Complete Sequence of the First Chimera Genome Constructed by Cloning the Whole Genome of Synechocystis Strain PCC6803 into the Bacillus subtilis 168 Genome

Genome synthesis of existing or designed genomes is made feasible by the first successful cloning of a cyanobacterium, Synechocystis PCC6803, in Gram-positive, endospore-forming Bacillus subtilis. Whole-genome sequence analysis of the isolate and parental B. subtilis strains provides clues for identifying single nucleotide polymorphisms (SNPs) in the 2 complete bacterial genomes in one cell.     

De Novo Assembly of the Complete Genome of an Enhanced Electricity-Producing Variant of Geobacter sulfurreducens Using Only Short Reads

State-of-the-art DNA sequencing technologies are transforming the life sciences due to their ability to generate nucleotide sequence information with a speed and quantity that is unapproachable with traditional Sanger sequencing. Genome sequencing is a principal application of this technology, where the ultimate goal is the full and complete sequence of the organism of interest. Due to the nature of the raw data produced by these technologies, a full genomic sequence attained without the aid of Sanger sequencing has yet to be demonstrated.We have successfully developed a four-phase strategy for using only next-generation sequencing technologies (Illumina and 454) to assemble a complete microbial genome de novo. We applied this approach to completely assemble the 3.7 Mb genome of a rare Geobacter variant (KN400) that is capable of unprecedented current production at an electrode. Two key components of our strategy enabled us to achieve this result. First, we integrated the two data types early in the process to maximally leverage their complementary characteristics. And second, we used the output of different short read assembly programs in such a way so as to leverage the complementary nature of their different underlying algorithms or of their different implementations of the same underlying algorithm.The significance of our result is that it demonstrates a general approach for maximizing the efficiency and success of genome assembly projects as new sequencing technologies and new assembly algorithms are introduced. The general approach is a meta strategy, wherein sequencing data are integrated as early as possible and in particular ways and wherein multiple assembly algorithms are judiciously applied such that the deficiencies in one are complemented by another.     

产纳豆激酶枯草芽孢杆菌种子液培养条件的优化

利用Plackett-Burman法(PB法),以紫外分光光度法作为测量方法对产纳豆激酶枯草芽孢杆菌种子液培养条件进行了优化。确定了产纳豆激酶枯草芽孢杆菌种子液的最佳氮源是大豆蛋白胨,优化出最佳种子液培养条件为牛肉膏0.5 g/100 mL、大豆蛋白胨1 g/100 mL、NaCl 0.75 g/100 mL、pH 7、培养温度37℃、培养时间8.5 h、接种量为3 mL/100 mL、装液量100 mL/250 mL。     

Complete Genome Sequence of Bacillus subtilis Strain QB928, a Strain Widely Used in B. subtilis Genetic Studies

The complete genome sequence of Bacillus subtilis strain QB928 was constructed to facilitate studies in the evolution of the genetic code. With a widespread use of the strain in Bacillus subtilis genetics studies, its complete genome sequence would facilitate deeper understanding of Bacillus subtilis genetics.     

Functional myo-inositol catabolic genes of Bacillus subtilis Natto are involved in depletion of pinitol in Natto (fermented soybean)

Soybeans are rich in pinitol (PI; 3-O-methyl-D-chiro-inositol), which improves health by treating conditions associated with insulin resistance, such as diabetes mellitus and obesity. Natto is a food made from soybeans fermented by strains of Bacillus subtilis natto. In the chromosome of natto strain OK2, there is a putative promoter region almost identical to the iol promoter for myo-inositol (MI) catabolic genes of B. subtilis 168. In the presence of MI, the putative iol promoter functioned to induce inositol dehydrogenase, the enzyme for the first-step reaction in the MI catabolic pathway. PI also induced inositol dehydrogenase and the promoter was indispensable for the utilization of PI as well as MI, suggesting that PI might be an alternative carbon source metabolized in a way involving the MI catabolic genes. Natto fermentation studies have revealed that the parental natto strain consumed PI while a mutant defective in the iol promoter did not do so at all. These results suggest that inactivating the MI catabolic genes might prevent PI consumption, retaining it in natto for enrichment of possible health-promoting properties.     

Complete Growth Inhibition of Bacillus subtilis by Nisin-Producing Lactococci in Fermented Soybeans

The growth inhibition by nisin-producing lactococci against Bacillus subtilis and its application to soybean miso fermentation were investigated. Lactococcus lactis subsp. lactis IFO12007 (nisin-producing, salt-intolerant) rapidly proliferated to more than 10⁹ cells/g in cooked soybeans without any excessive pH decrease. In spite of the mild decrease in pH, the growth of B. subtilis was completely inhibited; no living cells were detected in a soybean sample inoculated with 10⁶ cells/g and incubated for 24 to 72 h. This Lc. lactis was applied to soybean miso fermentation as a starter culture. It produced high nisin activity (1.28×10⁵ AU/g) in cooked soybean, resulting in the complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation, throughout the process of miso production. Over-acidification, which is undesirable for miso quality, was successfully prevented simply by adding salt which killed the salt-intolerant Lc. lactis. Furthermore, the nisin activity in miso disappeared with aging.     

Complete Genome Sequence of Bacillus thuringiensis Mutant Strain BMB171

Bacillus thuringiensis has been widely used as a biopesticide for a long time. Here we report the finished and annotated genome sequence of B. thuringiensis mutant strain BMB171, an acrystalliferous mutant strain with a high transformation frequency obtained and stocked in our laboratory.Bacillus thuringiensis is an insect pathogen which is widely used as a biopesticide due to its various endogenous crystal proteins and spores (12). To improve the virulence and practical effectiveness of B. thuringiensis, genetic transformation of different genes with beneficial traits is a fundamental procedure. Simultaneously, genetic transformation can facilitate functional genomic research. However, wild-type strains are not suitable to be used as recipient strains because of low transformation efficiency. This obstacle is mainly caused by the thick cell wall layer of B. thuringiensis together with multiple plasmids inside the cell, which harbor genes encoding insecticidal crystal proteins. We used the method of elevating the growth temperature and adding 0.05% sodium dodecyl sulfate to treat several parental strains and finally obtained mutant strain BMB171, with no resident plasmid, from wild-type crystalliferous strain YBT-1463 (9). The electrotransformation frequency of mutant BMB171 could reach up to 10⁷ transformants/μg DNA after optimization of the electrotransformation parameters (7), which was 4.8 × 10⁴-fold higher than that of the parental strain (8). Moreover, mutant strain BMB171 exhibited the same characteristics as YBT-1463, such as metabolic abilities and growth properties, as well as sensitivity to 10 antibiotics (8). Of course, BMB171 could produce parasporal crystals with characteristic geometric shapes through the expression of relevant cry genes carried by plasmids (7). Thus, B. thuringiensis mutant strain BMB171 has become a major recipient strain and is widely used for insecticidal crystal protein-encoding gene expression (14, 15), cell surface display (10, 13), gene function and regulation researches (2, 5), etc.The B. thuringiensis mutant strain BMB171 genome was sequenced by using a massive parallel pyrosequencing technology (454 GS-FLX). A total of 448,963 high-quality reads with an average read length of 391 bp were produced, providing about 32-fold coverage of the genome. Assembly was performed using the Newbler software of the 454 suite package (454 Life Sciences), which resulted in 193 large (defined as >500 bp) contigs. The relationship of contigs was determined by multiplex PCR, and gaps were filled through sequencing of PCR products by primer walking or shotgun sequencing with an ABI 3730 sequencer. The Phred/Phrap/Consed software package (3) was used for final sequence assembly and quality assessment. Protein-coding genes were predicted by combining the results of Glimmer 3.02 (1) and ZCURVE (4), followed by manual inspection. Both tRNA and rRNA genes were identified by tRNAscan-SE (11) and RNAmmer (6), respectively. Functional annotation was performed by searching against a protein database of the microbial genome developed in house.The 5.64-Mb genome of B. thuringiensis mutant strain BMB171 contains two replicons: a circular chromosome (5.33 Mb) encoding 5,088 open reading frames (ORFs) and a circular plasmid (0.31 Mb), which is named pBMB171, encoding 276 predicted ORFs. The G+C content of the chromosome is 35.3%, while that of the plasmid is 33.3%. The mutant strain BMB171 genome encodes 104 tRNAs and 14 rRNA operons. A previous study indicated that BMB171 is a plasmid-free mutant (9); however, our sequencing results demonstrated that a large plasmid still remains. The reason why the plasmid was not detected previously might be its large size and low copy number. We did not find any crystal protein genes in either chromosome or plasmid sequences, which was consistent with previous observations (9).In summary, the complete B. thuringiensis mutant strain BMB171 genome provides a better-defined genetic background for gene expression and regulation studies, especially crystal protein production and metabolic network construction.     

Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis

The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18.3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B.halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 σ factors which belong to the extracytoplasmic function family, 10 are unique to B.halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.     

The High-Quality Complete Genome Sequence of the Opportunistic Fungal Pathogen Candida vulturna CBS 14366T

Mycopathologia - Candida vulturna is a new member of the Candida haemulonii species complex that recently received much attention as it includes the emerging multidrug-resistant pathogen Candida...     

Complete Genome Sequence of the Extremophilic Bacillus cereus Strain Q1 with Industrial Applications

Bacillus cereus strain Q1 was isolated from a deep-subsurface oil reservoir in the Daqing oil field in northeastern China. This strain is able to produce biosurfactants and to survive in extreme environments. Here we report the finished and annotated genome sequence of this organism.Bacillus cereus strain Q1 was isolated from a deep-subsurface oil reservoir in the Daqing oil field in northeastern China. This strain can facilitate oil recovery when added to an oil reservoir. This attribute may be partially due to its ability to produce biosurfactants, which assist microbial enhanced oil recovery by lowering interfacial tension at the oil-rock interface (data not shown).The complete genome sequence of B. cereus Q1 was determined by the whole-genome shotgun strategy. Draft assemblies were based on 55,790 high-quality reads. All libraries provided sixfold coverage of the genome. Gap closure was accomplished by primer walking on gap-spanning clones and direct sequencing of combinatorial PCR products. Open reading frame (ORF) predictions were obtained and annotation was performed as described previously (4). GenomeComp was used for genomic comparison with default parameters (5).The genome of B. cereus Q1 is composed of one circular chromosome of 5,214,195 bp and two circular plasmids (pBc239 [239,246 bp] and pBc53 [52,766 bp]) with mean G+C contents of 35.6, 33.5, and 35.1%, respectively. There are 5,657 predicted ORFs, 13 rRNA operons, and 94 tRNA genes for all 20 amino acids, covering 86% of the genome. Putative functions were assigned to 3,946 ORFs. Of the remainder, 1,561 showed similarity to hypothetical proteins and 140 had no detectable homologs in the public protein database (E value, <10⁻¹⁰). Twelve phage-related genes were identified, but no complete prophages were found. Whole-genome comparison showed that Q1 has extensive similarity to the genomes of other members of the B. cereus group and the greatest similarity to nonpathogenic strain B. cereus ATCC 10987 (5,224,283 bp).Genome analysis revealed that B. cereus Q1 contains several genes related to niche-specific adaptations. As a thermophilic bacterium, Q1 can easily adapt to geothermal oil reservoirs. Three thermophily-associated genes (BC1015, BC1017, and BC1018) found in Q1 have orthologs in Moorella thermoacetica ATCC 39073. The latter genes encode the structural maintenance of chromosome protein, exonuclease SbcC, and subunit A of DNA topoisomerase VI, respectively. The presence of the genes involved in the utilization of l-fucose (BC2995 to BC3006) and d-mannose (BC5091 to BC5094, BC5097 to BC5102, and BC5105 to BC5111) helps Q1 use these carbohydrates as carbon sources under glucose-limited conditions. Q1 also contains the nitrate utilization gene cluster (BC2100 to BC2123), including a typical narGHJI operon that encodes membrane-bound nitrate reductase. The nitrate utilization gene cluster might play an important role in helping the strain use nitrate as a nitrogen source and survive under anaerobic or oxygen-limited conditions. Moreover, we found an operon that encodes proteins responsible for producing a novel type of lantibiotic (2), which we designated cereicidin. All of the above-mentioned genes were not found in the other B. cereus group bacteria.One of the notable features of Q1 is its ability to produce biosurfactants. The dhb operon (dhbACEBF), which is involved in nonribosomal peptide synthesis and encodes the biosynthetic template for the catecholic siderophore in B. subtilis (1), was identified in Q1. Downstream of the operon, the sfp gene, which encodes phosphopantetheinyl transferase and is required for production of the lipopeptide antibiotic surfactin in B. subtilis (3), was found. No surfactin synthetase gene (srfAA, srfAB, or srfAC) was found, but the mbtH gene involved in mycobactin synthesis and a gene (BC2300) with an unknown function were found in the region between the dhb operon and the sfp gene. We therefore speculated that these three genes located downstream of the dhbF gene might belong to the dhb operon, which is involved in antibiotic-siderophore-surfactin biosynthesis.The B. cereus Q1 genome provides an excellent platform for the further improvement of this organism for biosurfactant production and extends our understanding of the evolutionary relationships among B. cereus group organisms.     

Complete physical map of the Bacillus subtilis 168 chromosome constructed by a gene-directed mutagenesis method

All the SfiI sites and most of the NotI sites were located precisely on the chromosome of Bacillus subtilis 168 by a novel method, termed gene-directed mutagenesis. The stepwise elimination of these restriction sites by this method allowed not only the physical connection of the restriction fragments but also the accurate determination of the position of the restriction sites themselves. The resulting physical map of the 4165 x 10(3) base-pair B. subtilis chromosome has been correlated with the genetic map by determination of the exact location of known genes. The complete physical map provides a rapid and accurate way for mapping of new genes as well as analysis of large DNA rearrangements on the chromosome. The novel strategy is, in principle, applicable to the analysis of the genome of other organisms.     

Genome homology and divergence in the SP beta-related bacteriophages of Bacillus subtilis

Chromosomal organization in related temperate Bacillus subtilis bacteriophages SP beta, phi 3T, rho 11, Z, and E was compared. DNA-DNA hybridization studies done in conjunction with available restriction fragment maps of SP beta, phi 3T, and rho 11 demonstrated that DNA homology between these three phages extended over most of their respective genomes, although each contained unique chromosomal segments, phi 3T, rho 11, Z, and E, but not SP beta, possessed apparently homologous structural genes (thyP) for thymidylate synthetase. DNA from all thyP-containing phages transformed thymine auxotrophs of B. subtilis SP beta lysogens to prototrophy. This transformation commonly involved incorporation of the thyP gene into SP beta prophage within a region corresponding to the middle of the viral chromosome. Chimeric plasmids containing the thyP gene from phi 3T or cloned fragments of SP beta DNA were used in DNA-DNA hybridization studies to locate the thymidylate synthetase gene near the center of the phi 3T chromosome, and to demonstrate that the organization of this region resembled the analogous portion of the SP beta genome. Profiles of virion structural proteins from the five phages were also very similar, further suggesting functional homology between these viruses. However, despite these evidences of relatedness, populations of fragments generated by digesting SP beta, phi 3T, rho 11, Z, and E DNA with restriction enzymes were quite dissimilar.     

Production of sedoheptulose by Bacillus subtilis

Wild type strains of Bacillus subtilis produced sedoheptulose from d-ribose but not from d-glucose, B. subtilis mutants deficient in transketolase produced sedoheptulose when d-glucose was used as a carbon source. The addition of d-ribose to the culture medium increased the amount of sedoheptulose accumulated, reaching about 20 mg/ml of culture broth. The mutant strains reverted to wild type strains at a high frequency during cell growth, and therefore the accumulation of sedoheptulose was caused by the genetic instability of the mutant: d-ribose formed from d-glucose by the mutant strain was converted into sedoheptulose by revertant cells that appeared during cultivation.