Extensive intra-phylotype diversity in lactobacilli and bifidobacteria from the honeybee gut

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 A. 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.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1476-6) contains supplementary material, which is available to authorized users.     

Evidence for a gram-positive, eubacterial root of the tree of life

Directed indels, insertions, and deletions within paralogous genes, have the potential to root the tree of life. Here we apply a newly developed rooting algorithm, top-down rooting, to indels found in informational and operational gene sets, introduce new computational tools for indel analyses, and present evidence (P < .01) that the root of the tree of life is not present in its traditional location, between the Eubacteria and the Archaebacteria. Using indels contained in the dihydroorotate dehydrogenase/uroporphyrinogen decarboxylase gene pair and in the ribosomal protein S12/beta prime subunit of the RNA polymerase gene pair, we exclude the root from within the clade consisting of the Firmicutes plus the Archaebacteria and their most recent common ancestor. These results, plus previous directed indel studies excluding the root from the eukaryotes, restrict the root to just four possible sites. One potential root is on the branch leading to the double-membrane prokaryotes, another is on the branch leading to the Actinobacteria, another is within the Actinobacteria, and the fourth is on the branch leading to the Firmicutes-Archaea clade. These results imply (1) that the cenancestral population was not hyperthermophilic, but moderate thermophily cannot be excluded for the root on the branch leading to the Firmicutes-Archaea clade, (2) that the cenancestral population was surrounded by ester lipids and a peptidoglycan layer, and (3) that parts of the mevalonate synthesis pathway were present in the population ancestral to the Bacilli and the Archaebacteria, including geranylgeranylglyceryl phosphate synthase, an enzyme thought to be partially responsible for the unique sn-1 stereochemistry of the archaeal glycerol phosphate backbone.     

A Comprehensive Evolutionary Scenario of Cell Division and Associated Processes in the Firmicutes

The cell cycle is a fundamental process that has been extensively studied in bacteria. However, many of its components and their interactions with machineries involved in other cellular processes are poorly understood. Furthermore, most knowledge relies on the study of a few models, but the real diversity of the cell division apparatus and its evolution are largely unknown. Here, we present a massive in-silico analysis of cell division and associated processes in around 1,000 genomes of the Firmicutes, a major bacterial phylum encompassing models (i.e. Bacillus subtilis, Streptococcus pneumoniae, and Staphylococcus aureus), as well as many important pathogens. We analyzed over 160 proteins by using an original approach combining phylogenetic reconciliation, phylogenetic profiles, and gene cluster survey. Our results reveal the presence of substantial differences among clades and pinpoints a number of evolutionary hotspots. In particular, the emergence of Bacilli coincides with an expansion of the gene repertoires involved in cell wall synthesis and remodeling. We also highlight major genomic rearrangements at the emergence of Streptococcaceae. We establish a functional network in Firmicutes that allows identifying new functional links inside one same process such as between FtsW (peptidoglycan polymerase) and a previously undescribed Penicilin-Binding Protein or between different processes, such as replication and cell wall synthesis. Finally, we identify new candidates involved in sporulation and cell wall synthesis. Our results provide a previously undescribed view on the diversity of the bacterial cell cycle, testable hypotheses for further experimental studies, and a methodological framework for the analysis of any other biological system.     

16S rDNA metabarcoding of the bacterial community associated with workers of Pheidole rugaticeps Emery (Hymenoptera: Formicidae)

Insect microbiota are receiving increasing attention from researchers, particularly with the continued advances in next generation sequencing (NGS) techniques. However, there is a paucity of data on the microbiota of ants that scavenge around human settlements. In this study, we characterized the bacterial communities of Pheidole rugaticeps Emery that were collected scavenging on other household insects using Illumina MiSeq high-throughput sequencing of the bacterial 16S ribosomal DNA gene. P. rugaticeps DNA was extracted from the insect samples using a HiYield? Genomic DNA isolation kit according to the manufacturer’s protocols and amplified using polymerase chain reaction (PCR). The PCR products were sequenced with the Illumina MiSeq platform according to the standard protocols to amplify the V3–V4 of the 16S rDNA gene. The results for the 16S rDNA genes were analysed using QIIME 2 Core ? 2020.6, and a 16S rDNA metabarcoding dataset was presented. A total of 46,651 reads were obtained from three genomic samples. A total of 368 amplicon sequence variants (ASV) comprising 165 genera were revealed and classified into 17 phyla. Proteobacteria (57.47%) and Firmicutes (33.14%) were the most abundant taxa, while Acinetobacter (37.10%) was the most abundant genus in all three sampling groups. Pathogenic bacteria species, such as Acinetobacter baumannii (15%) and Pseudomonas aeruginosa (2.92%), were identified from P. rugaticeps samples collected from a hospital environment. However, this study recommends more studies on the microbiota of Pheidole ants with different feeding habits and habitats to establish their core microbiome.     

A generation‐time effect on the rate of molecular evolution in bacteria

Molecular evolutionary rate varies significantly among species and a strict global molecular clock has been rejected across the tree of life. Generation time is one primary life‐history trait that influences the molecular evolutionary rate. Theory predicts that organisms with shorter generation times evolve faster because of the accumulation of more DNA replication errors per unit time. Although the generation‐time effect has been demonstrated consistently in plants and animals, the evidence of its existence in bacteria is lacking. The bacterial phylum Firmicutes offers an excellent system for testing generation‐time effect because some of its members can enter a dormant, nonreproductive endospore state in response to harsh environmental conditions. It follows that spore‐forming bacteria would—with their longer generation times—evolve more slowly than their nonspore‐forming relatives. It is therefore surprising that a previous study found no generation‐time effect in Firmicutes. Using a phylogenetic comparative approach and leveraging on a large number of Firmicutes genomes, we found sporulation significantly reduces the genome‐wide spontaneous DNA mutation rate and protein evolutionary rate. Contrary to the previous study, our results provide strong evidence that the evolutionary rates of bacteria, like those of plants and animals, are influenced by generation time.     

Toward defining the autoimmune microbiome for type 1 diabetes

Several studies have shown that gut bacteria have a role in diabetes in murine models. Specific bacteria have been correlated with the onset of diabetes in a rat model. However, it is unknown whether human intestinal microbes have a role in the development of autoimmunity that often leads to type 1 diabetes (T1D), an autoimmune disorder in which insulin-secreting pancreatic islet cells are destroyed. High-throughput, culture-independent approaches identified bacteria that correlate with the development of T1D-associated autoimmunity in young children who are at high genetic risk for this disorder. The level of bacterial diversity diminishes overtime in these autoimmune subjects relative to that of age-matched, genotype-matched, nonautoimmune individuals. A single species, Bacteroides ovatus, comprised nearly 24% of the total increase in the phylum Bacteroidetes in cases compared with controls. Conversely, another species in controls, represented by the human firmicute strain CO19, represented nearly 20% of the increase in Firmicutes compared with cases overtime. Three lines of evidence are presented that support the notion that, as healthy infants approach the toddler stage, their microbiomes become healthier and more stable, whereas, children who are destined for autoimmunity develop a microbiome that is less diverse and stable. Hence, the autoimmune microbiome for T1D may be distinctly different from that found in healthy children. These data also suggest bacterial markers for the early diagnosis of T1D. In addition, bacteria that negatively correlated with the autoimmune state may prove to be useful in the prevention of autoimmunity development in high-risk children.     

Transposons Tn916 and Tn925 can transfer from Enterococcus faecalis to Leuconostoc oenos

Abstract The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oenos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Lactococcus lactis LM2301. Further analysis of L. lactis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no efficient conjugal or transformation systems previously described for this microorganism.     

Changes in assembly processes in soil bacterial communities following a wildfire disturbance

Although recent work has shown that both deterministic and stochastic processes are important in structuring microbial communities, the factors that affect the relative contributions of niche and neutral processes are poorly understood. The macrobiological literature indicates that ecological disturbances can influence assembly processes. Thus, we sampled bacterial communities at 4 and 16 weeks following a wildfire and used null deviation analysis to examine the role that time since disturbance has in community assembly. Fire dramatically altered bacterial community structure and diversity as well as soil chemistry for both time-points. Community structure shifted between 4 and 16 weeks for both burned and unburned communities. Community assembly in burned sites 4 weeks after fire was significantly more stochastic than in unburned sites. After 16 weeks, however, burned communities were significantly less stochastic than unburned communities. Thus, we propose a three-phase model featuring shifts in the relative importance of niche and neutral processes as a function of time since disturbance. Because neutral processes are characterized by a decoupling between environmental parameters and community structure, we hypothesize that a better understanding of community assembly may be important in determining where and when detailed studies of community composition are valuable for predicting ecosystem function.     

精神分裂症患者肠道菌群特征与认知损害的关系

观察精神分裂症患者肠道菌群特征,并分析其与认知损害的关系。

选择2020年3月至2022年3月我院收治的175例精神分裂症患者作为研究对象。所有患者入院时均接受轻度认知损害筛查量表（sMCI）评估,根据认知损害发生情况,分为认知损害组和非认知损害组。统计两组患者一般资料和肠道菌群特征,分析精神分裂症患者肠道菌群特征与认知损害的关系。

175例精神分裂症患者中出现认知损害91例（52.00%）,未出现认知损害84例（48.00%）。认知损害组患者sMCI评分低于非认知损害组（P＜0.05）。两组患者肠道菌群α多样性比较差异无统计学意义（均P＞0.05）。两组对象肠道菌群的特征性物种在门水平为拟杆菌门（LDA = 2.54）,厚壁菌门（LDA = 4.25）;在纲水平为拟杆菌纲（LDA = 3.57）,梭菌纲（LDA = 4.31）;在科水平为产碱杆菌科（LDA = 4.23）和梭菌科（LDA = 2.76）;在属水平为萨特菌属（LDA = 3.18）。认知损害组患者肠道拟杆菌门丰度高于非认知损害组,厚壁菌门丰度低于非认知损害组;拟杆菌纲丰度高于非认知损害组,梭菌纲丰度低于非认知损害组;产碱杆菌科丰度高于非认知损害组,梭菌科丰度低于非认知损害组;萨特菌属丰度高于非认知损害组（均P＜0.05）。Logistics回归分析显示,高丰度的拟杆菌门、拟杆菌纲、产碱杆菌科、萨特菌属是精神分裂症患者发生认知损害的危险因素（OR＞1,均P＜0.05）;高丰度的厚壁菌门、梭菌纲是精神分裂症患者发生认知损害的保护因素（OR＜1,均P＜0.05）。

精神分裂症患者肠道菌群结构显著失衡,且部分菌群丰度与认知损害密切相关。