Macroevolutionary Dynamics in Micro-organisms: Generalists Give Rise to Specialists Across Biomes in the Ubiquitous Bacterial Phylum Myxococcota.

Prokaryotes dominate the Tree of Life, but our understanding of the macroevolutionary processes generating this diversity is still limited. Habitat transitions are thought to be a key driver of prokaryote diversity. However, relatively little is known about how prokaryotes successfully transition and persist across environments, and how these processes might vary between biomes and lineages. Here, we investigate biome transitions and specialization in natural populations of a focal bacterial phylum, the Myxococcota, sampled across a range of replicated soils and freshwater and marine sediments in Cornwall (UK). By targeted deep sequencing of the protein-coding gene rpoB, we found >2,000 unique Myxococcota lineages, with the majority (77%) classified as biome specialists and with only <5% of lineages distributed across the salt barrier. Discrete character evolution models revealed that specialists in one biome rarely transitioned into specialists in another biome. Instead, evolved generalism mediated transitions between biome specialists. State-dependent diversification models found variation in speciation rates across the tree, but this variation was independent of biome association or specialization. Our findings were robust to phylogenetic uncertainty, different levels of species delineation, and different assumed amounts of unsampled diversity resulting in an incomplete phylogeny. Overall, our results are consistent with a "jack-of-all-trades" tradeoff where generalists suffer a cost in any individual environment, resulting in rapid evolution of niche specialists and shed light on how bacteria could transition between biomes.

A new test suggests hundreds of amino acid polymorphisms in humans are subject to balancing selection.

The role that balancing selection plays in the maintenance of genetic diversity remains unresolved. Here, we introduce a new test, based on the McDonald-Kreitman test, in which the number of polymorphisms that are shared between populations is contrasted to those that are private at selected and neutral sites. We show that this simple test is robust to a variety of demographic changes, and that it can also give a direct estimate of the number of shared polymorphisms that are directly maintained by balancing selection. We apply our method to population genomic data from humans and provide some evidence that hundreds of nonsynonymous polymorphisms are subject to balancing selection.

Accessory microbiomes.

In microbiome research, considerable effort has been invested in finding core microbiomes, which have been hypothesized to contain the species most important for host function. Much less attention has been paid to microbiome members that are present in only a subset of hosts. Such accessory microbiomes must in large part consist of species that have no effect on fitness, but some will have deleterious effects on fitness (pathogens), and it is also possible that some accessory microbiome members benefit an ecologically distinct subset of hosts. This short paper discusses what we know about accessory microbiomes, specifically by comparing it with the concept of accessory genomes.

Testing for the fitness benefits of natural transformation during community-embedded evolution.

Natural transformation is a process where bacteria actively take up DNA from the environment and recombine it into their genome or reconvert it into extra-chromosomal genetic elements. The evolutionary benefits of transformation are still under debate. One main explanation is that foreign allele and gene uptake facilitates natural selection by increasing genetic variation, analogous to meiotic sex. However, previous experimental evolution studies comparing fitness gains of evolved transforming- and isogenic non-transforming strains have yielded mixed support for the 'sex hypothesis.' Previous studies testing the sex hypothesis for natural transformation have largely ignored species interactions, which theory predicts provide conditions favourable to sex. To test for the adaptive benefits of bacterial transformation, the naturally transformable wild-type Acinetobacter baylyi and a transformation-deficient ∆comA mutant were evolved for 5 weeks. To provide strong and potentially fluctuating selection, A. baylyi was embedded in a community of five other bacterial species. DNA from a pool of different Acinetobacter strains was provided as a substrate for transformation. No effect of transformation ability on the fitness of evolved populations was found, with fitness increasing non-significantly in most treatments. Populations showed fitness improvement in their respective environments, with no apparent costs of adaptation to competing species. Despite the absence of fitness effects of transformation, wild-type populations evolved variable transformation frequencies that were slightly greater than their ancestor which potentially could be caused by genetic drift.

A comprehensive list of bacterial pathogens infecting humans.

There exists an enormous diversity of bacteria capable of human infection, but no up-to-date, publicly accessible list is available. Combining a pragmatic definition of pathogenicity with an extensive search strategy, we report 1513 bacterial pathogens known to infect humans described pre-2021. Of these, 73 % were regarded as established (have infected at least three persons in three or more references) and 27 % as putative (fewer than three known cases). Pathogen species belong to 10 phyla and 24 classes scattered throughout the bacterial phylogeny. We show that new human pathogens are discovered at a rapid rate. Finally, we discuss how our results could be expanded to a database, which could provide a useful resource for microbiologists. Our list is freely available and archived on GitHub and Zenodo and we have provided walkthroughs to facilitate access and use.

Copper selects for siderophore-mediated virulence in Pseudomonas aeruginosa.

BACKGROUND: Iron is essential for almost all bacterial pathogens and consequently it is actively withheld by their hosts. However, the production of extracellular siderophores enables iron sequestration by pathogens, increasing their virulence. Another function of siderophores is extracellular detoxification of non-ferrous metals. Here, we experimentally link the detoxification and virulence roles of siderophores by testing whether the opportunistic pathogen Pseudomonas aeruginosa displays greater virulence after exposure to copper. To do this, we incubated P. aeruginosa under different environmentally relevant copper regimes for either two or twelve days. Subsequent growth in a copper-free environment removed phenotypic effects, before we quantified pyoverdine production (the primary siderophore produced by P. aeruginosa), and virulence using the Galleria mellonella infection model. RESULTS: Copper selected for increased pyoverdine production, which was positively correlated with virulence. This effect increased with time, such that populations incubated with high copper for twelve days were the most virulent. Replication of the experiment with a non-pyoverdine producing strain of P. aeruginosa demonstrated that pyoverdine production was largely responsible for the change in virulence. CONCLUSIONS: We here show a direct link between metal stress and bacterial virulence, highlighting another dimension of the detrimental effects of metal pollution on human health.

Signatures of selection in core and accessory genomes indicate different ecological drivers of diversification among Bacillus cereus clades.

Bacterial clades are often ecologically distinct, despite extensive horizontal gene transfer (HGT). How selection works on different parts of bacterial pan-genomes to drive and maintain the emergence of clades is unclear. Focusing on the three largest clades in the diverse and well-studied Bacillus cereus sensu lato group, we identified clade-specific core genes (present in all clade members) and then used clade-specific allelic diversity to identify genes under purifying and diversifying selection. Clade-specific accessory genes (present in a subset of strains within a clade) were characterized as being under selection using presence/absence in specific clades. Gene ontology analyses of genes under selection revealed that different gene functions were enriched in different clades. Furthermore, some gene functions were enriched only amongst clade-specific core or accessory genomes. Genes under purifying selection were often clade-specific, while genes under diversifying selection showed signs of frequent HGT. These patterns are consistent with different selection pressures acting on both the core and the accessory genomes of different clades and can lead to ecological divergence in both cases. Examining variation in allelic diversity allows us to uncover genes under clade-specific selection, allowing ready identification of strains and their ecological niche.

Determining the prevalence, identity and possible origin of bacterial pathogens in soil.

Soil biomes are vast, exceptionally diverse and crucial to the health of ecosystems and societies. Soils also contain an appreciable, but understudied, diversity of opportunistic human pathogens. With climate change and other forms of environmental degradation potentially increasing exposure risks to soilborne pathogens, it is necessary to gain a better understanding of their ecological drivers. Here we use the Galleria mellonella insect virulence model to selectively isolate pathogenic bacteria from soils in Cornwall (UK). We find a high prevalence of pathogenic soil bacteria with two genera, Providencia and Serratia, being especially common. Providencia alcalifaciens, P. rustigianii, Serratia liquefaciens and S. plymuthica strains were studied in more detail using phenotypic virulence and antibiotic resistance assays and whole-genome sequencing. Both genera displayed low levels of antibiotic resistance and antibiotic resistance gene carriage. However, Serratia isolates were found to carry the recently characterized metallo-ß-lactamase blaSPR-1 that, although not conferring high levels of resistance in these strains, poses a potential risk of horizontal transfer to other pathogens where it could be fully functional. The Galleria assay can be a useful approach to uncover the distribution and identity of pathogenic bacteria in the environment, as well as uncover resistance genes with an environmental origin.

Transposon mutagenesis in Pseudomonas fluorescens reveals genes involved in blue pigment production and antioxidant protection.

Pseudomonas fluorescens Ps_77 is a blue-pigmenting strain able to cause food product discoloration, causing relevant economic losses especially in the dairy industry. Unlike non-pigmenting P. fluorescens, blue pigmenting strains previously were shown to carry a genomic region that includes homologs of trpABCDF genes, pointing at a possible role of the tryptophan biosynthetic pathway in production of the pigment. Here, we employ random mutagenesis to first identify the genes involved in blue-pigment production in P. fluorescens Ps_77 and second to investigate the biological function of the blue pigment. Genetic analyses based on the mapping of the random insertions allowed the identification of eight genes involved in pigment production, including the second copy of trpB (trpB_1) gene. Phenotypic characterization of Ps_77 white mutants demonstrated that the blue pigment increases oxidative-stress resistance. Indeed, while Ps_77 was growing at a normal rate in presence of 5 mM of H2O2, white mutants were completely inhibited. The antioxidative protection is not available for non-producing bacteria in co-culture with Ps_77.

Staphylococcus cornubiensis sp. nov., a member of the Staphylococcus intermedius Group (SIG).

We here describe a novel species in the Staphylococcus intermedius Group (SIG) which is phenotypically similar to Staphylococcus pseudintermedius but is genomically distinct from it and other SIG members, with an average nucleotide identity of 90.2 % with its closest relative S. intermedius. The description of Staphylococcus cornubiensis sp. nov. is based on strain NW1T (=NCTC 13950T=DSM 105366T) isolated from a human skin infection in Cornwall, UK. Although pathogenic, NW1T carries no known virulence genes or mobilizable antibiotic resistance genes and further studies are required to assess the prevalence of this species in humans as well as its potential presence in companion animals.

Improved detection of Staphylococcus intermedius group in a routine diagnostic laboratory.

The Staphylococcus intermedius group (SIG) includes zoonotic pathogens traditionally associated with dog bites. We describe a simple scheme for improved detection of SIG using routine laboratory methods, report its effect on isolation rates, and use sequencing to confirm that, apart from one atypical SIG strain, most isolates are Staphylococcus pseudintermedius.

Sociobiology of the myxobacteria.

Cooperation is integral to much of biological life but can be threatened by selfish evolutionary strategies. Diverse cooperative traits have evolved among microbes, but particularly sophisticated forms of sociality have arisen in the myxobacteria, including group motility and multicellular fruiting body development. Myxobacterial cooperation has succeeded against socially destructive cheaters and can readily re-evolve from some socially defective genotypes. However, social harmony does not extend far. Spatially structured natural populations of the model species Myxococcus xanthus have fragmented into a large number of socially incompatible genotypes that exclude, exploit, and/or antagonize one another, including genetically similar neighbors. Here, we briefly review basic social evolution concepts as they pertain to microbes, discuss potential benefits of myxobacterial social traits, highlight recent empirical studies of social evolution in M. xanthus, and consider their implications for how myxobacterial cooperation and conflict evolve in the wild.

Improvement in Staphylococcus and Bacillus strain differentiation by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry profiling by using microwave-assisted enzymatic digestion.

RATIONALE: Distinguishing between individual bacterial strains below the species level is a challenge to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bacterial profiling. We propose a quick method for improving strain differentiation of two Staphylococcus and one Bacillus species. METHODS: An alternative procedure to the extraction protocol recommended by Bruker Daltonics was developed. Ethanol-sterilized cells of six S. aureus and six S. haemolyticus strains were digested by trypsin using 2-min microwave irradiation and were then analyzed. Twenty-eight strains belonging to two ecotypes of B. subtilis were subjected to the same procedure to extend the scope of the method. RESULTS: S. aureus and S. haemolyticus strains, only partially distinguishable by the standard sample preparation procedure, were subjected to microwave-assisted tryptic digestion. The repeatability of the procedure was checked in three experiments accomplished at weekly intervals. Clear distinction of the strains was achieved by cluster analysis. The differentiation of B. subtilis ecotypes was also improved significantly by the digestion method. The discriminatory power of the novel method was supported by an increase in the number of strain-specific peaks, as compared to the standard method. CONCLUSIONS: The method modulates the discriminatory power of MALDI-TOF MS profiling. The differentiation of a set of S. aureus, S. haemolyticus and B. subtilis strains was improved significantly after microwave-accelerated tryptic digestion of the cellular material.

Collection of Annotated Acinetobacter Genome Sequences.

The genus Acinetobacter contains environmental species as well as opportunistic pathogens of humans. Several species are competent for natural transformation, an important mechanism of horizontal gene transfer. Here, we present the genome sequences of 19 Acinetobacter strains used in past and upcoming studies of natural transformation.

Adaptive radiations in natural populations of prokaryotes: innovation is key.

Prokaryote diversity makes up most of the tree of life and is crucial to the functioning of the biosphere and human health. However, the patterns and mechanisms of prokaryote diversification have received relatively little attention compared to animals and plants. Adaptive radiation, the rapid diversification of an ancestor species into multiple ecologically divergent species, is a fundamental process by which macrobiological diversity is generated. Here, we discuss whether ecological opportunity could lead to similar bursts of diversification in bacteria. We explore how adaptive radiations in prokaryotes can be kickstarted by horizontally acquired key innovations allowing lineages to invade new niche space that subsequently is partitioned among diversifying specialist descendants. We discuss how novel adaptive zones are colonized and exploited after the evolution of a key innovation and whether certain types of are more prone to adaptive radiation. Radiation into niche specialists does not necessarily lead to speciation in bacteria when barriers to recombination are absent. We propose that in this scenario, niche-specific genes could accumulate within a single lineage, leading to the evolution of an open pangenome.

The effect of metal remediation on the virulence and antimicrobial resistance of the opportunistic pathogen Pseudomonas aeruginosa.

Anthropogenic metal pollution can result in co-selection for antibiotic resistance and potentially select for increased virulence in bacterial pathogens. Metal-polluted environments can select for the increased production of siderophore molecules to detoxify non-ferrous metals. However, these same molecules also aid the uptake of ferric iron, a limiting factor for within-host pathogen growth, and are consequently a virulence factor. Anthropogenic methods to remediate environmental metal contamination commonly involve amendment with lime-containing materials. However, whether this reduces in situ co-selection for antibiotic resistance and siderophore-mediated virulence remains unknown. Here, using microcosms containing non-sterile metal-contaminated river water and sediment, we test whether liming reduces co-selection for these pathogenicity traits in the opportunistic pathogen Pseudomonas aeruginosa. To account for the effect of environmental structure, which is known to impact siderophore production, microcosms were incubated under either static or shaking conditions. Evolved P. aeruginosa populations had greater fitness in the presence of toxic concentrations of copper than the ancestral strain and showed increased resistance to the clinically relevant antibiotics apramycin, cefotaxime and trimethoprim, regardless of lime addition or environmental structure. Although we found virulence to be significantly associated with siderophore production, neither virulence nor siderophore production significantly differed between the four treatments. Furthermore, liming did not mitigate metal-imposed selection for antibiotic resistance or virulence in P. aeruginosa. Consequently, metal-contaminated environments may select for antibiotic resistance and virulence traits even when treated with lime.

Copper reduces the virulence of bacterial communities at environmentally relevant concentrations.

Increasing environmental concentrations of metals as a result of anthropogenic pollution are significantly changing many microbial communities. While there is evidence metal pollution can result in increased antibiotic resistance, the effects of metal pollution on the virulence of bacterial communities remains largely undetermined. Here, we experimentally test whether metal stress alters the virulence of bacterial communities. We do this by incubating three wastewater influent communities under different environmentally relevant copper concentrations for three days. We then quantify the virulence of the community phenotypically using the Galleria mellonella infection model, and test if differences are due to changes in the rate of biomass accumulation (productivity), copper resistance, or community composition (quantified using 16S amplicon sequencing). The virulence of the communities was found to be reduced by the highest copper concentration, but not to be affected by the lower concentration. As well as reduced virulence, communities exposed to the highest copper concentration were less diverse and had lower productivity. This work highlights that metal pollution may decrease virulence in bacterial communities, but at a cost to diversity and productivity.

Bacterial colonisation dynamics of household plastics in a coastal environment.

Accumulation of plastics in the marine environment has widespread detrimental consequences for ecosystems and wildlife. Marine plastics are rapidly colonised by a wide diversity of bacteria, including human pathogens, posing potential risks to health. Here, we investigate the effect of polymer type, residence time and estuarine location on bacterial colonisation of common household plastics, including pathogenic bacteria. We submerged five main household plastic types: low-density PE (LDPE), high-density PE (HDPE), polypropylene (PP), polyvinyl chloride (PVC) and polyethylene terephthalate (PET) at an estuarine site in Cornwall (U.K.) and tracked bacterial colonisation dynamics. Using both culture-dependent and culture-independent approaches, we found that bacteria rapidly colonised plastics irrespective of polymer type, reaching culturable densities of up to 1000 cells cm3 after 7 weeks. Community composition of the biofilms changed over time, but not among polymer types. The presence of pathogenic bacteria, quantified using the insect model Galleria mellonella, increased dramatically over a five-week period, with Galleria mortality increasing from 4% in week one to 65% in week five. No consistent differences in virulence were observed between polymer types. Pathogens isolated from plastic biofilms using Galleria enrichment included Serratia and Enterococcus species and they harboured a wide range of antimicrobial resistance genes. Our findings show that plastics in coastal waters are rapidly colonised by a wide diversity of bacteria independent of polymer type. Further, our results show that marine plastic biofilms become increasingly associated with virulent bacteria over time.

Prevalence and mobility of integrative and conjugative elements within a Streptomyces natural population.

Horizontal Gene Transfer (HGT) is a powerful force generating genomic diversity in bacterial populations. HGT in Streptomyces is in large part driven by conjugation thanks to plasmids, Integrative and Conjugative elements (ICEs) and Actinomycete ICEs (AICEs). To investigate the impact of ICE and AICE conjugation on Streptomyces genome evolution, we used in silico and experimental approaches on a set of 11 very closely related strains isolated from a millimeter scale rhizosphere population. Through bioinformatic searches of canonical conjugation proteins, we showed that AICEs are the most frequent integrative conjugative elements, with the central chromosome region being a hotspot for integrative element insertion. Strains exhibited great variation in AICE composition consistent with frequent HGT and/or gene loss. We found that single insertion sites can be home to different elements in different strains (accretion) and conversely, elements belonging to the same family can be found at different insertion sites. A wide variety of cargo genes was present in the AICEs with the potential to mediate strain-specific adaptation (e.g., DNA metabolism and resistance genes to antibiotic and phages). However, a large proportion of AICE cargo genes showed hallmarks of pseudogenization, consistent with deleterious effects of cargo genes on fitness. Pock assays enabled the direct visualization of conjugal AICE transfer and demonstrated the transfer of AICEs between some, but not all, of the isolates. Multiple AICEs were shown to be able to transfer during a single mating event. Although we did not obtain experimental evidence for transfer of the sole chromosomal ICE in this population, genotoxic stress mediated its excision from the chromosome, suggesting its functionality. Our results indicate that AICE-mediated HGT in Streptomyces populations is highly dynamic, with likely impact on strain fitness and the ability to adapt to environmental change.

Isolation by distance in the spore-forming soil bacterium Myxococcus xanthus.

Genetic differentiation between spatially separated populations within a species is commonly observed in plants and animals, but its existence in microbes has long been a contentious issue. Traditionally, many microbial ecologists have reasoned that microbes are not limited by dispersal as a result of their immense numbers and microscopic size. In this view, the absence of barriers to gene flow between populations would prevent differentiation of populations by genetic drift and hinder local adaptation. Myxococcus xanthus is a globally distributed, spore-forming bacterium that offers a robust test for genetic differentiation among populations because sporulation is expected to enhance dispersal. Using multi-locus sequence data, we show here that both diversity and the degree of differentiation between populations increase as a function of distance in M. xanthus. Populations are consistently differentiated at scales exceeding 10(2)-10(3) km, and isolation by distance, the divergence of populations by genetic drift due to limited dispersal, is responsible. Our results provide new insights into how genetic diversity within species of free-living microbes is distributed from centimeter to global scales.