues in cobalamin biosynthesis that need to be elucidated for future bioprocess improvements. In the present work, we review the current state of development and challenges for cobalamin (vitamin B12) biosynthesis, describing the major and novel prospective strains, and the studies of environmental factors and genetic tools effecting on the fermentation process are reported.

Four granular sequencing batch reactors (GSBRs) were inoculated with four denitrifying Pseudomonas strains carrying nosZ to study the process of granule formation, the operational conditions of the bioreactors, and the carbon concentration needed for nitrate removal. The selected Pseudomonas strains were P. stutzeri I1, P. fluorescens 376, P. denitrificans Z1, and P. fluorescens PSC26, previously reported as denitrifying microorganisms carrying the nosZ gene. Pseudomonas denitrificans Z1 produced fluffy, low-density granules, with a decantation speed below 10 m h−1 . However, P. fluorescens PSC26, P. stutzeri I1, and P. fluorescens 376 formed stable granules, with mean size from 7 to 15 mm, related to the strain and carbon concentration. P. stutzeri I1 and P. fluorescens 376 removed nitrate efficiently with a ratio in the range of 96%, depending on the source and concentration of organic matter. Therefore, the findings suggest that the inoculation of GSBR systems with denitrifying strains of Pseudomonas spp. containing the nosZ gene enables the formation of stable granules, the efficient removal of nitrate, and the transformation of nitrate into nitrogen gas, a result of considerable environmental interest to avoid the generation of nitrous oxide., LIFE16 ENV/ES/000196 project

RESUMEN La batata (Ipomoea batatas L.) se cultiva en todo el mundo como fuente de carbohidratos, y su producción comercial requiere un alto aporte de fertilizantes químicos, lo cual eleva los costos de producción. Los inoculantes microbianos, se emplean como una fuente alternativa de nutrición vegetal. El objetivo de esta investigación fue evaluar el efecto de Pseudomonas denitrificans IBVS2 y Azotobacter vinelandii IBVS13 con diferentes niveles fertilización química nitrogenada en el cultivo de batata en la microrregión del Valle del Sinú en el Caribe Colombiano. Para los montajes de los experimentos se utilizó un diseño completamente aleatorizado, ocho tratamientos y tres repeticiones usando como material vegetal plántulas obtenidas in vitro endurecidas en invernadero. Los resultados demostraron que la cepa Azotobacter vinelandii IBVS13 con un 75% de fertilización nitrogenada (FN) mejoró la capacidad de acumulación de materia seca en los tubérculos de batata, generando incrementos de 6,65 t/ha respecto al testigo químico y 3,18 t/ha en relación con el testigo absoluto, garantizando un incremento del rendimiento. Así mismo, el contenido de proteína bruta aumentó 13,93% al realizar la inoculación de las plantas con esta cepa. En el mismo sentido, la cepa Pseudomonas denitrificans IBVS2+ fertilización nitrogenada 50% presentó aumentos en la variable de fibra cruda 31,75% respecto al testigo absoluto, contribuyendo de manera eficaz como bioestimulante microbiano en la agricultura. ABSTRACT Sweet potatoes (Ipomoea batatas L.) are grown worldwide as a source of carbohydrates, and their commercial production requires a high contribution of chemical fertilizers, which increases production costs. Microbial inoculants are used as an alternative source of plant nutrition. The objective of this research was to evaluate the effect of Pseudomonas denitrificans IBVS2 and Azotobacter vinelandii IBVS13 with different levels of nitrogen chemical fertilization in the sweet potato crop in the microregion of the Sinú Valley in the Colombian Caribbean. A completely randomized design was used for the experiment development, eight treatments was evaluated and three repetitions were carried out. In vitro hardened seedlings was used as a plant material. The results showed that the Azotobacter vinelandii IBVS1 3 strain with 75% nitrogen fertilization (FM) improved the accumulation capacity of dry matter in sweet potato roots, generating increases of 6.65 t / ha compared to the chemical control and 3.18 t / ha in relation to the absolute control, guaranteeing an increase in yield. The crude protein content was increased in 13.93% when inoculating the plants with this strain. In the same way, with the inoculation of strain Pseudomonas denitrificans IBVS2 + 50% nitrogen fertilization the crude fiber variable was increased in 31.75% compared to the absolute control, contributing effectively as a microbial biostimulant in agriculture.

In this study, whole-cell biosensors that detect and indicate the concentration of 3-hydroxypropionic acid (3-HP) by producing a green fluorescent signal were developed using a transcription factor (TF)-responsive 3-HP inducible promoter identified in the Pseudomonas denitrificans 3-HP degradation pathway. Upon forming a complex with 3-HP, the MmsR TF protein, a LysR-type transcriptional regulator (LTTR), binds to the PmmsA promoter and controls the expression of the PmmsA-regulated gfp reporter gene in response to the concentration of 3-HP. Furthermore, by enhancing the expression of MmsR and through mutagenesis of the PmmsA promoter region, three well-performing biosensors were developed that covered a wide dynamic range of 3-HP (0.01-100 mM when added externally) with ~100-fold signal change upon induction in P. denitrificans. The 3-HP biosensor machinery, composed of MmsR, the PmmsA promoter and gfp could also function well in E. coli and P. putida. The developed 3-HP biosensors should be useful for engineering 3-HP-producing strains and the enzymes associated with its production.

The assessment of the impact of the bacteria Pseudomonas denitrificans, Pseudomonas fluorescens, Bacillus subtilis and yeast Yarrowia lipolytica on commercial poly(ether urethanes) Tecoflex® and Tecothane® is presented. The polyurethane samples were incubated with pure cultures of the microorganisms at 30 °C for five months. The changes in the chemical structure of the polymers were evaluated using loss of weight and contact angle measurements, infrared spectroscopy (ATR-FTIR), mass spectrometry (Py-MS), differential scanning calorimetry (DSC) and the thermogravimetric analysis (TG). In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were applied for imaging changes in surface morphology of the poly(ether urethanes). Comparative analysis of these polyurethane features before and after incubation with the microorganism cultures showed that Tecoflex® was less stable than Tecothane®. This can be explained by the presence of aromatic rings within the diphenylmethane diisocyanate group in the chemical structure of the latter. Bacterial strains of Bacillus subtilis and Pseudomonas fluorescens showed a much more prominent destructive effect compared to the strain of yeast Y. lipolytica.

The use of acetate as carbon feedstock can enhance sustainability and economics of the current bio-productions. This study explored the potential of acetate for the production of 3-hydroxypropionic acid by engineered Pseudomonas denitrificans. Heterologous mcr (encoding malonyl-CoA reductase) from Chloroflexus aurantiacus and endogenous accABCD (encoding acetyl-CoA carboxylase) were overexpressed in P. denitrificans. Carbon flux to 3-HP synthesis at the malonyl-CoA node was promoted by suppressing fatty acid synthesis through addition of cerulenin or deletion of fabF gene. In addition, stimulation of glyoxylate shunt and/or TCA cycle were attempted. Recombinant P. denitrificans overexpressing mcr and accABCD produced 19.3 mM 3-HP with cerulenin addition, and 14.2 mM with fabF deletion, respectively. Furthermore, the non-growing cells devoid of fabF could continuously produce 3-HP up to 40.4 mM without losing its production activity for 22 h. This study demonstrates that acetate is a good substrate for 3-HP production by recombinant P. denitrificans.

Исследования проводили с целью поиска новых перспективных микробных продуцентов витамина В12 для его синтеза. Осуществляли скрининг восьми «диких» штаммов микроорганизмов, выделенных из растениеводческой продукции, как возможных продуцентов кобаламинов: 1 - Pseudomonas aeruginosa, 1 - Bacillus subtilis, 2 - Bacillus circulans, 3 - Pseudomonas fluorescens, 1 -Pseudomonasdenitrificans. На первом этапе их оценивали по накоплению биомассы в одинаковых условиях культивирования. Преимуществом по величине этого показателя в аэробных условиях обладали штаммы B. subtilis В1 и P. denitrificans Р5: выход сухой биомассы в среднем составил соответственно 11,2 г/л и 12,6 г/л. На втором этапе изучали динамику накопления биомассы бактерий по показателю оптической плотности в зависимости от используемых источников углеводов (сахароза, глюкоза, мальтоза). Результаты на средах с глюкозой и мальтозой статистически не различались: соответственно 2,52 и 2,46 отн. ед. для P. denitrificans Р5 и 2,50 и 2,49 отн. ед. для B. subtilis В1. На питательных средах с добавлением сахарозы накапливалось достоверно меньше биомассы. На третьем этапе работы после окончания ферментации проводили измерение концентрации кобаламинов в клеточных экстрактах c помощью ВЭЖХ. Штамм B. subtilis В1 продуцирует 0,037 мг/мл цианокобаламина, P. denitrificans Р5 - 0,042 мг/мл. Учитывая технологическую сложность процесса культивирования B. subtilis (чередование аэробных и анаэробных фаз) для дальнейших исследований предпочтительнее использовать штамм P. denitrificans.

Glycine betaine is an important donor of methyl groups in various metabolic processes of the cell and acts as an osmoprotector when exposed to various abiotic stresses in pro- and eukaryotic organisms. Moreover, exogenous application of betaine activates the production of vitamin B12 in industrial strains-producers. In this work, we have developed a new technology for microbiological betaine synthesis that can be used in biotechnology to activate B12 biosynthesis during large-scale fermentation of Pseudomonas denitrificans.

Excessive nitrate in aquaculture systems has attracted wide attention. To isolate novel aerobic denitrifying strain and characterize its nitrogen removal processes, a facultative anaerobic denitrification bacterium, identified as Pseudomonas denitrificans G1, was isolated from marine sediments. Strain G1 could grow and remove 90–98% of nitrate and 97–99% of nitrite under an aerobic or anaerobic condition in 24 h, with the total nitrogen removal rate of 33–38% (87–100 mg/L). The highest denitrification rate could reach 15.1 mg/(L·h). The suitable condition for the denitrification of G1 is C/N ratio 5–22, dissolved oxygen 0–4.68 mg/L, salinity 0–30 g NaCl/L, pH 7–9.5. Under the aerobic condition, G1 grew fast; however, the mass spectrographic analysis showed that the gas product was N2O. Under the anaerobic conditions, G1 grow relatively slowly, but could also achieve effective denitrification and the final product was N2. In denitrification of aquaculture wastewater, strain G1 can remove 60.57% of nitrate and 36.36% of total nitrogen; meanwhile, there was a slight accumulation of ammonia nitrogen. P. denitrificans strain G1 has potential in denitrification processes for the treatment of aquaculture wastewater. However, the regulation of reaction conditions and gas products needs to be further studied.

Bio-production of 1,3-propanediol (1,3-PDO) from glycerol was studied using Pseudomonas denitrificans as host, which aerobically synthesizes coenzyme B12, an essential cofactor of glycerol dehydratase (GDHt). P. denitrificans was transformed with the 1,3-PDO synthesis pathway composed of GDHt and 1,3-PDO oxidoreductase (PDOR), and its putative 3-hydroxypropionaldehyde (3-HPA) dehydrogenase(s), leading to the production of 3-hydroxypropioninc acid form the intermediary 3-HPA, was identified and deleted. In addition, to improve the availability of NADH for PDOR, oxidation of NADH in the electron transport chain was disturbed by deletion of the nuo operon and/or ndh gene. Finally, acetate formation pathway was eliminated. One resulting strain could produce 68.95 mM 1,3-PDO with the yield of 0.92 mol 1,3-PDO/mol glycerol on flask scale and 440 mM with the yield of 0.89 mol 1,3-PDO/mol glycerol in a fed-batch bioreactor experiment. This study demonstrates that P. denitrificans is a promising recombinant host for the production of 1,3-PDO from glycerol.

It is well known that activated sludge treatment systems generate a lot of surplus sludge having environmental and economic impacts. Although several approaches have been proposed for the treatment/reuse of the excess of sludge, there are few studies focused on decreasing the biomass yield without affecting the metabolic activity. This work reports the effect of low magnitude electrical fields (0.07 to 0.2 V/cm) on the growth yield of a pure strain of Pseudomonas denitrificans (used as model microorganism). Cell potentials between 0.2 and 0.57 V were measured during 24 h to the aerobic culture; biomass production and substrate consumption were evaluated at regular intervals. Results indicated that the substrate (lactate) consumption efficiency increased with the applied potential, up to 100%, while the yield diminished 31% (0.34 g biomass/g lactate consumed) at 0.7 V vs. NHE. Bioenergetics showed that the fraction of electron equivalents toward biomass synthesis decreased from 0.68 (when no potential was applied) to 0.47 at 0.57 V, pointing out the redirection of the energy flow toward maintenance to cope with the stress caused by the imposed voltage. Therefore, the electrical stimulus could be used as control of biomass growth in aerobic wastewater treatment lines.

MmsR (33.3 kDa) is a putative LysR-type transcriptional activator of Pseudomonas denitrificans. With the help of 3-hydroxypropionic acid (3-HP), an important platform chemical, MmsR positively regulates the expression of mmsA, which encodes methylmalonylsemialdehyde dehydrogenase, the enzyme involved in valine degradation. In the present study, the cellular function of MmsR and its binding to the regulatory DNA sequence of mmsA expression were investigated both in vivo and in vitro. Transcription of the mmsA was enhanced >140-fold in the presence of 3-HP. In the MmsR-responsive promoter region, two operators showing dyad symmetry, designated O1 and O2 and centered at the −79 and −28 positions, respectively, were present upstream of the mmsA transcription start site. An electrophoretic mobility shift assay indicated that MmsR binds to both operator sites for transcription activation, probably in cooperative manner. When either O1 or O2 or both regions were mutated, the inducibility by the MmsR-3-HP complex was significantly reduced or completely removed, indicating that both sites are required for transcription activation. A 3-HP sensor was developed by connecting the activation of MmsR to a green fluorescent readout. A more than 50-fold induction by 25 mM 3-HP was observed.

The relationship between the morphological character of Pseudomonas denitrificans and vitamin B12 synthesis based on real-time capacitance measurement and online specific oxygen consumption rate (Q O2) control was established for enhancing vitamin B12 production. Results demonstrated that the threshold Q O2 value lower than 2.0 mmol/gDCW/l would greatly stimulate the state transfer from the cell number growth phase to the cell elongation phase and promote rapid vitamin B12 biosynthesis, while the vitamin B12 biosynthesis rate could also be inhibited when the rate of cell’s length-to-width ratio (ratio-LW) was higher than 10:1. Furthermore, the optimal morphology controlling strategy was achieved based on online Q O2 control, which increases the appropriate active cell numbers at the former phase, and then control the elongation of ratio-LW no more than 10:1 at the vitamin B12 biosynthesis phase. The maximal vitamin B12 production reached 239.7 mg/l at 168 h, which was improved by 14.7 % compared with the control (208 mg/l). This online controlling strategy would be effectively applied for improving industrial vitamin B12 fermentation.

Enhancement in denitrification rate with reduced intermediates accumulation has been a great challenge for nitrogen removal from wastewater. In this paper, it was found that the denitrification performance of two denitrifiers, Paracoccus denitrificans and Pseudomonas denitrificans, were both improved in the presence of Shewanella oneidensis MR-1. Data showed the introducing of S. oneidensis MR-1 promoted nitrate reduction rate by 40.3%–50.0% in Par. denitrificans reactors. Meanwhile, it also increased nitrite and nitrous oxide reduction rates by 32.5%–111.0% and 68.8%–694.0% respectively in Pse. denitrificans reactors. Further investigation illustrated S. oneidensis MR-1 generally increased the electron competition capability of nitrogen oxide (NOx) reductases in varying degrees. As the electron distribution to the downstream NOx reductases increased more than that of the upstream ones, it led to reduced intermediates accumulation. Finally, the effectiveness of S. oneidensis MR-1 on augment denitrification performance was also validated in carbon-limited condition.

espanolLas rizobacterias de vida libre o asociativas promueven el crecimiento vegetal, fi jan nitrogeno y sintetizan fi tohormonas. La combinacion de estos mecanismos contribuye a mejorar la efi ciencia en el uso de fertilizantes nitrogenados y aumentar la productividad de los cultivos. El objetivo de esta investigacion fue evaluar el efecto de Azotobacter vinelandii DVS9 y Pseudomonas denitrifi cans DVS10 bajo diferentes niveles de fertilizacion quimica nitrogenada (50% y 75%) sobre el rendimiento y calidad nutricional de los tuberculos de Dioscorea rotundata. El estudio se realizo en el municipio de Morindo, Cordoba (Colombia), empleando un diseno experimental completamente aleatorizado con un arreglo factorial aumentado 3x2+2, con 3 repeticiones. Los resultados obtenidos indican que la inoculacion de la cepa Pseudomonas denitrifi cans DVS10 redujo un 25% la fertilizacion nitrogenada el cultivo de name espino, asimismo, esta bacteria produjo incrementos signifi cativos en los contenidos de proteina y fi bra cruda del tuberculo. EnglishThe free-living or associative rhizobacteria promote plant growth, fix nitrogen and synthesize phytohormones. The combination of these mechanisms contributes to improve the efficiency in the use of nitrogen fertilizers and increase the productivity of crops. The objective of this investigation was to evaluate the effect of Azotobacter vinelandii DVS9 and Pseudomonas denitrificans DVS10under different levels of nitrogen chemical fertilization (50% and 75%) on the yield and nutritional quality of the tubers of Dioscorea rotundata. The study was conducted in the municipality of Morindo, Cordoba (Colombia), using a completely randomized experimental design with an increased factorial arrangement 3x2 + 2, with 3 repetitions. The results obtained indicate that the inoculation of the strain Pseudomonas denitrificans DVS10 reduced the nitrogen fertilization by 25%, the cultivation of yam hawthorn, likewise, this bacterium produced significant increases in the protein and crude fiber contents of the tuber.

Coenzyme B12 is an essential cofactor for many enzymes such as glycerol dehydratase, methionine synthase and methylmalonyl-CoA mutase. Herein, we revisited the B12 biosynthetic gene clusters (I and II) in Pseudomonas denitrificans, a well-known industrial producer of the coenzyme B12, to understand the regulation of gene expression and improve the production of coenzyme B12. There were eight operons, seven in cluster I and one in cluster II, and four operons were regulated by B12-responsive riboswitches with a switch-off concentration at ∼5 nM coenzyme B12. DNA sequences of the four riboswitches were partially removed, individually or in combination, to destroy the structures of riboswitches, but no improvement was observed. However, when the whole length of riboswitches in cluster I were completely removed and promoters regulated by the riboswitches were replaced with strong constitutive ones, B12 biosynthesis was improved by up to 2-fold. Interestingly, modification of the promoter region for cluster II, where many (>10) late genes of B12 biosynthesis belong, always resulted in a significant, greater than 6-fold reduction in B12 biosynthesis.

Objective: The purpose of this study was to investigate the chemical composition of essential oil through GC-MS and look for new antibacterial drug agents from T. foenum graecum. Methods: Bacterial strains Sarcina lutea IFO 3232, Bacillus subtilis IFO 3026, Xanthomonas campestris IAM 1671, Escherichia coli IFO 3007, Klebsiella pneumoniae ATTC 10031, Proteus vulgaris MTCC 321 and Pseudomonas denitrificans KACC 32026 were used to measure the antibacterial activity by disc diffusion assay and therefore the chemical composition of essential oil through GC-MS. Results: GC–MS analysis of the essential oil from T. foenum graecum revealed the presence of fourteen different compounds of that Decane, 5, 6-bis (2, 2 dimethylpropylidene), (E,Z)-(19.58%); Hexadecanoic acid, methyl ester (18.81%); 5, 10-Diethoxy-2, 3, 7, 8-tetrahydro-1H,6H-dipyrrolo[1, 2-a;1’, 2’-d] pyrazine (5.81%) and Octadecanoic acid, methyl ester (3.28%) were the major compounds and therefore the minor compounds were Oxiraneoctanoic acid, 3-octyl, methyl ester; Hexadecanoic acid, ethyl ester; 6-Octadecenoic acid; Cis-Calamenene; 2-Pentadecanone, 6, 10, 14-trimethyl; 1, 2, 3, 4 Tetrahydroisoquinolin6-ol-1-carboxylic acid and Murolan-3, 9(11)-diene-10-peroxy. Essential oil (300 µg/disk) of T. foenum-graecum displayed antibacterial activity against Bacillus subtilis IFO 3026, Sarcina lutea IFO 3232, Xanthomonas campestris IAM 1671, Proteus vulgaris MTCC 321 and Pseudomonas denitrificans KACC 32026 with their respective zones of inhibition of 6.5 ± 0 to 8 ± 1.7 mm and the essential oil (150 µl/disk) displayed far greater potential antibacterial activity compare to those extracts against all experimental bacteria with their respective zones of inhibition of 8 ± 0 to 15 ± 0.7 mm. Conclusion: This experiment showed a great potential of antibacterial activity against gram positive and gram negative bacteria. The results of this study counsel that T. foenum-graecum might have potential use as antibacterial agents.

The performances of betaine on the metabolic processes of vitamin B12-producing Pseudomonas denitrificans were investigated in this paper. The results showed that betaine was an indispensable methyl-group donor for vitamin B12 biosynthesis, but large amounts of the extracellular glycine accompanied by betaine metabolism would impose a severe restriction on the cell growth of P. denitrificans. By further using a comparative metabolomics approach coupled with intracellular free amino acids analysis for the fermentation processes with betaine addition (10 g/l) or not, it was found that betaine could highly strengthen the formation of some key precursors and intermediates facilitating vitamin B12 biosynthesis, such as δ-aminolevulinic acid (ALA, the first precursor of vitamin B12), glutamate (an intermediate of ALA via C5 pathway), glycine (an intermediate of ALA via C4 pathway), and methionine (directly participating in the methylation reaction involved in vitamin B12 biosynthetic pathway). Therefore, the performances of betaine on P. denitrificans metabolic processes were not only serving as a decisive methyl-group donor for vitamin B12 biosynthesis, but also playing a powerfully promoting role in the generation of vitamin B12 precursors and intermediates.

The aerobic Pseudomonas denitrificans is widely used for industrial and commercial vitamin B12 fermentation, due to its higher productivity compared to the anaerobic vitamin B12-producing microorganisms. This paper aimed to develop a cost-effective fermentation medium for industrial vitamin B12 production by P. denitrificans in 120,000-l fermenter. It was found that maltose syrup (a low-cost syrup from corn starch by means of enzymatic or acid hydrolysis) and corn steep liquor (CSL, a by-product of starch industry) were greatly applicable to vitamin B12 production by P. denitrificans. Under the optimal fermentation medium performed by response surface methodology, 198.27 ± 4.60 mg/l of vitamin B12 yield was obtained in 120,000-l fermenter, which was close to the fermentation with the refined sucrose (198.80 mg/l) and was obviously higher than that obtained under beet molasses utilization (181.75 mg/l). Therefore, maltose syrups and CSL were the efficient and economical substrates for industrial vitamin B12 fermentation by P. denitrificans.

An efficient fermentation-strengthening approach was developed to improve the anaerobic production of vitamin B12 by cultivation process optimization with Propionibacterium freudenreichii. The effects of the byproduct propionic acid and the precursor 5,6-dimethylbenzimidazole (DMB) on vitamin B12 biosynthesis were investigated. Byproduct inhibition experiments showed that maintaining propionic acid concentration in broth below 10-20 g/L in the early stage and 20-30 g/L in the late stage can efficiently improve vitamin B12 biosynthesis. Batch fermentation indicated the occurrence of feed-back inhibition in intracellular intermediate biosynthesis. In addition, the incorporation of the precursor DMB depended on the fermentation level of the vitamin B12 intermediate. High vitamin B12 concentration (58.8 mg/L) and production (0.37 mg/g) were obtained with an expanded bed adsorption bioreactor by using the propionic acid and DMB control method. The optimum concentration and production of 59.5 and 0.59 mg/L h for vitamin B12 production were respectively achieved after five continuous batches.

3-Hydroxypropionic acid (3-HP) can be produced via the biological route involving two enzymatic reactions: dehydration of glycerol to 3-hydroxypropanal (3-HPA) and then oxidation to 3-HP. However, commercial production of 3-HP using recombinant microorganisms has been hampered with several problems, some of which are associated with the toxicity of 3-HPA and the efficiency of NAD+ regeneration. We engineered α-ketoglutaric semialdehyde dehydrogenase (KGSADH) from Azospirillum brasilense for the second reaction to address these issues. The residues in the binding sites for the substrates, 3-HPA and NAD+, were randomized, and the resulting libraries were screened for higher activity. Isolated KGSADH variants had significantly lower Km values for both the substrates. The enzymes also showed higher substrate specificities for aldehyde and NAD+, less inhibition by NADH, and greater resistance to inactivation by 3-HPA than the wild-type enzyme. A recombinant Pseudomonas denitrificans strain with one of the engineered KGSADH variants exhibited less accumulation of 3-HPA, decreased levels of inactivation of the enzymes, and higher cell growth than that with the wild-type KGSADH. The flask culture of the P. denitrificans strain with the mutant KGSADH resulted in about 40% increase of 3-HP titer (53 mM) compared with that using the wild-type enzyme (37 mM).

3-hydroxypropionic acid (3-HP) is an important platform chemical used as a precursor for production of added-value compounds such as acrylic acid. Metabolically engineered yeast, Escherichia coli, cyanobacteria and other microorganisms have been developed for the biosynthesis of 3-HP. Attempts to overproduce this compound in recombinant Pseudomonas denitrificans revealed that 3-HP is consumed by this microorganism using the catabolic enzymes encoded by genes hpdH, hbdH and mmsA. 3-HP-inducible systems controlling the expression of these genes have been predicted in proteobacteria and actinobacteria. In this study, we identify and characterise 3-HP-inducible promoters and their corresponding LysR-type transcriptional regulators from Pseudomonas putida KT2440. A newly-developed modular reporter system proved possible to demonstrate that PpMmsR/P mmsA and PpHpdR/P hpdH are orthogonal and highly inducible by 3-HP in E. coli (12.3- and 23.3-fold, respectively) and Cupriavidus necator (51.5- and 516.6-fold, respectively). Bioinformatics and mutagenesis analyses revealed a conserved 40-nucleotide sequence in the hpdH promoter, which plays a key role in HpdR-mediated transcription activation. We investigate the kinetics and dynamics of the PpHpdR/P hpdH switchable system in response to 3-HP and show that it is also induced by both enantiomers of 3-hydroxybutyrate. These findings pave the way for use of the 3-HP-inducible system in synthetic biology and biotechnology applications.

Sulfur, most abundantly found in the environment as sulfate (SO42-), is an essential element in metabolites required by all living cells, including amino acids, co-factors and vitamins. However, current understanding of the cellular delivery of SO42- at the molecular level is limited. CysZ has been described as a SO42- permease, but its sequence family is without known structural precedent. Based on crystallographic structure information, SO42- binding and flux experiments, we provide insight into the molecular mechanism of CysZ-mediated translocation of SO42- across membranes. CysZ structures from three different bacterial species display a hitherto unknown fold and have subunits organized with inverted transmembrane topology. CysZ from Pseudomonas denitrificans assembles as a trimer of antiparallel dimers and the CysZ structures from two other species recapitulate dimers from this assembly. Mutational studies highlight the functional relevance of conserved CysZ residues.

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This study investigated how hydraulic retention time (HRT) and COD/N ratio affect nitrogen-converting consortia in constantly aerated granules treating high-ammonium digester supernatant. Three HRTs (10, 13, 19 h) were tested at COD/N ratios of 4.5 and 2.3. Denaturing gradient gel electrophoresis and relative real-time PCR were used to characterize the microbial communities. When changes in HRT and COD/N increased nitrogen loading, the ratio of the relative abundance of aerobic to anaerobic ammonium-oxidizers decreased. The COD/N ratio determined the species composition of the denitrifiers; however, Thiobacillus denitrificans, Pseudomonas denitrificans and Azoarcus sp. showed a high tolerance to the environmental conditions and occurred in the granules from all reactors. Denitrifier genera that support granule formation were identified, such as Pseudomonas, Shinella, and Flavobacterium. In aerated granules, nirK-possessing bacteria were more diverse than nirS-possessing bacteria. At a low COD/N ratio, N2O-reducer diversity increased because of the presence of bacteria known as aerobic denitrifiers.

This study examined the role and physiological relevance of 3-hydroxyisobutyrate dehydrogenase-I (3HIBDHI) of Pseudomonas denitrificans ATCC 13867 in the degradation of 3-hydroxypropionic acid (3-HP) during 3-HP production. The gene encoding 3HIBDH-I of P. denitrificans ATCC 13867 was cloned and expressed in Escherichia coli BL21 (DE3). The recombinant 3HIBDH-I was then purified on a Ni-NTA-HP column and characterized for its choice of substrates, cofactors, metals, reductants, and the optimal temperature and pH. The recombinant 3HIBDH-I showed a high catalytic constant (k cat/K m) of 604.1 ± 71.1 mM/S on (S)-3-hydroxyisobutyrate, but no detectable activity on (R)-3-hydroxyisobutyrate. 3HIBDH-I preferred NAD+ over NADP+ as a cofactor for its catalytic activity. The k cat/K m determined for 3-HP was 15.40 ± 1.43 mM/S in the presence of NAD+ at 37°C and pH 9.0. In addition to (S)-3-hydroxyisobutyrate and 3-HP, 3HIBDH-I utilized l-serine, methyl-d,l-serine, and methyl-(S)-(+)-3-hydroxy-2-methylpropionate; on the other hand, the k cat/K m values determined for these substrates were less than 5.0mM/S. Ethylenediaminetetraacetic acid, 2-mercaptoethanol, dithiothreitol and Mn2+ increased the activity of 3HIBDHI significantly, whereas the presence of Fe2+, Hg2+ and Ag+ in the reaction mixture at 1.0 mM completely inhibited its activity. This study revealed the characteristics of 3HIBDH-I and its significance in 3-HP degradation.

BACKGROUND Previous research proved that fermentation via dissolved oxygen (DO) step-wise reduction was favorable for vitamin B12 productivity by Pseudomonas denitrificans, but the high-production mechanism is still unclear. Based on this fact, P. denitrificans metabolic characteristics under different DO supply models were investigated in a 5 L fermenter by assaying the activities of the key enzymes involved in central carbon metabolism and the vitamin B12 biosynthetic pathway. RESULTS Compared with fermentation processes under the DO-stat strategy and DO step-wise increase, it was found that DO step-wise reduction for P. denitrificans fermentation could significantly strengthen the Embden–Meyerhof–Parnas flux and weaken the tricarboxylic acid cycle and hexose monophosphate pathway, but also facilitate the activities of δ-aminolevulinic acid synthase and porphobilinogen synthase. CONCLUSIONS When P. denitrificans fermentation was implemented using DO step-wise reduction, the metabolic characteristics indicated that more precursors for vitamin B12 biosynthesis could be generated. As a result, a significant improvement in vitamin B12 productivity was achieved (69.09 ± 4.48 mg L−1). © 2013 Society of Chemical Industry

3-Hydroxypropionic acid (3-HP) can be produced from glycerol through two sequential enzymatic reactions that are catalyzed by a coenzyme B12 -dependent glycerol dehydratase and an NAD(P)(+) -dependent aldehyde dehydrogenase (ALDH), respectively. Pseudomonas denitrificans synthesizes coenzyme B12 under aerobic conditions, where NAD(P)(+) is regenerated efficiently. Hence, it is considered an ideal host for the production of 3-HP from glycerol under aerobic conditions. In this study, recombinant strains of P. denitrificans were developed and their potential for the production of 3-HP from glycerol was evaluated. When the enzymes, glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GdrAB), of Klebsiella pneumoniae were expressed heterologously, P. denitrificans could produce 3-HP at 37.7 mmol/L with 62% (mol/mol) yield on glycerol. Glucose was required as the carbon and energy sources for cell growth. The overexpression of heterologous ALDH was not essential; however, the titer and yield of 3-HP were improved to 54.7 mmol/L and 67% (mol/mol), respectively, when an ALDH gene (puuC) from K. pneumoniae was overexpressed. One serious drawback hindering the use of P. denitrificans as a recombinant host for 3-HP production is that it oxidizes 3-HP to malonate and utilizes 3-HP as a carbon source for growth. This is the first report on the development and use of recombinant P. denitrificans for 3-HP production from glycerol.

BACKGROUND Fermentation of the industrial vitamin B12 by Pseudomonas denitrificans usually utilizes sucrose or maltose as the sole carbon source, which results in increased medium costs. In order to decrease the fermentation cost, it is crucial and essential to employ a low-cost and convenient raw material as an alternative medium substrate for industrial vitamin B12 production. RESULTS The results obtained in chemically defined medium showed that glutamate and sucrose were favorable for cell growth and vitamin B12 biosynthesis of P. denitrificans. Due to containing a mass of ingredients such as sucrose, glutamate and betaine, beet molasses was consequently chosen as the main medium substrate for industrial P. denitrificans fermentation in a 120 000 L fermenter. Vitamin B12 production reached 181.75 mg L–1. CONCLUSIONS Beet molasses is a by-product of the sugar industry and is thus very inexpensive, and it was proved that beet molasses was an efficient and economical medium substrate for industiral vitamin B12 fermentation by P. denitrificans. © 2013 Society of Chemical Industry

Vitamin B12 is a complex biomolecule that acts as a cofactor for a variety of enzymes in microbial metabolism. Pseudomonas denitrificans is exclusively used as an industrial strain for the production of vitamin B12 under aerobic conditions. However, only a few strains of Pseudomonas have been reported to possess the capability of producing this vitamin and they are strongly patent-protected. To improve the applicability of the vitamin B12-producing microorganisms, a new isolate was obtained from municipal waste samples and characterized for its biological properties. The new isolate, designated as SP2, was identified to be a Pseudomonas species based on the sequence homology of its 16S rDNA. Pseudomonas species SP2 had essential genes for vitamin B12 synthesis such as cobB and cobQ and produced a similar amount of vitamin B12 (10.6 ± 0.05 μg/mL) as P. denitrificans ATCC 13867 in 24 h flask culture. SP2 grew well under aerobic condition with the maximum specific growth rate (µ max ) of 0.91 ± 0.03/h, but showed a poor growth under micro-aerobic conditions. SP2 was resistant to antibiotics like streptomycin, carbenicillin, ampicillin, cefpodoxime, colistin, nalidixic acid and sparfloxacin. The ability of SP2 to grow faster and produce vitamin B12 under aerobic conditions makes it a promising host for the production of some biochemicals requiring a coenzyme B12-dependent enzyme, such as glycerol dehydratase.

δ-aminolevulinate (ALA) is an important intermediate involved in tetrapyrrole synthesis (precursor for vitamin B12, chlorophyll and heme) in vivo. It has been widely applied in agriculture and medicine. On account of many disadvantages of its chemical synthesis, microbial production of ALA has been received much attention as an alternative because of less expensive raw materials, low pollution, and high productivity. Vitamin B12, one of ALA derivatives, which plays a vital role in prevention of anaemia has also attracted intensive works. In this review, recent advances on the production of ALA and vitamin B12 with novel approaches such as whole-cell enzyme-transformation and metabolic engineering are described. Furthermore, the direction for future research and perspective are also summarized.

BACKGROUND: There are two different routes for vitamin B12 biosynthesis, which results in discrepancies and uncertainties of the dissolved oxygen (DO) concentration for vitamin B12 fermentation. In this paper, the DO control strategy was explored for industrial vitamin B12 fermentation by Pesudomonas denitrificans in 120000-L fermenter. RESULTS: A DO-stat strategy was first successfully scaled up from a 9000 L fermenter to a 120 000 L fermenter. Then a multi-stage DO control strategy was further established in the 120 000 L fermenter, in which the DO level was shifted from 8–10% (20–48 h) to 2–5% (49–106 h) and below 2% (107–168 h) by gradually reducing the rate of aeration and agitation. As a result, 198.80 mg L−1 of vitamin B12 was obtained, which was significantly higher than those obtained under the fermentations with one-stage DO control. CONCLUSIONS: The comparatively low DO level was favorable for vitamin B12 biosynthesis, but it would have an extremely negative effect on cell growth. Compared with the low DO level maintained at all times of the fermentation process, a multi-stage DO control strategy could not only increase the biomass but also improve vitamin B12 biosynthesis. Copyright © 2012 Society of Chemical Industry

The network topology and metabolic fluxes of central carbon metabolism in the industrial vitamin B-12 producing strain Pseudomonas denitrificans were characterized under oxygen limiting levels. Cultivations were carried out with 100% [1-C-13] or 20% [U-C-13] glucose as substrates under different oxygen supply conditions. The labeling patterns of the proteinogenic amino acids of exponentially growing cells were used to accurately estimate the fluxes in the central carbon metabolism of P. denitrificans. Metabolic flux analysis showed that glucose was mostly catabolized by the Entner-Doudoroff and pentose phosphate pathways. Up to 33% of glucose was consumed via the PP pathway under high specific oxygen uptake rate (SOUR) conditions. This amount was 77.9% higher than that under low oxygen uptake conditions. Quantitative evidence was also found for reversible serine hydroxymethyl transferase and threonine aldolase activities. Metabolic flux and cofactor analyses further showed that higher SOUR accelerated the supply of precursors and methyl groups. SOUR also provided more NADPH for higher vitamin B12 production under the same glucose consumption.

Statistical experiment design and data analysis were used to establish the major factors in a chemically defined medium and to develop an ammonium control strategy to optimize the specific vitamin B 12 production rate (Yp) of Pseudomonas denitrificans . Through Plackett-Burman design, the major factors of glucose, ammonium sulfate and KCl were selected as the significant factors affecting vitamin B 12 biosynthesis and these were further optimized by central composite design with response surface methodology. The maximum Yp of 34.2 μg/gDCW/h was obtained in batch cultivation under the estimated optimal initial composition of glucose (93.6 g/l), (NH 4 ) 2 SO 4 (7.93 g/l) and KCl (1.24 g/l). Ammonium control strategies in fed-batch fermentation showed that when ammonium concentration was maintained at 40 mmol/l, the maximum Yp reached 36.0 ± 1.31 μg/gDCW/h, which was 57.2% higher than that of the control (22.9 ± 0.83 μg/gDCW/h). This ammonium control strategy successfully enhanced the industrial production, resulting in a stable high vitamin B 12 production of 212.02 ± 3.03 mg/l and Yp of 37.1 μg/gDCW/h. Key words : Statistical designs, Pseudomonas denitrificans, chemically defined medium, ammonium controlling strategy, vitamin B 12 .

Paracoccus pantotrophus expresses two nitrate reductases—membrane bound nitrate reductase (Nar) and periplasmic nitrate reductase (Nap). In growth experiments with two denitrifying species (Paracoccus pantotrophus and Alcaligenes eutrophus) that have both Nap and Nar and two species (Pseudomonas denitrificans and Pseudomonas fluorescens) with Nar only, it was found that diauxic lag is shorter for bacteria that express Nap. In P. pantotrophus, napEDABC encodes the periplasmic nitrate reductase. To analyze the effect of Nap on diauxic lag, the nap operon was deleted from P. pantotrophus. The growth experiments with nap− mutant resulted in increased diauxic lag when switched from aerobic to anoxic respiration, suggesting Nap is responsible for shorter lags and helps in adaptation to anoxic metabolism after transition from aerobic conditions. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009

A comparative study of microbial reduction of nitrate ions by immobilized Pseudomonas denitrificans cells in the presence and in the absence of a constant electric field was carried out. The experiments were carried out in a continuous stirred tank reactor at a broad range of dilution rates. The cathode potential was maintained constant around the standard potential value of the nitrate/nitrite redox couple. The residual outlet concentrations of nitrate, when the electric field was applied, were much lower than those obtained without the electric field, all other conditions being equal. It was demonstrated that the electric field had a significant effect on the nitrate reduction rate allowing the use of much higher inlet flow rates compared to the flow rate in the absence of an electric field.

The ring contraction process that occurs during cobalamin (vitamin B(12)) biosynthesis is mediated via the action of two enzymes, CobG and CobJ. The first of these generates a tertiary alcohol at the C-20 position of precorrin-3A by functioning as a monooxygenase, a reaction that also forms a gamma lactone with the acetic acid side chain on ring A. The product, precorrin-3B, is then acted upon by CobJ, which methylates at the C-17 position and promotes ring contraction of the macrocycle by catalyzing a masked pinacol rearrangement. Here, we report the characterization of CobG enzymes from Pseudomonas denitrificans and Brucella melitensis. We show that both contain a [4Fe-4S] center as well as a mononuclear non-heme iron. Although both enzymes are active in vivo, the P. denitrificans enzyme was found to be inactive in vitro. Further analysis of this enzyme revealed that the mononuclear non-heme iron was not reducible, and it was concluded that it is rapidly inactivated once it is released from the bacterial cell. In contrast, the B. melitensis enzyme was found to be fully active in vitro and the mononuclear non-heme iron was reducible by dithionite. The reduced mononuclear non-heme was able to react with the oxygen analogue NO, but only in the presence of the substrate precorrin-3A. The cysteine residues responsible for binding the Fe-S center were identified by site-directed mutagenesis. A mechanism for CobG is presented.

A novel Gram-negative, aerobic, motile, short rod-shaped bacterium, designated J64T, was isolated from black sand collected from Soesoggak, Jeju Island, Korea. Cells grew at 4-37 degrees C, at pH 5.5-10.0 and with 0-10 % NaCl. The strain was found to be oxidase- and catalase-positive. Phylogenetic analyses showed that strain J64T belongs to the genus Pseudomonas, forming a monophyletic group with Pseudomonas pachastrellae, Pseudomonas pertucinogena and 'Pseudomonas denitrificans'. The 16S rRNA gene sequence similarity between strain J64T and type strains of all Pseudomonas species with validly published names was below 96.6 %. Low levels of DNA-DNA relatedness were found with respect to type strains of P. pachastrellae and P. pertucinogena, supporting the classification of strain J64T within a novel species of the genus Pseudomonas. Strain J64T contained C(18 : 1)omega7c (37.2 %), C(16 : 0) (20.4 %), summed feature 3 (17.4 %; comprising iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c) and C(12 : 0) (7.6 %) as major cellular fatty acids. On the basis of the phenotypic and phylogenetic data, strain J64T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas sabulinigri sp. nov. is proposed. The type strain is J64T (=KCTC 22137T =JCM 14963T).

The strategy of betaine control for vitamin B12 large-scale fermentation by Pseudomonas denitrificans was investigated in this paper. The results obtained in shake-flask experiments demonstrated that betaine could greatly stimulate vitamin B12 biosynthesis but had an inhibition to cell growth. Based on the influence of betaine on the fermentation of P. denitrificans, betaine feeding was a beneficial strategy to solve the inconsistency between cell growth and vitamin B12 production. As a result, an effective and economical strategy of betaine feeding was established for vitamin B12 fermentation in 120-m3 fermenter, in which betaine was continuously fed to maintain betaine concentration of the broth at the range of 5–7 g/l during 50–140 h of fermentation.

A taxonomic study was carried out on strain C10-2T, a moderately halophilic denitrifier isolated from activated sludge samples collected in China. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain C10-2T belonged to the genus Pseudomonas, with highest sequence similarity of 98.1 and 97.7 % to 'Pseudomonas denitrificans' IAM 12023 and Pseudomonas pertucinogena JCM 11590T, respectively. The gyrB, rpoD and rpoB gene sequence similarity between strain C10-2T and P. pertucinogena JCM 11590T was respectively 84.1, 79.0 and 88.2 %. Phylogenetic trees based on these housekeeping genes showed that strain C10-2T and P. pertucinogena JCM 11590T form a clade at the periphery of the genus Pseudomonas. The DNA-DNA hybridization value between strain C10-2T and P. pertucinogena JCM 11590T was 30-32 %. The G+C content of the chromosomal DNA was 61.2 mol%. The combined genotypic and phenotypic data show that strain C10-2T represents a novel species of the genus Pseudomonas, for which the name Pseudomonas xiamenensis sp. nov. is proposed, with the type strain C10-2T (=CGMCC 1.6446 =JCM 13530T =MCCC 1A00089T).

Previous research has confirmed that cobalt ion and dimethylbenzimidazole (DMBI) are the precursors of vitamin B12 biosynthesis, and porphobilinogen synthase (PBG synthase) is a zinc-requiring enzyme. In this paper, the effects of Zn2+, Co2+ and DMBI on vitamin B12 production by Pseudomonas denitrificans in shake flasks were studied. Present experimental results demonstrated that the addition of the above mentioned three components to the fermentation medium could significantly stimulate the biosynthesis of vitamin B12. The concentrations of zinc sulphate, cobaltous chloride and DMBI in the fermentation medium were further optimized with rotatable orthogonal central composite design and statistical analysis by Data Processing System (DPS) software. As a result, vitamin B12 production was increased from 69.36 ± 0.66 to 78.23 ± 0.92 μg/ml.

The mechanism of the ring contraction process during vitamin B(12) biosynthesis by the anaerobe Propionibacterium shermanii was investigated under both aerobic and anaerobic conditions by means of feeding experiments with delta-amino[1-(13)C]levulinic acid (a biosynthetic intermediate of tetrapyrrole) and delta-amino[1-(13)C,1,1,4-(18)O(3)]levulinic acid in combination with (13)C-NMR spectroscopy. We showed that the characteristic mechanism of the ring contraction process (the generation of precorrin-3x from formation of the gamma-lactone from the ring A acetate group at C1 and hydroxylation at C20 by molecular oxygen catalyzed by CobG, and the migration of ring D by cleavage of the carbon-oxygen bond at C1 of precorrin-3x) in the aerobe Pseudomonas denitrificans was not seen in P. shermanii under aerobic conditions, and the mechanism of the ring contraction process in P. shermanii was the same irrespective of the presence or absence of oxygen.

We present a model for diauxic growth of denitrifying bacteria in which nitrate reductase synthesis kinetics dominate the overall growth kinetics. The model is based on the assumption of the existence of a nitrate respiration operon, thereby linking the rate of nitrate uptake to the activity of nitrate reductase. We show that this approach can model diauxic growth of Pseudomonas denitrificans by conducting experiments in which nitrate reductase activity was measured during both lag and ensuing exponential growth phases. We consistently observed the pattern of low nitrate reductase enzyme activity during the lag phase, increasing before the onset of growth. By fitting model parameters we were able to successfully match experimental data for growth, nitrate uptake, and enzyme activity level.

Bacteria were isolated from the rhizosphere of cotton, wheat, alfalfa, and tomato grown in field locations within a semi-arid region of Uzbekistan. Strains were identified as Pseudomonas denitrificans, P. rathonis, Bacillus laevolacticus, Bacillus amyloliquefaciens, and Arthrobacter simplex. The isolated strains produced different enzymes, phytohormone auxin and were antagonists against specific plant-pathogenic fungi. Most of the strains are tolerant with respect to salt and temperature. All of the bacterial strains isolated in this study have been found to increase plant growth of wheat and maize in pot experiments.

The crystallographic structure of the Pseudomonas denitrificans S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase (SUMT), which is encoded by the cobA gene, has been solved by molecular replacement to 2.7A resolution. SUMT is a branchpoint enzyme that plays a key role in the biosynthesis of modified tetrapyrroles by controlling flux to compounds such as vitamin B(12) and sirohaem, and catalysing the transformation of uroporphyrinogen III into precorrin-2. The overall topology of the enzyme is similar to that of the SUMT module of sirohaem synthase (CysG) and the cobalt-precorrin-4 methyltransferase CbiF and, as with the latter structures, SUMT has the product S-adenosyl-L-homocysteine bound in the crystal. The roles of a number of residues within the SUMT structure are discussed with respect to their conservation either across the broader family of cobalamin biosynthetic methyltransferases or within the sub-group of SUMT members. The D47N, L49A, F106A, T130A, Y183A and M184A variants of SUMT were generated by mutagenesis of the cobA gene, and tested for SAM binding and enzymatic activity. Of these variants, only D47N and L49A bound the co-substrate S-adenosyl-L-methionine. Consequently, all the mutants were severely restricted in their capacity to synthesise precorrin-2, although both the D47N and L49A variants produced significant quantities of precorrin-1, the monomethylated derivative of uroporphyrinogen III. The activity of these variants is interpreted with respect to the structure of the enzyme.