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1. One-step genome engineering in bee gut bacterial symbionts

   https://doi.org/10.1128/mbio.01392-24  

   Lariviere, Patrick J; Ashraf, A_H_M Zuberi; Navarro-Escalante, Lucio; Leonard, Sean P; Miller, Laurel G; Moran, Nancy A; Barrick, Jeffrey E (September 2024, mBio)

   Kaltenpoth, Martin (Ed.)

   ABSTRACT Mechanistic understanding of interactions in many host-microbe systems, including the honey bee microbiome, is limited by a lack of easy-to-use genome engineering approaches. To this end, we demonstrate a one-step genome engineering approach for making gene deletions and insertions in the chromosomes of honey bee gut bacterial symbionts. Electroporation of linear or non-replicating plasmid DNA containing an antibiotic resistance cassette flanked by regions with homology to a symbiont genome reliably results in chromosomal integration. This lightweight approach does not require expressing any exogenous recombination machinery. The high concentrations of large DNAs with long homology regions needed to make the process efficient can be readily produced using modern DNA synthesis and assembly methods. We use this approach to knock out genes, including genes involved in biofilm formation, and insert fluorescent protein genes into the chromosome of the betaproteobacterial bee gut symbiontSnodgrassella alvi. We are also able to engineer the genomes of multiple strains ofS. alviand another species,Snodgrassella communis, which is found in the bumble bee gut microbiome. Finally, we use the same method to engineer the chromosome of another bee symbiont,Bartonella apis, which is an alphaproteobacterium. As expected, gene knockout inS. alviusing this approach isrecA-dependent, suggesting that this straightforward procedure can be applied to other microbes that lack convenient genome engineering methods. IMPORTANCEHoney bees are ecologically and economically important crop pollinators with bacterial gut symbionts that influence their health. Microbiome-based strategies for studying or improving bee health have utilized wild-type or plasmid-engineered bacteria. We demonstrate that a straightforward, single-step method can be used to insert cassettes and replace genes in the chromosomes of multiple bee gut bacteria. This method can be used for investigating the mechanisms of host-microbe interactions in the bee gut community and stably engineering symbionts that benefit pollinator health. 

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2. Comparative genomics of Lactobacillaceae from the gut of honey bees, Apis mellifera , from the Eastern United States

   https://doi.org/10.1093/g3journal/jkac286  

   Bradford, Emma L; Wax, Noah; Bueren, Emma K; Walke, Jenifer B; Fell, Richard; Belden, Lisa K; Haak, David C (November 2022, G3 Genes|Genomes|Genetics)

   Engel, P (Ed.)

   Abstract Lactobacillaceae are an important family of lactic acid bacteria that play key roles in the gut microbiome of many animal species. In the honey bee (Apis mellifera) gut microbiome, many species of Lactobacillaceae are found, and there is functionally important strain-level variation in the bacteria. In this study, we completed whole-genome sequencing of 3 unique Lactobacillaceae isolates collected from hives in Virginia, USA. Using 107 genomes of known bee-associated Lactobacillaceae and Limosilactobacillus reuteri as an outgroup, the phylogenetics of the 3 isolates was assessed, and these isolates were identified as novel strains of Apilactobacillus kunkeei, Lactobacillus kullabergensis, and Bombilactobacillus mellis. Genome rearrangements, conserved orthologous genes (COG) categories and potential prophage regions were identified across the 3 novel strains. The new A. kunkeei strain was enriched in genes related to replication, recombination and repair, the L. kullabergensis strain was enriched for carbohydrate transport, and the B. mellis strain was enriched in transcription or transcriptional regulation and in some genes with unknown functions. Prophage regions were identified in the A. kunkeei and L. kullabergensis isolates. These new bee-associated strains add to our growing knowledge of the honey bee gut microbiome, and to Lactobacillaceae genomics more broadly. 

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3. Global Composition of the Bacteriophage Community in Honey Bees

   https://doi.org/10.1128/msystems.01195-21  

   Busby, Taylor J.; Miller, Craig R.; Moran, Nancy A.; Van Leuven, James T. (April 2022, mSystems)

   Gambino, Michela (Ed.)

   ABSTRACT The microbial communities in animal digestive systems are critical for host development and health. They stimulate the immune system during development, synthesize important chemical compounds like hormones, aid in digestion, competitively exclude pathogens, etc. Compared to the bacterial and fungal components of the microbiome, we know little about the temporal and spatial dynamics of bacteriophage communities in animal digestive systems. Recently, the bacteriophages of the honey bee gut were characterized in two European bee populations. Most of the bacteriophages described in these two reports were novel, harbored many metabolic genes in their genomes, and had a community structure that suggests coevolution with their bacterial hosts. To describe the conservation of bacteriophages in bees and begin to understand their role in the bee microbiome, we sequenced the virome of Apis mellifera from Austin, TX, and compared bacteriophage compositions among three locations around the world. We found that most bacteriophages from Austin are novel, sharing no sequence similarity with anything in public repositories. However, many bacteriophages are shared among the three bee viromes, indicating specialization of bacteriophages in the bee gut. Our study, along with the two previous bee virome studies, shows that the bee gut bacteriophage community is simple compared to that of many animals, consisting of several hundred types of bacteriophages that primarily infect four of the dominant bacterial phylotypes in the bee gut. IMPORTANCE Viruses that infect bacteria (bacteriophages) are abundant in the microbial communities that live on and in plants and animals. However, our knowledge of the structure, dynamics, and function of these viral communities lags far behind our knowledge of their bacterial hosts. We sequenced the first bacteriophage community of honey bees from the United States and compared the U.S. honey bee bacteriophage community to those of samples from Europe. Our work is an important characterization of an economically critical insect species and shows how bacteriophage communities can contain highly conserved individuals and be highly variable in composition across a wide geographic range. 

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4. No apparent correlation between honey bee forager gut microbiota and honey production

   https://doi.org/10.7717/peerj.1329  

   Horton, Melissa A.; Oliver, Randy; Newton, Irene L. (January 2015, PeerJ)

   null (Ed.)

   One of the best indicators of colony health for the European honey bee ( Apis mellifera ) is its performance in the production of honey. Recent research into the microbial communities naturally populating the bee gut raise the question as to whether there is a correlation between microbial community structure and colony productivity. In this work, we used 16S rRNA amplicon sequencing to explore the microbial composition associated with forager bees from honey bee colonies producing large amounts of surplus honey (productive) and compared them to colonies producing less (unproductive). As supported by previous work, the honey bee microbiome was found to be dominated by three major phyla: the Proteobacteria, Bacilli and Actinobacteria, within which we found a total of 23 different bacterial genera, including known “core” honey bee microbiome members. Using discriminant function analysis and correlation-based network analysis, we identified highly abundant members (such as Frischella and Gilliamella ) as important in shaping the bacterial community; libraries from colonies with high quantities of these Orbaceae members were also likely to contain fewer Bifidobacteria and Lactobacillus species (such as Firm-4). However, co-culture assays, using isolates from these major clades, were unable to confirm any antagonistic interaction between Gilliamella and honey bee gut bacteria. Our results suggest that honey bee colony productivity is associated with increased bacterial diversity, although this mechanism behind this correlation has yet to be determined. Our results also suggest researchers should not base inferences of bacterial interactions solely on correlations found using sequencing. Instead, we suggest that depth of sequencing and library size can dramatically influence statistically significant results from sequence analysis of amplicons and should be cautiously interpreted. 

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5. Temperature dependence of parasitic infection and gut bacterial communities in bumble bees

   https://doi.org/10.1111/1462-2920.14805  

   Palmer‐Young, Evan_C; Ngor, Lyna; Burciaga_Nevarez, Rodrigo; Rothman, Jason_A; Raffel, Thomas_R; McFrederick, Quinn_S (November 2019, Environmental Microbiology)

   Summary High temperatures (e.g., fever) and gut microbiota can both influence host resistance to infection. However, effects of temperature‐driven changes in gut microbiota on resistance to parasites remain unexplored. We examined the temperature dependence of infection and gut bacterial communities in bumble bees infected with the trypanosomatid parasiteCrithidia bombi. Infection intensity decreased by over 80% between 21 and 37°C. Temperatures of peak infection were lower than predicted based on parasite growthin vitro, consistent with mismatches in thermal performance curves of hosts, parasites and gut symbionts. Gut bacterial community size and composition exhibited slight but significant, non‐linear, and taxon‐specific responses to temperature. Abundance of total gut bacteria and of Orbaceae, both negatively correlated with infection in previous studies, were positively correlated with infection here. Prevalence of the bee pathogen‐containing family Enterobacteriaceae declined with temperature, suggesting that high temperature may confer protection against diverse gut pathogens. Our results indicate that resistance to infection reflects not only the temperature dependence of host and parasite performance, but also temperature‐dependent activity of gut bacteria. The thermal ecology of gut parasite‐symbiont interactions may be broadly relevant to infectious disease, both in ectothermic organisms that inhabit changing climates, and in endotherms that exhibit fever‐based immunity. 

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