Whole-genome sequence analysis of Bombella intestini LMG 28161T, a novel acetic acid bacterium isolated from the crop of a red-tailed bumble bee, Bombus lapidarius

The whole-genome sequence of Bombella intestini LMG 28161(T), an endosymbiotic acetic acid bacterium (AAB) occurring in bumble bees, was determined to investigate the molecular mechanisms underlying its metabolic capabilities. The draft genome sequence of B. intestini LMG 28161(T) was 2.02 Mb. Metabolic carbohydrate pathways were in agreement with the metabolite analyses of fermentation experiments and revealed its oxidative capacity towards sucrose, D-glucose, D-fructose and D-mannitol, but not ethanol and glycerol. The results of the fermentation experiments also demonstrated that the lack of effective aeration in small-scale carbohydrate consumption experiments may be responsible for the lack of reproducibility of such results in taxonomic studies of AAB. Finally, compared to the genome sequences of its nearest phylogenetic neighbor and of three other insect associated AAB strains, the B. intestini LMG 28161(T) genome lost 69 orthologs and included 89 unique genes. Although many of the latter were hypothetical they also included several type IV secretion system proteins, amino acid transporter/permeases and membrane proteins which might play a role in the interaction with the bumble bee host

Phylogenetic Analysis of a Spontaneous Cocoa Bean Fermentation Metagenome Reveals New Insights into Its Bacterial and Fungal Community Diversity

This is the first report on the phylogenetic analysis of the community diversity of a single spontaneous cocoa bean box fermentation sample through a metagenomic approach involving 454 pyrosequencing. Several sequence-based and composition-based taxonomic profiling tools were used and evaluated to avoid software-dependent results and their outcome was validated by comparison with previously obtained culture-dependent and culture-independent data. Overall, this approach revealed a wider bacterial (mainly γ-Proteobacteria) and fungal diversity than previously found. Further, the use of a combination of different classification methods, in a software-independent way, helped to understand the actual composition of the microbial ecosystem under study. In addition, bacteriophage-related sequences were found. The bacterial diversity depended partially on the methods used, as composition-based methods predicted a wider diversity than sequence-based methods, and as classification methods based solely on phylogenetic marker genes predicted a more restricted diversity compared with methods that took all reads into account. The metagenomic sequencing analysis identified Hanseniaspora uvarum, Hanseniaspora opuntiae, Saccharomyces cerevisiae, Lactobacillus fermentum, and Acetobacter pasteurianus as the prevailing species. Also, the presence of occasional members of the cocoa bean fermentation process was revealed (such as Erwinia tasmaniensis, Lactobacillus brevis, Lactobacillus casei, Lactobacillus rhamnosus, Lactococcus lactis, Leuconostoc mesenteroides, and Oenococcus oeni). Furthermore, the sequence reads associated with viral communities were of a restricted diversity, dominated by Myoviridae and Siphoviridae, and reflecting Lactobacillus as the dominant host. To conclude, an accurate overview of all members of a cocoa bean fermentation process sample was revealed, indicating the superiority of metagenomic sequencing over previously used techniques

Comparison of different community composition analysis methods on rank species.

*<p>Detected at 30 h.</p>**<p>No analysis performed at 30 h.</p>***<p>Only phylogenetic marker gene(s) sequences available in databases.</p><p>Species are only considered as present in the ecosystem if they could be detected by all five taxonomic profiling tools. The relative species abundances, predicted by the different classification tools, are expressed as a range that represent the lowest and highest values obtained. For the other methods, the presence of a species is denoted by “X”. <i>A.</i>: <i>Acetobacter</i>; <i>E.</i>: <i>Escherichia</i>; <i>En.</i>: <i>Enterobacter</i>; <i>Er.</i>: <i>Erwinia</i>; <i>G.</i>: <i>Gluconobacter</i>; <i>Ga.</i>: <i>Gluconacetobacter</i>; <i>K.</i>: <i>Klebsiella</i>; <i>L.</i>: <i>Lactobacillus</i>; <i>Leuc.</i>: <i>Leuconostoc</i>; <i>Lc.</i>: <i>Lactococcus</i>; <i>O.</i>: <i>Oenococcus</i>; <i>P.</i>: <i>Pediococcus</i>; <i>Pe.</i>: <i>Pectobacterium</i>; <i>S.</i>: <i>Salmonella</i>; <i>St.</i>: <i>Streptococcus</i>; <i>T.</i>: <i>Tatumella</i>.</p

Bacterial composition analysis on ranks class and order by using different taxonomic profiling tools.

<p>Classes within the orders <i>Bacilli</i> and <i>γ-Proteobacteria</i> are shown on the left y-axis; classes within the orders <i>Actinobacteria</i>, <i>α-Proteobacteria</i>, and others are shown on the right y-axis. ‘SmashCommunity RG’ depicts SmashCommunity reference genomes, ‘SmashCommunity 16S (1)’ depicts the SmashCommunity 16S rRNA gene-based method using the meta_rrna approach, ‘SmashCommunity 16S (2)’ depicts the SmashCommunity 16S rRNA gene-based method using the 16S rRNA gene sequence database approach.</p

Bacterial composition analysis on rank genus of the low-abundance members by using different taxonomic profiling tools.

<p>‘SmashCommunity RG’ depicts SmashCommunity reference genomes, ‘SmashCommunity 16S (1)’ depicts the SmashCommunity 16S rRNA gene-based method using the meta_rrna approach, ‘SmashCommunity 16S (2)’ depicts the SmashCommunity 16S rRNA gene-based method using the 16S rRNA gene sequence database approach.</p

Rarefaction analysis of the genera found with data sets A and B.

<p>The rarefaction curves represent an estimation of the number of genera associated with different sampling sizes. As the results of the two 16S rRNA gene-based methods of the SmashCommunity platform were similar, only one method (based on similarity with the 16S rRNA gene sequence database of the SmashCommunity platform) is shown. As the plateau phase of the SmashCommunity reference genomes platform and MEGAN was reached at 25,000 reads, the X-axis is limited to this number of reads.</p