Comparative Genomic Analysis of 45 Type Strains of the Genus Bifidobacterium: A Snapshot of Its Genetic Diversity and Evolution

Bifidobacteria are well known for their human health-promoting effects and are therefore widely applied in the food industry. Members of the Bifidobacterium genus were first identified from the human gastrointestinal tract and were then found to be widely distributed across various ecological niches. Although the genetic diversity of Bifidobacterium has been determined based on several marker genes or a few genomes, the global diversity and evolution scenario for the entire genus remain unresolved. The present study comparatively analyzed the genomes of 45 type strains. We built a robust genealogy for Bifidobacterium based on 402 core genes and defined its root according to the phylogeny of the tree of bacteria. Our results support that all human isolates are of younger lineages, and although species isolated from bees dominate the more ancient lineages, the bee was not necessarily the original host for bifidobacteria. Moreover, the species isolated from different hosts are enriched with specific gene sets, suggesting host-specific adaptation. Notably, bee-specific genes are strongly associated with respiratory metabolism and are potential in helping those bacteria adapt to the oxygen-rich gut environment in bees. This study provides a snapshot of the genetic diversity and evolution of Bifidobacterium, paving the way for future studies on the taxonomy and functional genomics of the genus.     

Genomic Encyclopedia of Bacteria and Archaea: Sequencing a Myriad of Type Strains

Microbes hold the key to life. They hold the secrets to our past (as the descendants of the earliest forms of life) and the prospects for our future (as we mine their genes for solutions to some of the planet''s most pressing problems, from global warming to antibiotic resistance). However, the piecemeal approach that has defined efforts to study microbial genetic diversity for over 20 years and in over 30,000 genome projects risks squandering that promise. These efforts have covered less than 20% of the diversity of the cultured archaeal and bacterial species, which represent just 15% of the overall known prokaryotic diversity. Here we call for the funding of a systematic effort to produce a comprehensive genomic catalog of all cultured Bacteria and Archaea by sequencing, where available, the type strain of each species with a validly published name (currently∼11,000). This effort will provide an unprecedented level of coverage of our planet''s genetic diversity, allow for the large-scale discovery of novel genes and functions, and lead to an improved understanding of microbial evolution and function in the environment.     

Genomic Encyclopedia of Type Strains,Phase I: The one thousand microbial genomes (KMG-I) project

The Genomic Encyclopedia of Bacteria and Archaea (GEBA) project was launched by the JGI in 2007 as a pilot project with the objective of sequencing 250 bacterial and archaeal genomes. The two major goals of that project were (a) to test the hypothesis that there are many benefits to the use the phylogenetic diversity of organisms in the tree of life as a primary criterion for generating their genome sequence and (b) to develop the necessary framework, technology and organization for large-scale sequencing of microbial isolate genomes. While the GEBA pilot project has not yet been entirely completed, both of the original goals have already been successfully accomplished, leading the way for the next phase of the project.Here we propose taking the GEBA project to the next level, by generating high quality draft genomes for 1,000 bacterial and archaeal strains. This represents a combined 16-fold increase in both scale and speed as compared to the GEBA pilot project (250 isolate genomes in 4+ years). We will follow a similar approach for organism selection and sequencing prioritization as was done for the GEBA pilot project (i.e. phylogenetic novelty, availability and growth of cultures of type strains and DNA extraction capability), focusing on type strains as this ensures reproducibility of our results and provides the strongest linkage between genome sequences and other knowledge about each strain. In turn, this project will constitute a pilot phase of a larger effort that will target the genome sequences of all available type strains of the Bacteria and Archaea.     

Genomic Evolution of 11 Type Strains within Family Planctomycetaceae

The species in family Planctomycetaceae are ideal groups for investigating the origin of eukaryotes. Their cells are divided by a lipidic intracytoplasmic membrane and they share a number of eukaryote-like molecular characteristics. However, their genomic structures, potential abilities, and evolutionary status are still unknown. In this study, we searched for common protein families and a core genome/pan genome based on 11 sequenced species in family Planctomycetaceae. Then, we constructed phylogenetic tree based on their 832 common protein families. We also annotated the 11 genomes using the Clusters of Orthologous Groups database. Moreover, we predicted and reconstructed their core/pan metabolic pathways using the KEGG (Kyoto Encyclopedia of Genes and Genomes) orthology system. Subsequently, we identified genomic islands (GIs) and structural variations (SVs) among the five complete genomes and we specifically investigated the integration of two Planctomycetaceae plasmids in all 11 genomes. The results indicate that Planctomycetaceae species share diverse genomic variations and unique genomic characteristics, as well as have huge potential for human applications.     

Characterization of Bifidobacterium Strains Using Box Primers

Twenty-five Bifidobacterium strains isolated from infants' faeces were identified by Rep-PCR. Using BOX-PCR, characteristic bands of Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis and Bifidobacterium adolescentis were found in 40 strains of bidfidobacteria. These bands were not found in lactobacilli. By computerized numerical analysis strains were grouped in two major clusters. Strains of B. bifidum fell into a well-differentiated cluster that joined the cluster of the remaining species at 0.771 of similarity. The predominant species among the isolated strains were Bifidobacterium bifidum, Bifidobacterium longum andBifidobacterium breve . In another set of experiments, DNA was extracted from bacteria harvested from fermented milks to which different concentrations of bifidobacteria had been added. In all cases characteristic bands in the agarose gel belonging to lactobacilli and streptococci were detected. Bifidobacterium was detected only when 10⁸CFU/ml were added to the fermented milks. On the basis of our results, we propose this methodology as another tool in the polyphasic taxonomy.     

Comparison of the Phospholipid Composition of Bifidobacterium and Lactobacillus Strains

Phospholipid composition of 10 Bifidobacterium strains of human intestinal origin and of 9 Lactobacillus strains was determined by quantitative two-dimensional thin-layer chromatography. Phospholipids of three Bifidobacterium strains from honey bees and of two strains from bovine rumen liquor were qualitatively investigated. Diphosphatidylglycerol and phosphatidylglycerol were present in strains of both genera. All Bifidobacterium strains contained as specific phospholipids a new polyglycerolphospholipid, compound 15, and its lyso derivatives, earlier detected in B. bifidum var. pennsylvanicus. Also, lyso compounds of diphosphatidylglycerol and alanyl phosphatidylglycerol were only present in this genus in variable amounts. Lysyl phosphatidylglycerol was the only ninhydrin-positive phospholipid in seven Lactobacillus strains. In L. delbrückii and L. helveticus it was absent and partially replaced by an unidentified ninhydrin-negative phospholipid. The differences in phospholipid composition between bifidobacteria and lactobacilli may be another argument to differentiate these two genera.     

Investigation of the Evolutionary Development of the Genus Bifidobacterium by Comparative Genomics

The Bifidobacterium genus currently encompasses 48 recognized taxa, which have been isolated from different ecosystems. However, the current phylogeny of bifidobacteria is hampered by the relative paucity of genotypic data. Here, we reassessed the taxonomy of this bacterial genus using genome-based approaches, which demonstrated that the previous taxonomic view of bifidobacteria contained several inconsistencies. In particular, high levels of genetic relatedness were shown to exist between particular Bifidobacterium taxa which would not justify their status as separate species. The results presented are here based on average nucleotide identity analysis involving the genome sequences for each type strain of the 48 bifidobacterial taxa, as well as phylogenetic comparative analysis of the predicted core genome of the Bifidobacterium genus. The results of this study demonstrate that the availability of complete genome sequences allows the reconstruction of a more robust bifidobacterial phylogeny than that obtained from a single gene-based sequence comparison, thus discouraging the assignment of a new or separate bifidobacterial taxon without such a genome-based validation.     

Genomic Comparison of Kingella kingae Strains

Kingella kingae is a betaproteobacterium from the order Neisseriales, and it is an agent of invasive infections in children. We sequenced the genome from the septic arthritis strain 11220434. It is composed of a 1,990,794-bp chromosome but no plasmid, and it contains 2,042 protein-coding genes and 52 RNA genes, including 3 rRNA genes.     

Carbohydrate Catabolism of Selected Strains in the Genus Agrobacterium

Radiorespirometric and enzyme analyses were used to reveal the glucose-catabolizing mechanisms functioning in single strains of seven presumed Agrobacterium species. The Entner-Doudoroff and pentose cycle pathways functioned in A. radiobacter, A. tumefaciens, A. rubi, and A. rhizogenes. Whereas both catabolic pathways were utilized to an almost equal degree in the A. radiobacter and A. tumefaciens strains, use of the Entner-Doudoroff pathway predominated in the A. rubi and A. rhizogenes strains. A stellulatum catabolized glucose almost solely through the Entner-Doudoroff pathway. In A. pseudotsugae and A. gypsophilae, glucose was metabolized mainly through the Emden-Meyerhof-Parnas pathway; the pentose phosphate pathway was also utilized.     

Highly Diverse Cyanobactins in Strains of the Genus Anabaena

Cyanobactins are small, cyclic peptides recently found in cyanobacteria. They are formed through proteolytic cleavage and posttranslational modification of short precursor proteins and exhibit antitumor, cytotoxic, or multi-drug-reversing activities. Using genome project data, bioinformatics, stable isotope labeling, and mass spectrometry, we discovered novel cyclic peptides, anacyclamides, in 27 Anabaena strains. The lengths of the anacylamides varied greatly, from 7 to 20 amino acids. Pronounced sequence variation was also detected, and only one amino acid, proline, was present in all anacyclamides. The anacyclamides identified included unmodified proteinogenic or prenylated amino acids. We identified an 11-kb gene cluster in the genome of Anabaena sp. 90, and heterologous expression in Escherichia coli confirmed that this cluster was responsible for anacyclamide production. The discovery of anacyclamides greatly increases the structural diversity of cyanobactins.Cyanobacteria are a prolific source of secondary metabolites with potential as drug leads or useful probes for cell biology studies (23). They include biomedically interesting compounds, such as the anticancer drug lead cryptophycin (15), and environmentally problematic hepatotoxic peptides, such as microcystins and nodularins produced by bloom-forming cyanobacteria (23). Many of these compounds contain nonproteinogenic amino acids and modified peptides and are produced by nonribosomal peptide synthesis (23, 26).The cyanobactins are a new group of cyclic peptides recently found in cyanobacteria (4). They are assembled through posttranslational proteolytic cleavage and head-to-tail macrocyclization of short precursor proteins. The cyanobactins are low-molecular-weight cyclic peptides that contain heterocyclized amino acids and can be prenylated or contain d-amino acids (3, 4). The cyanobactins that contain heterocyclized amino acids include patellamides, ulithiacyclamides, trichamide, tenuecyclamides, trunkamides, patellins, and microcyclamides and are synthesized in this manner (3, 4, 20, 24, 28). They possess antitumor, cytotoxic, and multi-drug-reversing activities and have potential as drug leads (4, 18, 20).Cyanobactins containing heterocyclized amino acids are found in a variety of cyanobacteria (4). A recent study demonstrated that the cyanobactin biosynthetic pathway is prevalent in planktonic bloom-forming cyanobacteria (14). However, the products of these gene clusters encoding new cyanobactins are unknown. Here we report discovery of a novel family of low-molecular-weight cyanobactins and show that these compounds are common in strains of the genus Anabaena. These anacyclamides exhibit pronounced length and sequence variation and contain unmodified or prenylated amino acids.     

链霉菌的rep-PCR基因指纹分析

对重复片段PCR(repPCR)基因指纹分析应用于链霉菌分子分型进行研究,结果表明repPCR基因指纹分析具有分辨率高、稳定、重现性好、简便易行等特点,在一定程度上与16S rDNA 序列比较结果相一致,是一种快速而有效的DNA指纹技术,能反映出链霉菌种和菌株水平的基因型、系统发育和分类学关系,可应用于种及以下水平的分类和快速鉴定,尤其适用于分析大量的菌株或分离株。     

Starch Gel Electrophoresis of Fructose-6-Phosphate Phosphoketolase in the Genus Bifidobacterium

Three groups of nomenspecies of the genus Bifidobacterium were distinguished by the different mobility of their fructose-6-phosphate phosphoketolase in starchgel electrophoresis; there is apparently a close relatedness between electrophoretic type of phosphoketolase and habitat.     

Biochemical Characterization of Ochratoxin A-Producing Strains of the Genus Penicillium

In order to explore the biochemical scope of ochratoxin A-producing penicillia, we screened 48 Penicillium verrucosum isolates for the production of secondary metabolites. Fungal metabolites were analyzed by high-pressure liquid or gas chromatography coupled to diode array detection or mass spectrometry. The following metabolites were identified: ochratoxins A and B, citrinin, verrucolones, verrucines, anacines, sclerotigenin, lumpidin, fumiquinazolines, alantrypinones, daldinin D, dipodazine, penigequinolines A and B, 2-pentanone, and 2-methyl-isoborneol. By use of average linking clustering based on binary (nonvolatile) metabolite data, the 48 isolates could be grouped into two large and clearly separated groups and a small outlying group of four non-ochratoxin-producing isolates. The largest group, containing 24 isolates, mainly originating from plant sources, included the type culture of P. verrucosum. These isolates produced ochratoxin A, verrucolones, citrinin, and verrucines and had a characteristic dark brown reverse color on yeast extract-sucrose agar medium. Almost all of a group of 20 isolates mainly originating from cheese and meat products had a pale cream reverse color on yeast extract-sucrose agar medium and produced ochratoxin A, verrucolones, anacines, and sclerotigenin. This group included the former type culture of P. nordicum. We also found that P. verrucosum isolates and three P. nordicum isolates incorporated phenylalanine into verrucine and lumpidin metabolites, a finding which could explain why those isolates produced relatively lower levels of ochratoxins than did most isolates of P. nordicum.