From the Winter 2021 issue of "Microcosm."

Microbiology Mysteries

Researchers are puzzled by the increased deaths of songbirds associated with a mysterious disease that is causing lethargy, neurological symptoms and crusty, oozing patches over the eyes of songbirds in the eastern United States. So far, tests have been more successful at eliminating potential sources of infection than at identifying the pathogen that is responsible. Salmonella, Trichomonas parasites and several families of viruses known to cause mass mortality in birds have all been ruled out. Some species, including the blue jay, European starling, common grackle, American robin, northern cardinal, house finch, house sparrow, eastern bluebird, red-bellied woodpecker, Carolina chickadee and Carolina wren, seem to be more affected than others. And evidence suggests that young birds are particularly susceptible to the illness. At this time, there is no known treatment or cure.

In the spring of 2021, portions of the disease outbreak area overlapped with the emergence of periodical cicadas belonging to the 17-year Brood X. Since birds eat cicadas, and cicadas can carry fungus (particularly Massospora) and/or accumulate pesticides or contaminants while living underground in the soil, some scientists initially hypothesized that the cicadas might be playing a role in the songbird deaths. However, sick birds have been observed in geographic locations where cicadas are rare, and the correlation does not seem to fit. More information is urgently needed to solve this mystery, but in the meantime, officials have urged people in the Mid-Atlantic region to discontinue feeding birds and providing water in bird baths in an attempt to slow transmission of the deadly disease.

Cyanobacteria, photosynthetic bacteria found in water and moist soil, are capable of producing toxins that are harmful to humans, wildlife and the environment, especially in high concentrations, or blooms, which result from overgrowth in warm, stagnant, nutrient-rich water. Remote-sensing and data obtained from water samples collected in the area showed that cyanotoxin concentrations (ranging from 0.36 to 124,460 μg L−1) were much higher than the provisional guideline value of 1.0 μg L−1 recommended for mammals and humans by the World Health Organization (WHO). At high concentrations, neurotoxins produced by cyanobacteria can cause paralysis, cardiac or respiratory failure and death, and reports of elephants seen walking in circles before suddenly collapsing to death in Botswana seem to support the cyanobacteria hypothesis.

Mysterious die-offs of African elephants are also making headlines. During the months of May-June 2020, approximately 350 elephant deaths occurred in Botswana, Africa. An additional 39 deaths were reported in the Botswana Moremi Game Reserve from January-March 2021. The casualties are especially concerning in light of the African Elephant's standing on The International Union for Conservation of Nature's (IUCN) Red List of Threatened Species.

Evidence points to a common cause of death, and microbes have been implicated. At a news conference in September 2020, the principal veterinary officer with Botswana's Department of Wildlife and National Parks stated that lab tests had identified cyanobacterial neurotoxins as the culprit.

While cyanobacteria contamination in pools of drinking water is a plausible explanation for the deaths of these elephants, many questions still remain. Other animals in the surrounding area, including scavengers who fed on the carcasses of the affected pachyderms, appeared to be unharmed. This observation, combined with the fact that the government has yet to release full test results from their analysis to the public, have led some scientists to suggest that the elephants may have been targeted. Although poaching has been ruled out as a possibility due to the fact that the dead elephants all had intact tusks, some have questioned whether the agency's tests were designed to rule out neurotoxins that may be available to farmers eager to prevent the giant herbivores from trampling and eating their crops. Additional data and transparency are needed to solve this microbiological mystery and prevent further die-offs of the endangered species.

Nature vs. Nurture? Heritability of the Microbiome

Commensal bacteria are known to be more similar between relatives than nonrelatives, but it is less clear whether gut microbiome traits are heritable or more likely to result from shared environments between related individuals. Research published in Science characterized changes in the microbiomes of 585 wild baboons from fecal samples collected over 14 years. After controlling for diet, age and socioecological variation, scientists found that nearly all (97%) gut microbiome taxa are heritable in baboons. Although baboons have a microbiome similar to humans, these results contradict previous work, which found few heritable taxa in humans. The magnitude of heritability was small (mean = 0.068), aligning the results of this study with prior observations that environmental effects have a larger impact on gut microbiome composition than additive genetic effects. Overall, the findings of this study indicate that it is important to consider host genetics when evaluating microbial landscapes and imply that host microbiome traits are subject to natural selection with the host genome.

While commensal bacteria are found in adult butterflies, resident microbial communities are almost entirely absent in butterfly larvae. It is known that parental exposure to environmental conditions can exert transgenerational effects on the phenotypes of offspring, but it is unclear whether the microbiome of the parent has any direct effect on the organism’s offspring. A study published in Applied and Environmental Microbiology tested the hypothesis that disturbance of parental microbial communities via antibiotic treatment would affect the ability of first-generation (F1) larvae to cope with transgenerational shifts in host plant species. In support of this hypothesis, researchers found that in 75 percent of experiments, larvae derived from antibiotic-treated parents gained less biomass than control larvae when feeding on a different plant species than their parents, but not when they fed on the same plant species as the parent generation.

The effect is linked to higher prophenoloxidase activity (a modified form of the compliment response that is found in some invertebrates) and downregulation of a major allergen gene (MA) involved in the detoxification of glucosinolates, secondary metabolites of plants that can be activated to produce toxic products, such as isothiocyanates, when damage occurs to the plant.

Evolution of Antimicrobial Resistance (AMR)

Compared to the magnitude of the ongoing threat of antimicrobial resistance (AMR), relatively little is known about the de novo emergence of resistance genes or the role of microbial physiology on resistance. It is known that AMR often results in a fitness cost to the microbe, which may be mitigated by compensatory mutations elsewhere in the genome. One of the better-studied examples of that fitness cost is rifampicin resistance. Rifampicin targets the bacterial RNA polymerase (RNAP). Rifampicin resistance is commonly due to mutations in the β subunit of RNAP. These RNAP mutations affect how different genes are transcribed, thereby influencing the amount of various proteins in the bacteria. Mutations in the β′ subunit of RNAP can compensate for the fitness cost of resistance mutations occurring in the β subunit.

In a study published in Antimicrobial Agents and Chemotherapy, researchers sought to identify molecular mechanisms underpinning the fitness cost of rifampicin resistance in Mycobacteria tuberculosis by subjecting a collection of rifampicin-resistant M. tuberculosis strains to genome-wide transcriptomic and proteomic profiling. Analysis revealed a signature of physiological changes that may alter the fitness cost of rifampicin-resistance. The most common clinically conferring rifampicin-resistance mutation, rpoB Ser450Leu, causes measurable fitness defects, which vary in different M. tuberculosis genetic backgrounds. Increased abundance of proteins involved in central carbon metabolism correlated with the fitness defects, leading the authors to conclude that posttranscriptional modulation of gene expression occurred in most of the strains carrying rpoB Ser450Leu. Based on the data of this study, researchers asserted that the fitness cost of rifampicin resistance and its compensation are mediated by differences in gene expression conferred by the rpoB mutations, suggesting that expression of the mycobactin biosynthetic cluster is the most likely cause of growth rate differences of resistant M. tuberculosis strains.

In order to better understand how de novo resistance genes emerge, a study published in PLOS Genetics tested whether random DNA sequences can generate novel antibiotic-resistance determinants. Scientists expressed over 100 million randomly generated sequences in E. coli and identified six de novo colistin-resistance-conferring peptides (Dcr) that were auxiliary activators of the two-component regulatory system, PmrAB. The newly identified peptides conferred resistance by modifying the cell envelope and causing reduced antibiotic uptake. This is the first example of random-expression libraries being used to select for peptides that lead to an AMR phenotype via direct peptide-protein interactions in vivo. The results of the study support the idea that noncoding DNA can serve as a substrate for de novo gene evolution and suggest that a new class of peptide could potentially evolve to be resistance-determinant in nature.

How Does Phage DNA Enter a Bacterial Host?

(Hint: It Isn't as Simple as You Learned in Your General Microbiology Class.)

When you learned about phage in your general microbiology class, you probably learned that the DNA is injected into the cell via a pre-assembled hypodermic-like structure, a dogma based on the well-characterized phage T4 that has a long, beautiful tail. However, phage T7 and many other phage have puny little tails that don't fit the hypodermic model. A paper published in Molecular Cell combined biochemical, structural, biophysical and modeling approaches to generate a composite model of the periplasmic channel used by the phage T7 DNA to transfer the phage DNA from the capsid to the cytoplasm of a bacterial host.

These studies showed that phage T7 has an "ejectosome" composed of phage proteins: gp14 forms an outer membrane channel connected to a periplasmic channel composed of gp15 bound to gp16, and a large cytoplasmic hub formed by gp16 has DNA-binding activity. This work nicely shows how structural studies can inform an understanding of functional questions that have been difficult to resolve.

Why Do Respiratory Infections Often Lead to Intestinal Symptoms?

Although influenza is primarily considered a respiratory disease, influenza virus infections are frequently associated with complications outside of the respiratory tract, including intestinal symptoms like nausea, vomiting and diarrhea. Previous studies had indicated that influenza alters the gut microbiome, but the cause, nature and consequences of influenza-associated intestinal symptoms had not been not understood.

In a study published in Infection and Immunity, researchers examined signs of intestinal injury and inflammation, altered gene expression and compromised intestinal barrier function in influenza A virus (IAV)-infected mice. They found that the virus altered the composition of gut microbiota and made 2 important changes: (1) a decrease in the production of short-chain fatty acids (SCFAs) derived from fermentative gut microbiota; and (2) up-regulation of inflammatory markers in the liver of the infected mice, suspected to result from translocation of bacterial products across the gut barrier. Scientists concluded that influenza virus infection can remotely impair intestinal barrier function and thereby trigger secondary enteric infections. Moreover, when IAV-infected mice were treated with SCFAs, systemic infection of Salmonella typhimurium fell, supporting this hypothesis.

New Insights into Microbes and Biogeochemical Cycles

It has been suggested that of all of Earth's biogeochemical cycles, the methane cycle is the most tightly linked to climate, and approximately 1 gigaton of methane is generated by methane-producing (methanogenic) archaea in oxygen-depleted environments every year. Methane-oxidizing microbes, or methanotrophs, play an important role in balancing atmospheric methane. A team of scientists recently discovered large (~1 Mbp), linear DNA sequences containing genes that expand redox and respiratory capacity. Results of the investigation are not yet peer-reviewed and are currently in preprint on the bioRxiv server. Data suggest that these sequences are novel extrachromosomal elements (ECEs) that coexist and replicate within Methanoperedens, a methane-oxidizing archaea. Because these elements have the ability to scavenge and "assimilate" genes from microorganisms in their environment, scientists have named them "Borgs" after the fictional "Star Trek" aliens who sequester the technology and knowledge of other alien species.

Still, Methanoperedens has yet to be cultured in a lab, and the findings are based on sequence data alone. Finding Borgs in cultured Methanoperedens is an important step to verifying the conclusions of this study. The team is currently investigating the role of DNA repeats present in these sequences in the hopes that Borgs could be a useful tool for gene-editing and the reduction of methane emissions.

Fe(II)-oxidizing microorganisms and Fe(III)-reducing microbes are key drivers of Earth's biogeochemical Fe cycle. A study published in Microbiology Spectrum identified the first single organism capable of both Fe(II) oxidation and anaerobic Fe(III) reduction at circumneutral pH. Researchers isolated a novel neutrophilic Fe(II)-oxidizing Rhodoferax bacterium from an iron-rich wetland in Japan. The novel strain, MIZ03, can grow chemolithoautrophically at nearly neutral pH (6.5-7.5) by oxidizing Fe(II), H2 or thiosulfate as the sole electron donor under (micro)aerobic conditions and can reduce Fe(III) or nitrate under anaerobic conditions. This discovery identified a novel model organism that will provide insights into the molecular mechanisms of microbial Fe redox-cycling and the Fe cycle in the environment.

How Do Fungi Breech Plant Surfaces?

To better understand how oomycetes breech plant surfaces during infection, a publication in Nature Microbiology analyzed how Phytophthora infestans invades potatoes. P. infestans was responsible for the devastating Irish Potato Famine and is still a global threat to potato and tomato crops. The authors used three-dimensional confocal imaging to monitor the invasion of etiolated stems of potato plantlets by strains of GFP-expressing Phytophthora. The authors observed that, following spore germination and germ tube growth, the oomycete hyphal tip indented and invaded host surfaces at an oblique angle of attack, suggesting a slicing mechanism of invasion. Fracture imaging confirmed that surface cracks in front of the growing hyphae allow pathogens to invade. This study indicates that Phytophthora hyphae slice through plant surfaces to initiate infection. It is the first description of this novel mechanism of plant invasion, which does not rely on appressorial structures.