Most biological nitrogen fixation is catalyzed by molybdenum-dependent nitrogenase, an enzyme complex comprising two component proteins that contains three different metalloclusters. Diazotrophs contain a common core of nitrogen fixation nif genes that encode the structural subunits of the enzyme and components required to synthesize the metalloclusters. However, the complement of nif genes required to enable diazotrophic growth varies significantly amongst nitrogen fixing bacteria and archaea. In this study, we identified a minimal nif gene cluster consisting of nine nif genes in the genome of Paenibacillus sp. WLY78, a gram-positive, facultative anaerobe isolated from the rhizosphere of bamboo. We demonstrate that the nif genes in this organism are organized as an operon comprising nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV and that the nif cluster is under the control of a σ70 (σA)-dependent promoter located upstream of nifB. To investigate genetic requirements for diazotrophy, we transferred the Paenibacillus nif cluster to Escherichia coli. The minimal nif gene cluster enables synthesis of catalytically active nitrogenase in this host, when expressed either from the native nifB promoter or from the T7 promoter. Deletion analysis indicates that in addition to the core nif genes, hesA plays an important role in nitrogen fixation and is responsive to the availability of molybdenum. Whereas nif transcription in Paenibacillus is regulated in response to nitrogen availability and by the external oxygen concentration, transcription from the nifB promoter is constitutive in E. coli, indicating that negative regulation of nif transcription is bypassed in the heterologous host. This study demonstrates the potential for engineering nitrogen fixation in a non-nitrogen fixing organism with a minimum set of nine nif genes.

An improved strategy was developed for the high-density culture of Magnetospirillum gryphiswaldense strain MSR-1 and large-scale magnetosome production in both 7.5-and 42-liter autofermentors. By using a nutrientbalanced feeding strategy and the replacement of carbon and nitrogen sources to reduce accumulation of Na ؉ and Cl ؊ ions, we reduced the factors that tend to inhibit cell growth, particularly the increase of osmotic potential. Semicontinuous culture was thereby achieved in the autofermentor for the first time. When the cells were harvested at 36 and 73 h, magnetosome yields (dry weight) as high as 168.3 and 83.5 mg/liter/day, respectively, were achieved. These values were, respectively, approximately 10 and 5 times higher than the yields achieved in previous studies and represent a significant improvement in magnetosome production efficiency.Biomineralized magnetosomes (chains of magnetite crystals found in prokaryotes) have attracted commercial interest because of their narrow size range, good dispersibility, and biomembrane enclosure. Previous studies have addressed a variety of applications and properties, including enzyme immobilization (5), gene delivery system (16), cell separation (19), drug carriers (11, 12), immunoassays (4, 14), protein and multisubunit enzyme complexes (7,20), and use of microorganisms per se for mineral recovery (13). Because of the highly restrictive culture conditions for magnetotactic bacteria, in terms of the dissolved oxygen concentration (dO 2 ) (3, 17), nutrients, etc., the yields of both magnetosomes and their host microorganisms under artificial culture tend to be low (10, 18). A long-standing research goal of our laboratory is improved large-scale production of cells and magnetosomes.In a previous study using fed-batch culture techniques (10), we achieved maximal cell density (optical density at 565 nm [OD 565 ] of 7.24, cell dry weight of 2.17 g/liter [0.87 g/liter/day], and magnetosome dry weight of 41.7 mg/liter [16.7 mg/liter/ day]). Through further optimization of culture temperature, pH, dO 2 , and nutrients, we achieved an OD 565 value of 12 in a 7.5-liter fermentor after 40 h of culture (unpublished data). In revising our previous feeding strategy, we focused on supplementation of carbon and nitrogen sources but ignored two possible factors that could inhibit cell growth: (i) nutrient limitation arising during fermentation process and (ii) the accumulation of Na ϩ and Cl Ϫ in a fermentor fed with sodium lactate and ammonium chloride. By replacing the carbon and nitrogen sources and using an optimally nutrient-balanced feeding strategy, in a 7.5-liter fermentor after 44 h, we achieved an OD 565 of 30.4, a cell dry weight of 7.59 g/liter (3.8 g/liter/ day), and a magnetosome dry weight of 225.53 mg/liter (112.77 mg/liter/day). In a larger (42-liter) fermentor, after 44 h, we achieved an OD 565 of 42, a cell dry weight of 9.16 g/liter (4.58 g/liter/day), and a magnetosome dry weight of 356.52 mg/liter (178.26 mg/liter/day). The efficiency of magnetosome produ...

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N2-fixing strains. The heterogeneity of the 31 genomes (15 N2-fixing and 16 non-N2-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N2-fixing strains. The nif cluster is under control of a σ70-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N2-fixing and non-N2-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N2-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N2-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N2-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.

Magnetotactic bacteria (MTB) synthesize unique organelles termed "magnetosomes," which are membraneenclosed structures containing crystals of magnetite or greigite. Magnetosomes form a chain around MamK cytoskeletal filaments and provide the basis for the ability of MTB to navigate along geomagnetic field lines in order to find optimal microaerobic habitats. Genomes of species of the MTB genus Magnetospirillum, in addition to a gene encoding the tubulin-like FtsZ protein (involved in cell division), contain a second gene termed "ftsZ-like," whose function is unknown. In the present study, we found that the ftsZ-like gene of Magnetospirillum gryphiswaldense strain MSR-1 belongs to a 4.9-kb mamXY polycistronic transcription unit. We then purified the recombinant FtsZ-like protein to homogeneity. The FtsZ-like protein efficiently hydrolyzed ATP and GTP, with ATPase and GTPase activity levels of 2.17 and 5.56 mol phosphorus per mol protein per min, respectively. The FtsZ-like protein underwent GTP-dependent polymerization into long filamentous bundles in vitro. To determine the role of the ftsZ-like gene, we constructed a ftsZ-like mutant (⌬ftsZ-like mutant) and its complementation strain (⌬ftsZ-like_C strain). Growth of ⌬ftsZ-like cells was similar to that of the wild type, indicating that the ⌬ftsZ-like gene is not involved in cell division. Transmission electron microscopic observations indicated that the ⌬ftsZ-like cells, in comparison to wild-type cells, produced smaller magnetosomes, with poorly defined morphology and irregular alignment, including large gaps. Magnetic analyses showed that ⌬ftsZ-like produced mainly superparamagnetic (SP) magnetite particles, whereas wildtype and ⌬ftsZ-like_C cells produced mainly single-domain (SD) particles. Our findings suggest that the FtsZ-like protein is required for synthesis of SD particles and magnetosomes in M. gryphiswaldense.Magnetotactic bacteria (MTB) can orient themselves along geomagnetic field lines and search for microaerophilic environments. These capabilities are based on unique prokaryotic organelles termed magnetosomes (3). Magnetosomes are nanometer-size magnetic particles of iron oxide (magnetite; Fe 3 O 4 ) or iron sulfide (greigite; Fe 3 S 4 ) (4, 5, 45), enclosed within intracytoplasmic vesicles of the magnetosome membrane (MM) (3, 43). Magnetosome formation is a complex process involving vesicle formation, iron transportation, nucleation and growth of magnetite crystals, and their assembly into chain-like structures. A model for magnetosome formation has been proposed by Komeili (18) and Schüler (44). According to this model, magnetosome vesicles are invaginated from the inner membrane, and protein sorting to the MM occurs concurrently. The protein MamA was suggested to activate magnetosome vesicles for magnetite biomineralization (19). With the help of the MamK and MamJ proteins, the membrane invaginations are then assembled into a chain structure. The bacterial actin-like MamK can form filaments required for maintaining magnetosome organization a...

Klebsiella oxytoca M5al is an excellent 1,3-propanediol (1,3-PD) producer, but too much lactic acid yielded greatly lessened the fermentation efficiency for 1,3-PD. To counteract the disadvantage, four lactate deficient mutants were obtained by knocking out the ldhA gene of lactate dehydrogenase (LDH) of K. oxytoca M5al. The LDH activities of the four mutants were from 3.85 to 6.92% of the parental strain. The fed-batch fermentation of 1,3-PD by mutant LDH3, whose LDH activity is the lowest, was studied. The results showed that higher 1,3-PD concentration, productivity, and molar conversion rate from glycerol to 1,3-PD can be gained than those of the wild type strain and no lactic acid is produced under both anaerobic and microaerobic conditions. Sucrose fed during the fermentation increased the conversion and sucrose added at the beginning increased the productivity. In fed-batch fermentation with sucrose as cosubstrate under microaerobic conditions, the 1,3-PD concentration, conversion, and productivity were improved significantly to 83.56 g l-1, 0.62 mol mol-1, and 1.61 g l(-1) h-1, respectively. Furthermore, 60.11 g l(-1) 2,3-butanediol was also formed as major byproduct in the broth.

Magnetotactic bacteria are difficult to grow under defined conditions in culture, which has presented a major obstacle to commercial application of magnetosomes. We studied the relationships among the cell growth, magnetosome formation, dissolved oxygen concentration (DO), and the ability to supply oxygen to the cells. Mass culture of Magnetospirillum gryphiswaldense MSR-1 for the production of magnetosomes was established in a 42-L fermentor under the following conditions: (1) sterile air was the sole gas supplied in the fermentor, and DO could be regulated at any level below 10% saturation by cascading the stir rate to DO, (2) to resolve the paradoxical situation that the cell growth requires higher DO whereas magnetosome formation requires low DO below the detectable range of regular oxygen electrode, DO was controlled to optimal level using the change of cell growth rate, rather than reading from the highly sensitive oxygen electrode, as the signal for determining appropriate DO, and (3) timing and rate of supplying the substrates were determined by measuring cell density and Na-lactate concentration. Under these conditions, cell density (OD565) of strain MSR-1 reached 7.24 after 60-h culture in a 42-L fermentor, and cell yield (dry weight) was 2.17 g/L, the highest yield so far being reported. The yield of magnetosomes (dry weight) was 41.7 mg/L and 16.7 mg/L/day, which were 2.8 and 2.7 times higher than the previously reported yields.

The function of the regulatory protein AveR in Streptomyces avermitilis was examined. An aveR deletion mutant abolished avermectin production and produced more oligomycin, and its phenotype was complemented by a single copy of the aveR gene. Removal of the C-terminal HTH domain of AveR abolished avermectin biosynthesis, indicating the importance of HTH domain for AveR function. Promoter titration and promoter probe assays suggested that the transcription of aveA1, encoding polypeptide AVES1 of avermectin PKS, was activated by AveR. Chromatin immunoprecipitation (ChIP) assay showed that the predicted promoter regions of both the ave cluster and the olm cluster were target sites of AveR, and the DNA-binding activity of AveR was dependent on its HTH domain. RT-PCR analysis revealed that the transcriptions of ave structural genes were dependent on AveR, but that of olm structural genes and putative pathway-specific regulatory genes increased in the aveR mutants. Consistent with these observations, overexpression of aveR successfully increased avermectin production. These results indicated that aveR encodes a pathway-specific activator essential for avermectin biosynthesis and it also negatively affects oligomycin biosynthesis.

Formation of HD from D2 has been demonstrated with nitrogenase preparations from Azotobacter vinelandii, Clostridium pasteurianum, Klebsiella pneumoniae, and Azospirillum sp. We conclude that the formation of HD from D2 is a general property of nitrogenases. However, the nitrogenases differ in their Ki values for D2 (N2 fixation) and in their rates of catalyzing HD formation; among the nitrogenases tested, C. pasteurianum nitrogenase had the lowest activity for formation of HD. When contaminating N2 was removed from the atmospheres above reaction mixtures, less than 1% of the total electron flux in the system was directed to HD formation; hence, we doubt that N2-independent HD formation is significant. A working hypothesis is suggested that operates without invoking an N2-independent reaction for forming HD.