The complete genome sequence of Pantoea ananatis AJ13355, an organism with great biotechnological potential

Pantoea ananatis AJ13355 is a newly identified member of the Enterobacteriaceae family with promising biotechnological applications. This bacterium is able to grow at an acidic pH and is resistant to saturating concentrations of L-glutamic acid, making this organism a suitable host for the production of L-glutamate. In the current study, the complete genomic sequence of P. ananatis AJ13355 was determined. The genome was found to consist of a single circular chromosome consisting of 4,555,536 bp [DDBJ: AP012032] and a circular plasmid, pEA320, of 321,744 bp [DDBJ: AP012033]. After automated annotation, 4,071 protein-coding sequences were identified in the P. ananatis AJ13355 genome. For 4,025 of these genes, functions were assigned based on homologies to known proteins. A high level of nucleotide sequence identity (99%) was revealed between the genome of P. ananatis AJ13355 and the previously published genome of P. ananatis LMG 20103. Short colinear regions, which are identical to DNA sequences in the Escherichia coli MG1655 chromosome, were found to be widely dispersed along the P. ananatis AJ13355 genome. Conjugal gene transfer from E. coli to P. ananatis, mediated by homologous recombination between short identical sequences, was also experimentally demonstrated. The determination of the genome sequence has paved the way for the directed metabolic engineering of P. ananatis to produce biotechnologically relevant compounds.     

Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species

Background

Bacillus licheniformis is a Gram-positive, spore-forming soil bacterium that is used in the biotechnology industry to manufacture enzymes, antibiotics, biochemicals and consumer products. This species is closely related to the well studied model organism Bacillus subtilis, and produces an assortment of extracellular enzymes that may contribute to nutrient cycling in nature.

Results

We determined the complete nucleotide sequence of the B. licheniformis ATCC 14580 genome which comprises a circular chromosome of 4,222,336 base-pairs (bp) containing 4,208 predicted protein-coding genes with an average size of 873 bp, seven rRNA operons, and 72 tRNA genes. The B. licheniformis chromosome contains large regions that are colinear with the genomes of B. subtilis and Bacillus halodurans, and approximately 80% of the predicted B. licheniformis coding sequences have B. subtilis orthologs.

Conclusions

Despite the unmistakable organizational similarities between the B. licheniformis and B. subtilis genomes, there are notable differences in the numbers and locations of prophages, transposable elements and a number of extracellular enzymes and secondary metabolic pathway operons that distinguish these species. Differences include a region of more than 80 kilobases (kb) that comprises a cluster of polyketide synthase genes and a second operon of 38 kb encoding plipastatin synthase enzymes that are absent in the B. licheniformis genome. The availability of a completed genome sequence for B. licheniformis should facilitate the design and construction of improved industrial strains and allow for comparative genomics and evolutionary studies within this group of Bacillaceae.     

The complete genome sequence of Clostridium indolis DSM 755T

Clostridium indolis DSM 755^T is a bacterium commonly found in soils and the feces of birds and mammals. Despite its prevalence, little is known about the ecology or physiology of this species. However, close relatives, C. saccharolyticum and C. hathewayi, have demonstrated interesting metabolic potentials related to plant degradation and human health. The genome of C. indolis DSM 755^T reveals an abundance of genes in functional groups associated with the transport and utilization of carbohydrates, as well as citrate, lactate, and aromatics. Ecologically relevant gene clusters related to nitrogen fixation and a unique type of bacterial microcompartment, the CoAT BMC, are also detected. Our genome analysis suggests hypotheses to be tested in future culture based work to better understand the physiology of this poorly described species.     

地衣芽孢杆菌DSM13分泌蛋白信号肽分析

地衣芽孢杆菌是重要的工业微生物,对于其分泌途径及信号肽进行预测和分析,有助于改善影响蛋白分泌的关键因素,高效生产异源蛋白。本研究首次在全基因范围内,利用SignalPv3.0等方法识别了地衣芽孢杆菌DSM13中各种分泌蛋白的信号肽。DSM13信号肽类型包括分泌型Sec信号肽、双精氨酸Tat信号肽、脂蛋白信号肽、IV型纤毛结构信号肽及生物信息素信号肽。同时分析了分泌途径组成,信号肽长度,氨基酸组成,各分泌信号肽特征,与枯草芽孢杆菌的异同以及重要工业酶制剂的分泌途径。该研究对使DSM13成为更有效分泌表达外源蛋白表达系统,具有重要的理论指导意义。     

The complete genome sequence of Bacillus thuringiensis Al Hakam

Bacillus thuringiensis is an insect pathogen that is widely used as a biopesticide (E. Schnepf, N. Crickmore, J. Van Rie, D. Lereclus, J. Baum, J. Feitelson, D. R. Zeigler, and D. H. Dean, Microbiol. Mol. Biol. Rev. 62:775-806, 1998). Here we report the finished, annotated genome sequence of B. thuringiensis Al Hakam, which was collected in Iraq by the United Nations Special Commission (L. Radnedge, P. Agron, K. Hill, P. Jackson, L. Ticknor, P. Keim, and G. Andersen, Appl. Environ. Microbiol. 69:2755-2764, 2003).     

Production of the Novel Two-Peptide Lantibiotic Lichenicidin by Bacillus licheniformis DSM 13

Background

Lantibiotics are small microbial peptide antibiotics that are characterized by the presence of the thioether amino acids lanthionine and methyllanthionine. Lantibiotics possess structural genes which encode inactive prepeptides. During maturation, the prepeptide undergoes posttranslational modifications including the introduction of rare amino acids as lanthionine and methyllanthione as well as the proteolytic removal of the leader. The structural gene (lanA) as well as the other genes which are involved in lantibiotic modification (lanM, lanB, lanC, lanP), regulation (lanR, lanK), export (lanT(P)) and immunity (lanEFG) are organized in biosynthetic gene clusters.

Methodology/Principal Findings

Sequence comparisons in the NCBI database showed that Bacillus licheniformis DSM 13 harbours a putative lantibiotic gene cluster which comprises two structural genes (licA1, licA2) and two modification enzymes (licM1, licM2) in addition to 10 ORFs that show sequence similarities to proteins involved in lantibiotic production. A heat labile antimicrobial activity was detected in the culture supernatant and a heat stabile activity was present in the isopropanol cell wash extract of this strain. In agar well diffusion assays both fractions exhibited slightly different activity spectra against Gram-positive bacteria. In order to demonstrate the connection between the lantibiotic gene cluster and one of the antibacterial activities, two Bacillus licheniformis DSM 13 mutant strains harbouring insertions in the structural genes of the modification enzymes licM1 and licM2 were constructed. These strains were characterized by a loss of activity in the isopropanol extract and substractive MALDI-TOF predicted masses of 3020.6 Da and 3250.6 Da for the active peptides.

Conclusions/Significance

In conclusion, B. licheniformis DSM 13 produces an antimicrobial substance that represents the two-peptide lantibiotic lichenicidin and that shows activity against a wide range of Gram-positive bacteria including methicillin resistant Staphylococcus aureus strains.     

The complete nucleotide sequence of the Bacillus licheniformis NM105 S-layer-encoding gene

A protein present on the cell surface of Bacillus licheniformis (Bl) NM105 was identified as an S-layer (OlpA in this paper), a protein present on many bacterial cell surfaces. Purification, SDS-PAGE and isoelectrofocusing showed one 94-kDa, slightly acidic (pI 6.5) protein band (defined as OlpA). The pure protein OlpA, has a tetragonal symmetry of its morphological subunits. Following Edman degradation, three 17-mer oligodeoxyribonucleotide (oligo) probes corresponding to the N-terminal sequence of OlpA were synthesized and used for gene cloning. The nucleotide (nt) sequence of the cloned gene (olpA) showed an ORF and encoded an 874 amino acid (aa) protein. In the promoter region of olpA, there appear to be -10 and -35 ∂^A-binding sites, as well as -10 and -35 regions specific for∂^H. The existence of these two potential promoters suggests that OlpA would be produced during both the vegetative and sporulating stages of growth. The ribosome-binding site (RBS) sequence perfectly matched its consensus sequence, suggesting a high efficiency of translation of olpA. A typical 29-aa leader peptide, characteristic of secretory proteins in Bacilli, is present in the OlpA pre-protein sequence. In olpA, there are two stem-loop structures in tandem, downstream from the stop codon. These stem-loops are probably involved in prolonged olpA expression, by extending the half life of the mRNA.     

Detailed modes of action and biochemical characterization of endo-arabinanase from Bacillus licheniformis DSM13

An endo-arabinanase (BLABNase) gene from Bacillus licheniformis DSM13 was cloned and expressed in Escherichia coli, and the biochemical properties of its encoded enzyme were characterized. The BLABNase gene consists of a single open reading frame of 987 nucleotides that encodes 328 amino acids with a predicted molecular mass of about 36 kDa. BLABNase exhibited the highest activity against debranched α-(1,5)-arabinan in 50 mM sodium acetate buffer (pH 6.0) at 55°C. Enzymatic characterization revealed that BLABNase hydrolyzes debranched or linear arabinans with a much higher activity than branched arabinan from sugar beet. Enzymatic hydrolysis pattern analyses demonstrated BLABNase to be a typical endo-(1,5)-α-s-arabinanase (EC 3.2.1.99) that randomly cleaves the internal α-(1,5)-linked L-arabinofuranosyl residues of a branchless arabinan backbone to release arabinotriose mainly, although a small amount of arabino-oligosaccharide intermediates is also liberated. Our results indicated that BLABNase acts preferentially along with the oligosaccharides longer than arabinopentaose, thus enabling the enzymatic production of various arabino-oligosaccharides.     

The complete genome sequence of Bacillus anthracis Ames "Ancestor"

The pathogenic bacterium Bacillus anthracis has become the subject of intense study as a result of its use in a bioterrorism attack in the United States in September and October 2001. Previous studies suggested that B. anthracis Ames Ancestor, the original Ames fully virulent plasmid-containing isolate, was the ideal reference. This study describes the complete genome sequence of that original isolate, derived from a sample kept in cold storage since 1981.     

Genetic relationship among Bacillus licheniformis rolling-circle-replicating plasmids and complete nucleotide sequence of pBL63.1, an atypical replicon

The degree of biodiversity among Bacillus licheniformis plasmids and their relation to other Bacillus subtilis group plasmids has been evaluated. To attain this goal we surveyed the diversity and linkage of replication modules in a collection of 21 naturally occurring plasmids of B. licheniformis strains, isolated from different geographical areas. On the basis of rep gene sequence analysis it was possible to group the B. licheniformis plasmids rep genes in two main cluster. Comparison with known rep genes from Bacillus rolling-circle-replicating (RCR) plasmids revealed the presence in B. licheniformis plasmids of replication genes with a DNA sequence peculiar to B. licheniformis species together with rep genes with a very high sequence similarity to B. subtilis plasmids. Furthermore, the molecular organization of an atypical replicon, pBL63.1, was shown. This plasmid did not display any significant similarity with known Bacillus RCR plasmids. The complete nucleotide sequence evidenced a replication module with an unexpected similarity with Rep proteins from RCR plasmids of bacterial species phylogenetically distantly related to Bacillus. pBL63.1 represents an exception to the low-level diversity hypothesis among Bacillus RC replicons.     

Enzymatic synthesis of apigenin glucosides by glucosyltransferase (YjiC) from Bacillus licheniformis DSM 13

Apigenin, a member of the flavone subclass of flavonoids, has long been considered to have various biological activities. Its glucosides, in particular, have been reported to have higher water solubility, increased chemical stability, and enhanced biological activities. Here, the synthesis of apigenin glucosides by the in vitro glucosylation reaction was successfully performed using a UDP-glucosyltransferase YjiC, from Bacillus licheniformis DSM 13. The glucosylation has been confirmed at the phenolic groups of C-4′ and C-7 positions ensuing apigenin 4′-O-glucoside, apigenin 7-O-glucoside and apigenin 4′,7-O-diglucoside as the products leaving the C-5 position unglucosylated. The position of glucosylation and the chemical structures of glucosides were elucidated by liquid chromatography/mass spectroscopy and nuclear magnetic resonance spectroscopy. The parameters such as pH, UDP glucose concentration and time of incubation were also analyzed during this study.     

Genome-Based Identification of Active Prophage Regions by Next Generation Sequencing in Bacillus licheniformis DSM13

Prophages are viruses, which have integrated their genomes into the genome of a bacterial host. The status of the prophage genome can vary from fully intact with the potential to form infective particles to a remnant state where only a few phage genes persist. Prophages have impact on the properties of their host and are therefore of great interest for genomic research and strain design. Here we present a genome- and next generation sequencing (NGS)-based approach for identification and activity evaluation of prophage regions. Seven prophage or prophage-like regions were identified in the genome of Bacillus licheniformis DSM13. Six of these regions show similarity to members of the Siphoviridae phage family. The remaining region encodes the B. licheniformis orthologue of the PBSX prophage from Bacillus subtilis. Analysis of isolated phage particles (induced by mitomycin C) from the wild-type strain and prophage deletion mutant strains revealed activity of the prophage regions BLi_Pp2 (PBSX-like), BLi_Pp3 and BLi_Pp6. In contrast to BLi_Pp2 and BLi_Pp3, neither phage DNA nor phage particles of BLi_Pp6 could be visualized. However, the ability of prophage BLi_Pp6 to generate particles could be confirmed by sequencing of particle-protected DNA mapping to prophage locus BLi_Pp6. The introduced NGS-based approach allows the investigation of prophage regions and their ability to form particles. Our results show that this approach increases the sensitivity of prophage activity analysis and can complement more conventional approaches such as transmission electron microscopy (TEM).     

Enrichment of the rebaudioside A concentration in Stevia rebaudiana extract with cyclodextrin glycosyltransferase from Bacillus licheniformis DSM 13

Stevia rebaudiana is a sweet herbaceous perennial plant, which is frequently used in the preparation of plant-based sweeteners. The demand for such sweeteners continues to increase due to purposeful nutrition and modern-day metabolic syndromes. More than 20 types of steviol glycosides provide a sweet taste, which are more than 300 times sweeter than sucrose. They are formed of two main components, namely stevioside and rebaudioside A. Only a handful of studies have dealt with Stevia rebaudiana leaf extracts, the conversion of pure stevioside into the preferred rebaudioside A is more common. The aim of this study was to enrich the rebaudioside A content of Stevia rebaudiana leaf extract using enzymatic bioconversion by applying fermented cyclodextrin glycosyltransferase from Bacillus licheniformis DSM13. Two differently processed plant materials, namely dried and lyophilized Stevia rebaudiana plants, were extracted and compared. Following the bioconversion, the rebaudioside A content was on average doubled. The maximum increase was fivefold with a 70–80% conversion of the stevioside.     

Chitinase from Bacillus licheniformis DSM13: expression in Lactobacillus plantarum WCFS1 and biochemical characterisation

The gene chi, coding for a GH18 chitinase from the Gram-positive bacterium Bacillus licheniformis DSM13 (ATCC 14580), was cloned into the inducible lactobacillal expression vectors pSIP403 and pSIP409, derived from the sakacin-P operon of Lactobacillus sakei, and expressed in the host strain Lactobacillus plantarum WCFS1. Both the complete chi gene including the original bacillal signal sequence as well as the mature chi gene were compared, however, no extracellular chitinase activity was detected with any of the constructs. The chitinase gene was expressed intracellularly as an active enzyme with these different systems, at levels of approximately 5mg of recombinant protein per litre of cultivation medium. Results obtained for the two different expression vectors that only differ in the promoter sequence were well comparable. To further verify the suitability of this expression system, recombinant, His-tagged chitinase Chi was purified from cell extracts of L. plantarum and characterised. The monomeric 65-kDa enzyme can degrade both chitin and chitosan, and shows properties that are very similar to those reported for the native chitinase purified from other B. licheniformis isolates. It shows good thermostability (half lives of stability of 20 and 8.4 days at 37 and 50°C, respectively), and good stability in the pH range of 5-10. The results presented lead the way to overproduction of chitinase in a food-grade system, which is of interest for the food and feed industry.     

Efficiency improvement of peptide identification for an organism without complete genome sequence, using expressed sequence tag database and tandem mass spectral data

We compared peptide identification by database (DB) search methods with de novo sequencing results for proteomics study in an organism without genome sequence information. When the former was done by searching the Expressed Sequence Tag (EST) DB of the sample organism or the NCBI nonredundant (nr) protein DB of green plants using either the MASCOT or SEQUEST software program, it was confirmed that the former is as accurate as the latter. Peptides identified from EST DB were twice as many as those from the nr protein DB, in spite of the fact that the EST DB has less data (26 222 EST) than the NCBI nr protein DB (224 238). This study demonstrates that EST DB with tandem mass spectra can be used reliably for high-throughput proteomics studies in an organism without genome information.     

Complete genome sequence of the industrial strain Bacillus megaterium WSH-002

Bacillus megaterium, an industrial strain, has been widely used in protein production and the vitamin C industry. Here we reported a finished, annotated, and compared 4.14-Mbp high-quality genome sequence of B. megaterium WSH-002, which is the companion strain for Ketogulonicigenium vulgare in the vitamin C industry and is stocked in our laboratory.     

Genome sequence of Bacillus licheniformis WX-02

Bacillus licheniformis is an important bacterium that has been used extensively for large-scale industrial production of exoenzymes and peptide antibiotics. B. licheniformis WX-02 produces poly-gamma-glutamate increasingly when fermented under stress conditions. Here its genome sequence (4,270,104 bp, with G+C content of 46.06%), which comprises a circular chromosome, is announced.