Bifidobacterium asteroides PRL2011 genome analysis reveals clues for colonization of the insect gut

Abstract

Bifidobacteria are known as anaerobic/microaerophilic and fermentative microorganisms, which commonly inhabit the gastrointestinal tract of various animals and insects. Analysis of the 2,167,301 bp genome of Bifidobacterium asteroides PRL2011, a strain isolated from the hindgut of Apis mellifera var. ligustica, commonly known as the honey bee, revealed its predicted capability for respiratory metabolism. Conservation of the latter gene clusters in various B. asteroides strains enforces the notion that respiration is a common metabolic feature of this ancient bifidobacterial species, which has been lost in currently known mammal-derived Bifidobacterium species. In fact, phylogenomic based analyses suggested an ancient origin of B. asteroides and indicates it as an ancestor of the genus Bifidobacterium. Furthermore, the B. asteroides PRL2011 genome encodes various enzymes for coping with toxic products that arise as a result of oxygen-mediated respiration.

Figure 1. Comparative genomic analysis of B. asteroides PRL2011 with other fully sequenced bifidobacterial genomes, as well as Gardnella vaginalis.

Panel a displays a Venn diagram of homologs shared between sequenced bifidobacterial-Gardnella genomes. Panel b shows the percentage of amino acid identity of the top-scoring self-matches for protein-coding genes in the analysed bacteria using the predicted proteome of B. asteroides PRL2011 as a reference. For each bacterium, the deduced protein-coding regions for each gene were compared with those derived from the B. asteroides PRL2011 genome. Panel c depicts a phylogenetic supertree based on the sequences of Bifidobacterium-Gardnella core proteins. Panel d indicates the generated phylogenetic tree based on 16S rRNA gene sequences from the same set of bacteria. Three other members of the Actinobacteria phylum, N. farcinia, T. whipplei and L. xyli, were also included in the analyses depicted in panels c and d, while the trees were rooted using L. salivarius as outgroup.

Figure 2. Oxygen consumption of bifidobacteria.

Panel a represents the oxygen uptake of different bifidobacterial species. Bifidobacteria were grown to mid-log phase in the absence of oxygen and placed in an oxygraph chamber. Lactococcus lactis subsp. lactis IL1403 was used as positive control. Panel b, shows the oxygen utilization of different B. asteroides cultures grown in the presence of 6.54–6.60 ppm of oxygen to mid-log phase in MRS plus succinate 1% as unique carbon source and without cysteine (curve 1), in MRS plus glucose 2% and cysteine (curve 2), in MRS plus citrate 1% as unique carbon source and without cysteine (curve 3), in MRS plus glucose 2% without cysteine (curve 4) in MRS with glucose 2% without cysteine and hemin 0.5 µg/ml (curve 5), and in MRS with glucose 2% without cysteine and protoporphyrin 10 ug/ml (curve 6).

Figure 3. The genes and encoded products of B. asteroides PRL2011 predicted to be involved in respiration and oxygen damage.

Panel a represents a circular genome atlas of B. asteroides PRL2011 (circle 1) with mapped orthologs (defined as reciprocal best FastA hits with more than 30% identity over at least 80% of both protein lengths) in seven publicly available Bifidobacterium genomes. From the outer circle, circle (2) shows B. breve UCC2003, circle (3) B. animalis subsp. lactis DSM 10140, circle (4) B. longum subsp. infantis ATCC 15697, circle (5) B. dentium Bd1, circle (6) B. longum NCC2705, circle (7) B. bifidum PRL2010, circle (8) B. adolescentis ATCC 15703. Circle(9) illustrates B. asteroides PRL2011 G+C% deviation followed by circle (10) that highlights B. asteroides PRL2011 GC skew (G−C/G+C). Moreover, the outer insets indicate the main genetic loci encoding enzymes involved in respiratory metabolism, which are mapped on the circular genome atlas of B. asteroides PRL2011. Panel b shows a schematic representation of a cell and metabolic pathways for respiration. The different ORFs of B. asteroides PRL2011 encoding the presumed enzymes involved in the respiratory chain are indicated.

Figure 4. Genomic diversity in the B. asteroides phylogenetic group with reference to the B. asteroides strain PRL2011 genome.

Panel a shows the comparative genomic hybridization data obtained using different members of the B. asteroides species. Each horizontal row corresponds to a probe on the array, and genes are ordered vertically according to their position on the PRL2011 genome. The columns represent the analysed strains, and strains are indicated by their strain codes. The colour code corresponding to the presence/absence is given at the top right of the figure: the gradient goes from black to yellow to indicate the presence, divergence or absence of a gene sequence. The predicted function of particular genes is shown on the right-hand margin. Black typed descriptions relate to most significant DNA regions that are absent in the investigated strains. Red typed descriptions represent DNA regions that encode enzymes predicted to be involved in respiration in PRL2011. Panel b details the presence (black) or absence (yellow) of key genes predicted to be involved in respiration within the B. asteroides phylogenetic group as well as in other genome sequenced bifidobacteria based on genomic data.

Figure 5. Identification of B. asteroides PRL2011 genes differentially expressed during growth in aerobic and anaerobic conditions.

Panel a displays the global transcription profiling of PRL2011 cells under different growth conditions. Panel b shows the whole genome transcriptome-based clustering data analysis. Panel c represents a heat-map indicating the change in transcription levels of putative respiratory chain-encoding genes upon cultivation of PRL2011 cells under different cultivation conditions. Lane 1, transcriptome of PRL2011 cells cultivated in the absence of oxygen and protoporphyrin; lane 2, transcriptome of PRL2011 cells in the presence of oxygen and protoporphyrin; lane 3, transcriptome of PRL2011 cells in the absence of oxygen and hemin; lane 4, transcriptome of PRL2011 cells in the presence of oxygen and hemin; lane 5, transcriptome of PRL2011 cells in the absence of oxygen and glucuronic acid; lane 6, transcriptome of PRL2011 cells in the presence of oxygen and glucuronic acid; lane 7, transcriptome of PRL2011 cells in the presence of iron chloride (3 mM) and under anaerobic conditions; lane 8, transcriptome of PRL2011 cells in the presence of iron chloride (3 mM) and under aerobic conditions. Each row represents a separate transcript and each column represents a separate sample. Colour legend is on the bottom of the figure. Green indicates DNA regions which are actively transcribed, while black represents DNA regions that exhibit no or very low transcriptional activity. In panel a, the arrows indicate the change in the transcription of PRL2011 genes encoding for putative respiratory chain.