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Evolution of symbiotic lineages and the origin of new traits
Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Molecular Evolution.ORCID iD: F-4815-2016
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis focuses on the genomic study of symbionts of two different groups of hymenopterans: bees and ants. Both groups of insects have major ecological impact, and investigating their microbiomes increases our understanding of their health, diversity and evolution.

The study of the bee gut microbiome, including members of Lactobacillus and Bifidobacterium, revealed genomic processes related to the adaptation to the gut environment, such as the expansion of genes for carbohydrate metabolism and the acquisition of genes for interaction with the host. A broader genomic study of these genera demonstrated that some lineages evolve under strong and opposite substitution biases, leading to extreme GC content values. A comparison of codon usage patterns in these groups revealed ongoing shifts of optimal codons.

In a separate study we analysed the genomes of several strains of Lactobacillus kunkeei, which inhabits the honey stomach of bees but is not found in their gut. We observed signatures of genome reduction and suggested candidate genes for host-interaction processes. We discovered a novel type of genome architecture where genes for metabolic functions are located in one half of the genome, whereas genes for information processes are located in the other half. This genome organization was also found in other Lactobacillus species, indicating that it was an ancestral feature that has since been retained. We suggest mechanisms and selective forces that may cause the observed organization, and describe processes leading to its loss in several lineages independently.

We also studied the genome of a species of Rhizobiales bacteria found in ants. We discuss its metabolic capabilities and suggest scenarios for how it may affect the ants’ lifestyle. This genome contained a region with homology to the Bartonella gene transfer agent (GTA), which is a domesticated bacteriophage used to transfer bacterial DNA between cells. We propose that its unique behaviour as a specialist GTA, preferentially transferring host-interaction factors, originated from a generalist GTA that transferred random segments of chromosomal DNA.

These bioinformatic analyses of previously uncharacterized bacterial lineages have increased our understanding of their physiology and evolution and provided answers to old and new questions in fundamental microbiology.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. , 96 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1415
Keyword [en]
symbiosis, host-association, Lactobacillus, Bifidobacterium, Rhizobiales, Bartonella, honeybees, ants, codon usage bias, genome architecture, genome organization, gene transfer agent, evolutionary genomics, comparative genomics
National Category
Evolutionary Biology Genetics Microbiology
Research subject
Biology with specialization in Molecular Evolution; Biology with specialization in Evolutionary Genetics; Biology with specialization in Microbiology
Identifiers
URN: urn:nbn:se:uu:diva-301939ISBN: 978-91-554-9672-2OAI: oai:DiVA.org:uu-301939DiVA: diva2:955801
Public defence
2016-10-14, B41, Biomedical Center (BMC), Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-09-22 Created: 2016-08-25 Last updated: 2016-09-22
List of papers
1. Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut
Open this publication in new window or tab >>Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut
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2015 (English)In: BMC Genomics, ISSN 1471-2164, E-ISSN 1471-2164, Vol. 16, 284Article in journal (Refereed) Published
Abstract [en]

Background: In the honeybee Apis mellifera, the bacterial gut community is consistently colonized by eight distinct phylotypes of bacteria. Managed bee colonies are of considerable economic interest and it is therefore important to elucidate the diversity and role of this microbiota in the honeybee. In this study, we have sequenced the genomes of eleven strains of lactobacilli and bifidobacteria isolated from the honey crop of the honeybee Apis mellifera. Results: Single gene phylogenies confirmed that the isolated strains represent the diversity of lactobacilli and bifidobacteria in the gut, as previously identified by 16S rRNA gene sequencing. Core genome phylogenies of the lactobacilli and bifidobacteria further indicated extensive divergence between strains classified as the same phylotype. Phylotype-specific protein families included unique surface proteins. Within phylotypes, we found a remarkably high level of gene content diversity. Carbohydrate metabolism and transport functions contributed up to 45% of the accessory genes, with some genomes having a higher content of genes encoding phosphotransferase systems for the uptake of carbohydrates than any previously sequenced genome. These genes were often located in highly variable genomic segments that also contained genes for enzymes involved in the degradation and modification of sugar residues. Strain-specific gene clusters for the biosynthesis of exopolysaccharides were identified in two phylotypes. The dynamics of these segments contrasted with low recombination frequencies and conserved gene order structures for the core genes. Hits for CRISPR spacers were almost exclusively found within phylotypes, suggesting that the phylotypes are associated with distinct phage populations. Conclusions: The honeybee gut microbiota has been described as consisting of a modest number of phylotypes; however, the genomes sequenced in the current study demonstrated a very high level of gene content diversity within all three described phylotypes of lactobacilli and bifidobacteria, particularly in terms of metabolic functions and surface structures, where many features were strain-specific. Together, these results indicate niche differentiation within phylotypes, suggesting that the honeybee gut microbiota is more complex than previously thought.

Keyword
Lactic acid bacteria, Lactobacillus spp, Firmicutes, Bifidobacteria, Comparative genomics, Phosphotransferase systems, Niche specialization
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-256855 (URN)10.1186/s12864-015-1476-6 (DOI)000355302300001 ()25880915 (PubMedID)
External cooperation:
Note

De två förstaförfattarna delar förstaförfattarskapet.

Available from: 2015-06-26 Created: 2015-06-26 Last updated: 2016-08-30Bibliographically approved
2. Functionally Structured Genomes in Lactobacillus kunkeei Colonizing the Honey Crop and Food Products of Honeybees and Stingless Bees
Open this publication in new window or tab >>Functionally Structured Genomes in Lactobacillus kunkeei Colonizing the Honey Crop and Food Products of Honeybees and Stingless Bees
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2015 (English)In: Genome Biology and Evolution, ISSN 1759-6653, Vol. 7, no 6, 1455-1473 p.Article in journal (Refereed) Published
Abstract [en]

Lactobacillus kunkeei is the most abundant bacterial species in the honey crop and food products of honeybees. The 16 S rRNA-genes of strains isolated from different bee species are nearly identical in sequence and therefore inadequate as markers for studies of coevolutionary patterns. Here, we have compared the 1.5Mb genomes of ten L. kunkeei strains isolated from all recognized Apis species and another two strains from Meliponini species. Agene flux analysis, including previously sequenced Lactobacillus species as outgroups, indicated the influence of reductive evolution. The genome architecture is unique in that vertically inherited core genes are located near the terminus of replication, whereas genes for secreted proteins and putative host-adaptive traits are located near the origin of replication. We suggest that these features have resulted from a genome-wide loss of genes, with integrations of novel genes mostly occurring in regions flanking the origin of replication. The phylogenetic analyses showed that the bacterial topology was incongruent with the host topology, and that strains of the same microcluster have recombined frequently across the host species barriers, arguing against codiversification. Multiple genotypes were recovered in the individual hosts and transfers of mobile elements could be demonstrated for strains isolated from the same host species. Unlike other bacteria with small genomes, short generation times and multiple rRNA operons suggest that L. kunkeei evolves under selection for rapid growth in its natural growth habitat. The results provide an extended framework for reductive genome evolution and functional genome organization in bacteria.

Keyword
genome organization, Lactobacillus kunkeei, honeybee, genome reduction, recombination
National Category
Biological Sciences
Identifiers
urn:nbn:se:uu:diva-261322 (URN)10.1093/gbe/evv079 (DOI)000358800100005 ()25953738 (PubMedID)
Available from: 2015-09-02 Created: 2015-09-01 Last updated: 2016-08-26Bibliographically approved
3. Functionally structured genome architectures in Lactobacillus – insights into their variability and evolution
Open this publication in new window or tab >>Functionally structured genome architectures in Lactobacillus – insights into their variability and evolution
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Bacterial genome architectures evolve in response to selective pressures on the interplay between replication and gene expression. Several genomes contain a higher fraction of genes coding for proteins involved in information processes near the origin of replication, which is thought to be due to selection for rapid growth. We recently described a novel type of genome architecture in Lactobacillus kunkeei (Tamarit, et al. 2015). In this genome, vertically inherited genes encoding proteins with roles in translation and replication have accumulated in the chromosomal half surrounding the terminus of replication, while species-specific genes, and genes encoding proteins with metabolic and transport functions have accumulated in the chromosomal half around the origin of replication. Here, we show that this pattern is present also in the closest relatives of L. kunkeei, and similar but not identical biased genome architectures are present in other groups within the Lactobacillaceae. Thus, the biased genome structure in L. kunkeei has emerged from an ancestral clustering of vertically inherited genes around the terminus of replication, while horizontally acquired genes have been inserted near the origin of replication. The genome bias has been lost independently in several groups due to insertions of mobile elements near the terminus of replication and/or major genome rearrangements. We propose chromosomal structuring in macrodomains in the Lactobacillaceae, and suggest that further exploration of its functional consequences and generality will provide valuable insights into the forces that shape genome organization in bacteria. 

Keyword
genome organization, replication axis
National Category
Evolutionary Biology
Research subject
Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-301781 (URN)
External cooperation:
Available from: 2016-08-25 Created: 2016-08-25 Last updated: 2016-08-31Bibliographically approved
4. Switches in Genomic GC Content Drive Shifts of Optimal Codons under Sustained Selection on Synonymous Sites
Open this publication in new window or tab >>Switches in Genomic GC Content Drive Shifts of Optimal Codons under Sustained Selection on Synonymous Sites
2016 (English)In: Genome Biology and Evolution, ISSN 1759-6653, E-ISSN 1759-6653Article in journal (Refereed) Published
Abstract [en]

The major codon preference model suggests that codons read by tRNAs in high concentrations are preferentially utilized in highly expressed genes. However, the identity of the optimal codons differs between species although the forces driving such changes are poorly understood. We suggest that these questions can be tackled by placing codon usage studies in a phylogenetic framework and that bacterial genomes with extreme nucleotide composition biases provide informative model systems. Switches in the background substitution biases from GC to AT have occurred in Gardnerella vaginalis (GC = 32%), and from AT to GC in Lactobacillus delbrueckii (GC=62%) and Lactobacillus fermentum (GC = 63%). We show that despite the large effects on codon usage patterns by these switches, all three species evolve under selection on synonymous sites. In G. vaginalis, the dramatic codon frequency changes coincide with shifts of optimal codons. In contrast, the optimal codons have not shifted in the two Lactobacillus genomes despite an increased fraction of GC-ending codons. We suggest that all three species are in different phases of an on-going shift of optimal codons, and attribute the difference to a stronger background substitution bias and/or longer time since the switch in G. vaginalis. We show that comparative and correlative methods for optimal codon identification yield conflicting results for genomes in flux and discuss possible reasons for the mispredictions. We conclude that switches in the direction of the background substitution biases can drive major shifts in codon preference patterns even under sustained selection on synonymous codon sites.

Keyword
Codon Usage, Lactobacillus, Bifidobacterium, GC content
National Category
Evolutionary Biology
Research subject
Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-300909 (URN)10.1093/gbe/evw201 (DOI)
External cooperation:
Available from: 2016-08-15 Created: 2016-08-15 Last updated: 2016-08-26
5. The genome of Rhizobiales bacteria in predatory ants indicates a role for urease in lifestyle switches
Open this publication in new window or tab >>The genome of Rhizobiales bacteria in predatory ants indicates a role for urease in lifestyle switches
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2016 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322Article in journal (Refereed) Submitted
Abstract [en]

Bacterial symbionts provide amino acids to herbivorous ants, but their role in carnivores is a puzzle. The most prevalent bacterial lineage in ants belongs to the order Rhizobiales. Sequence reads with similarity to Bartonella, a member of the Rhizobiales, were identified in the data collected in a genome project of the carnivorous ant Harpegnatos saltator. Here, we present an analysis of the closed 1.86 Mb genome of the Bartonella-like bacterium, here abbreviated Bhsal. A phylogenetic study showed that Bhsal diverged prior to the radiation of the Bartonella species. Uniquely present in the Bhsal genome is a gene for a giant protein of 6,177 amino acids with a repeated domain structure. We also identified genes for a multi- subunit urease protein complex, potentially involved in the hydrolysis of urea into ammonium. We hypothesize that the urease function protects Bhsal from the acidic environment of the ant gut. The urease genes are also present in Brucella, which has a fecal-oral transmission pathway, but they have been lost in Bartonella species, which use blood-borne transmission pathways. Taken together, the results suggest that the urease function has served an important role for transmission strategies and lifestyle changes in the host-associated members of the Rhizobiales. 

Keyword
symbiosis, genomics, Bartonella
National Category
Evolutionary Biology
Research subject
Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-301778 (URN)
External cooperation:
Available from: 2016-08-25 Created: 2016-08-25 Last updated: 2016-08-26
6. Origin and evolution of the Bartonella Gene Transfer Agent
Open this publication in new window or tab >>Origin and evolution of the Bartonella Gene Transfer Agent
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Gene transfer agents (GTAs) are bacteriophage particles that transfer bacterial DNA. Two GTAs have been identified in the Alphaproteobacteria: the RcGTA, which is widely distributed in a broad range of species; and the more recently evolved BaGTA, which is thought to have been important for the explosive radiation of the genus Bartonella. The BaGTA preferentially packages genes for host interaction factors amplified from an alternative, phage-derived origin of replication. Here, we show that the RcGTAs and the BaGTAs have non-overlapping phyletic distribution patterns in the Alphaproteobacteria. We identify BaGTA-like phage islands in Candidatus Tokpelaia hoelldoblerii, an early diverging lineage of Bartonella, as well as in three more distantly related alphaproteobacterial species. Moreover, we identify several BaGTA-like phage islands within the genus Bartonella, but unlike the BaGTA these are not conserved in either occurrence or genomic location. We thus hypothesize that the transfer of random DNA fragments with the aid of the GTA was an early innovation, which predated the amplification and targeted transfer of DNA segments flanking the phage-derived origin of replication. We propose a model for the gradual transformation of a prophage into a specialist GTA in a process driven by selection for transfer and recombination of host interaction factors within the bacterial population. 

Keyword
mobile elements, Phage domestication, GTA
National Category
Evolutionary Biology
Research subject
Biology with specialization in Molecular Evolution
Identifiers
urn:nbn:se:uu:diva-301779 (URN)
External cooperation:
Available from: 2016-08-25 Created: 2016-08-25 Last updated: 2016-08-31Bibliographically approved

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