How long is bacillus megaterium

After resuspension in 50 mM phosphate buffer pH 7. In order to visualize PHB inclusions inside B. After incubation in the dark at room temperature for 5—15 min, cells were washed to remove exess Nile Red, mounted on microscope slides and examined under the fluorescene microscope Olympus BX51 Microscope. Bacterial endospores were stained with malachite green. Briefly, a resuspended culture aliquot was smeared on a microscope slide and briefly left to air dry.

The smear was then flooded with malachite green and the dye heat fixed. Excess dye was washed off with water, and vegetative cells were counterstained with aqueous safranin for 30 s. Excess dye was again washed off, slides carefully blotted dry and observed under the oil immersion Zeiss Axiophot Microscope.

To confirm PHB bead formation inside recombinant B. After 24 h of growth, cells were harvested, washed with 50 mM potassium phosphate buffer pH 7. In vivo activity of the polyester synthase was obtained by analysing the polyester content of the respective bacterial cells. Protein samples including bead-bound proteins were routinely analysed by SDS-PAGE sodium dodecyl sulphate-polyacrylamide electrophoresis as described elsewhere [ 68 ].

The gels were stained with Coomassie brilliant blue G Protein concentrations were determined using the Bio-Rad Protein Assay. The IgG binding performance binding capacity and purification power of purified ZZ-displaying beads was assessed and quantified by two types of assays as previously described: binding and elution of purified IgG and purification of IgG from human serum [ 14 , 31 ].

Briefly, a defined amount of beads was incubated with the feed material IgG or serum , subjected to a series of washes and bound IgG eluted with glycine pH2. FEBS J. High cell density cultivation of recombinant Escherichia coli for prodrug of recombinant human GLPs production.

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Identification and characterization of a novel intracellular poly 3-hydroxybutyrate depolymerase from Bacillus megaterium.

Production system for biodegradable polyester polyhydroxybutyrate by Corynebacterium glutamicum. J Biosci Bioeng. Since various strains of B. ML and GD — performed most of the experiments; RA and AS — contributed to interpretation of data for the work; GiC, LB, and GrC — contributed to drafting the work and revising it critically; ER — contributed to discussions and suggestions during the work and contributed to writing the manuscript; RI — led the work and contributed to writing the manuscript.

All authors read and approved the final manuscript. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The exposure time is indicated. Baccigalupi, L. Venema and A. Campbell, R. A monomeric red fluorescent protein.

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Rosso, M. Plasmid 53, — Rusciano, G. Soil application of organic and inorganic fertilizers for crop and vegetable cultivation is the major source for soil nitrate-nitrogen nitrate-N , which increases agricultural productivity. However, the vegetable yields do not increase continuously with soil nitrate-N [ 1 ]. A large accumulation of nitrate in soil results in soil secondary salinization, having various adverse effects on soil productivity, and nitrate accumulation in vegetables [ 2 ].

What is more, the reduction of nitrate to nitrite can cause various human diseases [ 1 ]. Soil secondary salinization is a severe problem in intensively managed agricultural ecosystems [ 3 ]. It is required to develop a low-cost bioremediation method to remove nitrate from soil. In our previous study, Bacillus megaterium NCT-2 was isolated from the secondary nitrate-salinized soil in a greenhouse, which shows high nitrate-reducing capacity and salinity adaptation in secondary salinization soil [ 4 ].

In our field trails, the concentrations of NO 3 - in both soil and plant were reduced significantly when we used the NCT-2 strain mixed with straw powder to treat secondary salinization soil unpublished. Moreover, this strain showed significant phosphate-solubilizing ability of insoluble inorganic phosphates in the culture medium [ 5 ]. Strain NCT-2 has the potential to be utilized as a biofertilizer for bioremediation of the secondary nitrate-salinized soil and plant growth promotion [ 6 ].

The Gram-positive bacterium Bacillus megaterium is found in diverse habitats from soil to sediment, sea, and dried food. It was named after its big size with a volume approximately times than that of Escherichia coli [ 7 ]. Its big size made it ideal to be used in studies of cell structure, protein localization, sporulation, and membranes [ 8 , 9 ].

The genomes of them have been sequenced to gain insights into the metabolic versatility that facilitate biotechnological applications, not the bioremediation of secondary salinization soil [ 18 , 19 ]. Despite the previously published work sequenced the 5. The functional nitrate assimilation-related genes the nitrate reductase electron transfer subunit, the nitrate reductase catalytic subunit, the nitrite reductase [NAD P H] large subunit and small subunit, and the glutamine synthetase were identified [ 20 ].

The genes that could be involved in the full potential of strain NCT-2 in the bioremediation of secondary salinization soil remain unknown.

For this, we obtained its complete genome sequence by using a multiplatform strategy involving HiSeq and PacBio sequencing. Furthermore, we performed a comprehensive analysis of nitrogen metabolism and plant growth-promoting features. The comparative analysis might be helpful for use in soil bioremediation.

The whole genome of the B. The HiSeq clean reads were preliminarily assembled into contigs and then were used for hybrid error correction of the subreads from PacBio.

There were two rounds of error correction. The finished NCT-2 genome was submitted to GenBank, replacing the previous version of the draft genome [ 20 ].

The protein-coding genes were predicted by using Glimmer 3. The gene function annotation was accomplished by blasting the protein sequences against the database of Kyoto Encyclopedia of Genes and Genomes KEGG [ 28 ]. CGView was used to produce the maps of the circular genomes with gene feature information [ 31 ]. The whole genome-based phylogenetic analysis was performed by using the CVTree 3.

Fourteen genome sequences were obtained from GenBank. A phylogenetic tree was constructed by the neighbor-joining method using MEGA analysis [ 35 — 37 ]. In addition, FusionDB was used to analyze the functional repertories of B.

The PacBio platform yielded 48, polymerase reads with the average size of The complete genome was assembled by taking advantage of the higher accuracy short reads from the HiSeq platform and the long subreads from the PacBio platform. The genome consists of a circular chromosome of 5. The total genome size is 5. The whole genome contains 6, genes, including 5, coding sequences, RNA genes, and pseudo genes.

The general features of B. The genome GC contents for three B. The microbial genome size is positively correlated with their environment adaptability [ 40 ]. One typical characteristic of soil microorganisms is the high number of rRNAs, which is helpful for fast growth, successful sporulation, germination, and rapid response to changing the availability of nutrients [ 41 — 44 ].

These features indicate that strain NCT-2 has great ability of adaptation to various environments. Most strains of Bacillus megaterium carry multiple plasmids, such as strain QM B has seven resident plasmids [ 18 ], Bacillus megaterium strain has ten plasmids [ 45 ], and Bacillus megaterium NBRC has six plasmids.

The plasmids have significantly lower GC contents than the chromosome There are coding sequences and 23 RNA genes. Additional rRNA operons carried on plasmids slowed the growth rates of E. Further investigations are needed to clarify the role of plasmids in bacterial growth and adaptations to high-nitrate environments in bioremediation of the secondary salinization soils. We used CVTree 3. The obtained tree Figure 2 a indicated that B. The neighbor-joining phylogenetic tree shows that strain NCT-2 is closest to B.

Whole-genome alignment of B. The 3, genes annotated by GO were classified into biological processes, cellular components, and molecular functions Figure S4. The top five categories were catalytic activity 1, , metabolic process 1, , cellular process 1, , single-organism process 1, , and binding 1, Like most strains of B. There are also genes for cobalt-zinc-cadmium resistance, oxidative stress, and nitrosative stress.

The translated protein sequence of B. The submitted proteome containing 5, proteins matched to 3, FusionDB functions, while proteins could not be mapped to any function in their database. The functional similarities of B. Strain NCT-2 is most functionally similar to B. There were 1, functions shared by all of them.

The common functional annotations related to nitrogen metabolism were nitrite transporter NirC, nitrogen-fixing NifU domain protein, nitroreductase, nitrate transport protein, and 2-nitropropane dioxygenase. Notably, there are 3, functions shared among three strains of B.

Strain NCT-2 has most of the core genes and pathways, including vitamin biosynthesis and nitrogen metabolism. Furthermore, only strain NCT-2 carries the gene encoding for periplasmic nitrate reductase.

In our field experiment, strain NCT-2 shows high nitrate-reducing capacity in secondary salinization soil unpublished. The functional nitrate assimilation-related genes that are involved in the process of converting nitrate to glutamine have been identified [ 20 ]. The genes encoding nitrate and nitrite reductase were cloned and overexpressed in Escherichia coli [ 47 ]. Here, the whole genomic analysis also revealed the genes encoding sensor, transporter, and enzymes are involved in nitrogen metabolism.

The genes were scattered in the chromosome. Genes encoding nitrite-sensitive transcriptional repressor NsrR , which is directly sensitive to nitrosative stress, were found in both the chromosome and the plasmid Table S3 and Figure S6. In the process of nitrate and nitrite ammonification, assimilatory nitrate reductase NaRas and nitrite reductase NiRas catalyzed the reduction of nitrate to ammonia through nitrite [ 48 ].

Ammonium transporter Amt was also encoded in the genome. Ammonium is an important nitrogen source for plant growth. Bacterial Amt proteins act as passive channels for the uncharged gas ammonia NH 3 [ 50 ]. It means that B. In the face of nitrosative stress, genes encoding nitrite-sensitive transcriptional repressor NsrR were found in both the chromosome and the plasmid. NsrR played a pivotal role in the regulation of NirK nitrite reductase , which was expressed aerobically in response to the increasing concentration of NO 2 - and decreasing pH [ 51 ].

However, no functional NirK could be found. Instead, two nitric oxide reductase activation proteins NorD and NorQ for denitrifying reductase gene clusters were found but without nitric oxide reductase, making the function of denitrification highly unlikely. Thus, the genome analysis proposed that B. It is an effective bioremediation approach to remove nitrate from soils.

Our previous studies on the plant growth promotion of B. Inoculation with B. Glucose dehydrogenase can oxidize glucose to gluconic acid, which is the most frequent organic acid produced by phosphate-solubilizing bacteria [ 52 ]. Additionally, the phosphate starvation system for phosphate uptake encoded by pst S, pst C, pst A, and pst B was also found in the genome. The phosphate solubilization capacity of strain NCT-2 plays a positive role in promoting plant growth by dissolving unavailable P PO 4 3- in soil to plant available forms.

Many plant growth-promoting bacteria have the ability to synthesize plant auxins indoleacetic acid, IAA [ 53 , 54 ], which is a key regulator for plant growth and development, such as cell division and elongation, lateral root production, and flowering [ 55 ].

Large-scale genomic analysis of IAA synthesis pathways suggested that plenty of bacteria could synthesize IAA via multiple incomplete pathways, and Firmicutes genomes had the simplest Trp-dependent IAA synthetic system [ 56 ].

Both the phosphate solubilization and IAA synthesis play important roles in plant growth promotion of strain NCT-2 during biocontrol and bioremediation of the secondary salinization soils. From the genome perspective, we can see genes involved in cation transporters magnesium transport and copper transport system and stress response, such as osmotic stress, oxidative stress, and detoxification.

Glycine betaine, a very efficient osmoprotectant, can be synthesized or acquired from exogenous sources [ 57 ]. There are glycine betaine ABC transport systems opu A, opu C, and opu D for choline uptake and genes for the glycine betaine biosynthetic enzymes choline dehydrogenase, gbs B, and betaine-aldehyde dehydrogenase, gbs A in strain NCT-2 genome.

Moreover, the genome contains genes encoding for superoxide dismutase EC 1. It implied that NCT-2 has great ability of adaption to environments. A hybrid approach with multiple assembler was used to assemble the complete genome of B.

The deeper investigation identified clues associated with the features of the bioremediation of secondary salinization soil and plant growth promotion at the gene level, such as nitrogen metabolism, phosphate uptake, synthesis of organic acids and phosphatase for phosphate-solubilizing ability, and Trp-dependent IAA synthetic system. Furthermore, the genes involved in cation transporters, osmotic stress, and oxidative stress implied that NCT-2 has great ability of adaption to environments.

In summary, these results provide valuable genomic resources for further studies and applications of using B. All data generated or analyzed during this study are included in this published article and its supplementary information files. The genome sequence of B. The accession number for the B. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Xiaorui Liu and Pei Zhou conceptualized the study. Bin Wang and Xiaorui Liu performed formal analysis. Dan Zhang and Shaohua Chu took care of funding acquisition.

Yuee Zhi and Xiaorui Liu performed methodology. Pei Zhou acquired resources. Yuee Zhi and Pei Zhou performed supervision of the study. Bin Wang wrote the original draft. Dan Zhang and Xiaorui Liu reviewed and edited the manuscript.