A design-constraint trade-off underpins the diversity in ecologically important traits in species Escherichia coli

Bacterial species are internally diverse in genomic and multi-locus gene comparisons. The ecological causes of phenotypic and genotypic diversity within species are far less well understood. Here, we focus on the competitive fitness for growth on nutrients within Escherichia coli, an internally rich species. Competition experiments in nutrient-limited chemostats revealed that members of the ECOR collection exhibited a wide continuum of competitive abilities, with some fitter and some less fit than the lab strain MG1655. We observed an inverse relationship between competitiveness and the resistance of strains to detergent and antibiotic, consistent with the notion that membrane permeability and competitive fitness are linked by a trade-off between self-preservation and nutritional competence (SPANC); high permeability has a postulated cost in antibacterial sensitivity whereas a low permeability has a cost in nutrient affinity. Isolates moved along the markedly nonlinear trade-off curve by mutational adaptation; an ECOR strain sensitive to antibacterials and a good competitor was easily converted by mutation into a mutant with higher resistance but poorer competition in the presence of low antibiotic concentrations. Conversely, a resistant ECOR strain changed into a better competitor after a short period of selection under nutrient limitation. In both directions, mutations can affect porin proteins and outer membrane permeability, as indicated by protein analysis, gene sequencing and an independent assay of outer membrane permeability. The extensive, species-wide diversity of E. coli in ecologically important traits can thus be explained as an evolutionary consequence of a SPANC trade-off driven by antagonistic pleiotropy.     

Molecular and evolutionary bases of within-patient genotypic and phenotypic diversity in Escherichia coli extraintestinal infections

Although polymicrobial infections, caused by combinations of viruses, bacteria, fungi and parasites, are being recognised with increasing frequency, little is known about the occurrence of within-species diversity in bacterial infections and the molecular and evolutionary bases of this diversity. We used multiple approaches to study the genomic and phenotypic diversity among 226 Escherichia coli isolates from deep and closed visceral infections occurring in 19 patients. We observed genomic variability among isolates from the same site within 11 patients. This diversity was of two types, as patients were infected either by several distinct E. coli clones (4 patients) or by members of a single clone that exhibit micro-heterogeneity (11 patients); both types of diversity were present in 4 patients. A surprisingly wide continuum of antibiotic resistance, outer membrane permeability, growth rate, stress resistance, red dry and rough morphotype characteristics and virulence properties were present within the isolates of single clones in 8 of the 11 patients showing genomic micro-heterogeneity. Many of the observed phenotypic differences within clones affected the trade-off between self-preservation and nutritional competence (SPANC). We showed in 3 patients that this phenotypic variability was associated with distinct levels of RpoS in co-existing isolates. Genome mutational analysis and global proteomic comparisons in isolates from a patient revealed a star-like relationship of changes amongst clonally diverging isolates. A mathematical model demonstrated that multiple genotypes with distinct RpoS levels can co-exist as a result of the SPANC trade-off. In the cases involving infection by a single clone, we present several lines of evidence to suggest diversification during the infectious process rather than an infection by multiple isolates exhibiting a micro-heterogeneity. Our results suggest that bacteria are subject to trade-offs during an infectious process and that the observed diversity resembled results obtained in experimental evolution studies. Whatever the mechanisms leading to diversity, our results have strong medical implications in terms of the need for more extensive isolate testing before deciding on antibiotic therapies.     

Cultivable bacterial diversity from the human colon

Knowledge of the composition of the colonic microbiota is important for our understanding of how the balance of these microbes is influenced by diet and the environment, and which bacterial groups are important in maintaining gut health or promoting disease. Molecular methodologies have advanced our understanding of the composition and diversity of the colonic microbiota. Importantly, however, it is the continued isolation of bacterial representatives of key groups that offers the best opportunity to conduct detailed metabolic and functional studies. This also permits bacterial genome sequencing which will accelerate the linkage to functionality. Obtaining new human colonic bacterial isolates can be challenging, because most of these are strict anaerobes and many have rather exact nutritional and physical requirements. Despite this many new species are being isolated and described that occupy distinct niches in the colonic microbial community. This review focuses on these under-studied yet important gut anaerobes.     

Local selection across a latitudinal gradient shapes nucleotide diversity in balsam poplar, Populus balsamifera L

Molecular studies of adaptive evolution often focus on detecting selective sweeps driven by positive selection on a species-wide scale; however, much adaptation is local, particularly of ecologically important traits. Here, we look for evidence of range-wide and local adaptation at candidate genes for adaptive phenology in balsam poplar, Populus balsamifera, a widespread forest tree whose range extends across environmental gradients of photoperiod and growing season length. We examined nucleotide diversity of 27 poplar homologs of the flowering-time network-a group of genes that control plant developmental phenology through interactions with environmental cues such as photoperiod and temperature. Only one gene, ZTL2, showed evidence of reduced diversity and an excess of fixed replacement sites, consistent with a species-wide selective sweep. Two other genes, LFY and FRI, harbored high levels of nucleotide diversity and exhibited elevated differentiation between northern and southern accessions, suggesting local adaptation along a latitudinal gradient. Interestingly, FRI has also been identified as a target of local selection between northern and southern accessions of Arabidopsis thaliana, indicating that this gene may be commonly involved in ecological adaptation in distantly related species. Our findings suggest an important role for local selection shaping molecular diversity and reveal limitations of inferring molecular adaptation from analyses designed only to detect species-wide selective sweeps.     

肠杆菌科细菌的Rcs信号转导系统——毒力、应激

Rcs是肠杆菌科细菌中的一种复杂的双组分信号转导系统,能调节细菌荚膜异多糖酸合成,以及细菌鞭毛基因、抗酸性基因等的表达。Rcs不同于典型的双组分系统,其由3个蛋白构成,磷酸转移过程分3步进行。不同细菌中的Rcs功能有所区别,主要为调控细菌的毒力和应激。本文在简单介绍细菌双组分信号转导系统的基础上,重点对肠杆菌科细菌Rcs的组成、功能及磷酸转移机制进行综述。     

(p)ppGpp——“魔斑”核苷酸在细菌中的研究进展

鸟苷四磷酸（guanosine tetraphosphate,ppGpp）/鸟苷五磷酸（guanosine pentaphosphate,pppGpp）是细菌严谨反应的信号分子,其合成和水解由Rel/SpoT同系物（RelA/SpoT homologue,RSH）家族的蛋白质合成和水解活性控制。（p）ppGpp介导的严谨反应能够提高细菌对营养匮乏的适应能力和抗生素抗性。近年来发现（p）ppGpp与细菌生长和细胞分裂、抗生素合成等都密切相关,是细胞内重要的全局调控因子。（p）ppGpp在细菌细胞中有许多靶点,使其可以调节DNA复制、转录、细胞周期、核糖体生物合成以及抗生素合成基因簇的表达。然而,（p）ppGpp如何控制转录和其他代谢过程取决于细菌种类,并在不同的微生物中通过不同的机制调节相同的过程。因此,本文通过综述（p）ppGpp的合成/水解酶的种类和调节机制,（p）ppGpp对微生物代谢调控机制、对细胞周期的影响机制,以及（p）ppGpp对抗生素合成和耐受性的调控机制,为细菌耐药性研究和细胞生理学研究奠定基础。     

Occurrence of tetracycline resistance genes tet(M) and tet(S) in bacteria from marine aquaculture sites

Occurrence of tetracycline resistance genes encoding ribosomal protection proteins was examined in 151 tetracycline-resistant bacterial isolates from fish and seawater at coastal aquaculture sites in Japan and Korea. The tet(M) gene was detected in 34 Japanese and Korean isolates, which included Vibrio sp., Lactococcus garvieae, Photobacterium damsela subsp. piscicida, and unidentified Gram-positive bacteria. The majority of these bacterial isolates displayed high-level resistance with a minimum inhibitory concentrations (MICs) equal to or greater than 250 microg/ml of oxytetracycline and only four isolates had MICs less than 31.3 microg/ml. 16S rDNA RFLP typing of tet(M)-positive Vibrio isolates suggests that these are clonal populations of the same phylotype specific to a particular location. One Vibrio clone (phylotype III), however, is widely disseminated, being detected during different sampling years, at different locations, and in different fish species in both Japan and Korea. The tet(S) gene was detected in L. garvieae from yellowtail in Japan and in Vibrio sp. from seawater in Korea. This is the first report of tet(S) occurrence in Gram-negative facultative anaerobes. These results suggest that tet(M) and tet(S) genes are present in fish intestinal and seawater bacteria at aquaculture sites and could be an important reservoir of tetracycline resistance genes in the marine environment.     

Genomics of Adaptation during Experimental Evolution of the Opportunistic Pathogen Pseudomonas aeruginosa

Adaptation is likely to be an important determinant of the success of many pathogens, for example when colonizing a new host species, when challenged by antibiotic treatment, or in governing the establishment and progress of long-term chronic infection. Yet, the genomic basis of adaptation is poorly understood in general, and for pathogens in particular. We investigated the genetics of adaptation to cystic fibrosis-like culture conditions in the presence and absence of fluoroquinolone antibiotics using the opportunistic pathogen Pseudomonas aeruginosa. Whole-genome sequencing of experimentally evolved isolates revealed parallel evolution at a handful of known antibiotic resistance genes. While the level of antibiotic resistance was largely determined by these known resistance genes, the costs of resistance were instead attributable to a number of mutations that were specific to individual experimental isolates. Notably, stereotypical quinolone resistance mutations in DNA gyrase often co-occurred with other mutations that, together, conferred high levels of resistance but no consistent cost of resistance. This result may explain why these mutations are so prevalent in clinical quinolone-resistant isolates. In addition, genes involved in cyclic-di-GMP signalling were repeatedly mutated in populations evolved in viscous culture media, suggesting a shared mechanism of adaptation to this CF–like growth environment. Experimental evolutionary approaches to understanding pathogen adaptation should provide an important complement to studies of the evolution of clinical isolates.     

Variations in antibiotic resistance profile in Enterobacteriaceae isolated from wild Australian mammals

We carried out a retrospective analysis of 946 strains of Enterobacteriaceae isolated from wild Australian mammals between 1993 and 1997. The prevalence of resistance to fixed concentrations of 32 antimicrobial agents was determined, and the respective roles that taxonomic family of the host, state of origin and bacterial species play in defining prevalence and range of resistance were investigated. Our results demonstrated a low but widespread prevalence of antimicrobial resistance in wild isolates. Only amikacin, ciprofloxacin, meropenem and gentamicin inhibited growth in all 946 samples. There was extensive variation in the combination of antibiotics to which isolates were resistant, and multiple antibiotic resistance was common. Geographical location and host group significantly influenced the antibiotic resistance profile of an isolate, whereas bacterial species influenced both the resistance profile of an isolate and the number of antibiotics it was resistant to. The role of these factors in determining observed antibiotic resistance profiles suggests that any study measuring resistance in wild isolates should include the broadest possible range of bacterial species, host species and sampling locations. As such, this study provides an important new baseline for future measurements of antibiotic resistance in the Australian environment.     

At least two origins of fungicide resistance in grapevine downy mildew populations

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc1 complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.     

RNAi-mediated resistance to diverse isolates belonging to two virus species involved in Cassava brown streak disease

Cassava brown streak disease (CBSD) is emerging as one of the most important viral diseases of cassava (Manihot esculenta) and is considered today as the biggest threat to cassava cultivation in East Africa. The disease is caused by isolates of at least two phylogenetically distinct species of single-stranded RNA viruses belonging to the family Potyviridae, genus Ipomovirus. The two species are present predominantly in the coastal lowland [Cassava brown streak virus (CBSV); Tanzania and Mozambique] and highland [Cassava brown streak Uganda virus (CBSUV); Lake Victoria Basin, Uganda, Kenya and Malawi] in East Africa. In this study, we demonstrate that CBSD can be efficiently controlled using RNA interference (RNAi). Three RNAi constructs targeting the highland species were generated, consisting of the full-length (FL; 894 nucleotides), 397-nucleotide N-terminal and 491-nucleotide C-terminal portions of the coat protein (CP) gene of a Ugandan isolate of CBSUV (CBSUV-[UG:Nam:04]), and expressed constitutively in Nicotiana benthamiana. After challenge with CBSUV-[UG:Nam:04], plants homozygous for FL-CP showed the highest resistance, followed by the N-terminal and C-terminal lines with similar resistance. In the case of FL, approximately 85% of the transgenic plant lines produced were completely resistant. Some transgenic lines were also challenged with six distinct isolates representing both species: CBSV and CBSUV. In addition to nearly complete resistance to the homologous virus, two FL plant lines showed 100% resistance and two C-terminal lines expressed 50-100% resistance, whereas the N-terminal lines succumbed to the nonhomologous CBSV isolates. Northern blotting revealed a positive correlation between the level of transgene-specific small interfering RNAs detected in transgenic plants and the level of virus resistance. This is the first demonstration of RNAi-mediated resistance to CBSD and protection across very distant isolates (more than 25% in nucleotide sequence) belonging to two different species: Cassava brown streak virus and Cassava brown streak Uganda virus.     

In Vitro Communities Derived from Oral and Gut Microbial Floras Inhibit the Growth of Bacteria of Foreign Origins

The gastrointestinal (GI) tract is home to trillions of microbes. Within the same GI tract, substantial differences in the bacterial species that inhabit the oral cavity and intestinal tract have been noted. While the influence of host environments and nutritional availability in shaping different microbial communities is widely accepted, we hypothesize that the existing microbial flora also plays a role in selecting the bacterial species that are being integrated into the community. In this study, we used cultivable microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine this hypothesis. Microbes from these two areas were harvested and cultured using the same nutritional conditions, which led to two distinct microbial communities, each with about 20 different species as revealed by PCR-based denaturing gradient gel electrophoresis analysis. In vitro community competition assays showed that the two microbial floras exhibited antagonistic interactions toward each other. More interestingly, all the original isolates tested and their closely related species displayed striking community preferences: They persisted when introduced into the bacterial community of the same origin, while their viable count declined more than three orders of magnitude after 4 days of coincubation with the microbial flora of foreign origin. These results suggest that an existing microbial community might impose a selective pressure on incoming foreign bacterial species independent of host selection. The observed inter-flora interactions could contribute to the protective effect of established microbial communities against the integration of foreign bacteria to maintain the stability of the existing communities.     

Genotype-by-environment interactions and adaptation to local temperature affect immunity and fecundity in Drosophila melanogaster

Natural populations of most organisms harbor substantial genetic variation for resistance to infection. The continued existence of such variation is unexpected under simple evolutionary models that either posit direct and continuous natural selection on the immune system or an evolved life history "balance" between immunity and other fitness traits in a constant environment. However, both local adaptation to heterogeneous environments and genotype-by-environment interactions can maintain genetic variation in a species. In this study, we test Drosophila melanogaster genotypes sampled from tropical Africa, temperate northeastern North America, and semi-tropical southeastern North America for resistance to bacterial infection and fecundity at three different environmental temperatures. Environmental temperature had absolute effects on all traits, but there were also marked genotype-by-environment interactions that may limit the global efficiency of natural selection on both traits. African flies performed more poorly than North American flies in both immunity and fecundity at the lowest temperature, but not at the higher temperatures, suggesting that the African population is maladapted to low temperature. In contrast, there was no evidence for clinal variation driven by thermal adaptation within North America for either trait. Resistance to infection and reproductive success were generally uncorrelated across genotypes, so this study finds no evidence for a fitness tradeoff between immunity and fecundity under the conditions tested. Both local adaptation to geographically heterogeneous environments and genotype-by-environment interactions may explain the persistence of genetic variation for resistance to infection in natural populations.     

Host ecology shapes geographical variation for resistance to bacterial infection in Drosophila melanogaster

1. Geographically distinct host populations often experience very different ecological conditions. These variable ecological conditions impact the strength of selection that these hosts experience from their parasites. 2. Numerous studies have characterized geographical patterns of resistance to infection among natural populations in the context of host-parasite local adaptation, but what other factors might contribute to these differences? 3. Here, we determined whether 20 naturally isolated populations of Drosophila melanogaster collected along the East Coast of the United States varied for survival after being inoculated with one of two species of bacteria--Lactococcus lactis and Pseudomonas aeruginosa. We then asked whether host environment accounted for the observed patterns of resistance. 4. Resistance to both types of infection varied spatially. The hosts' natural environment was predictive of the observed spatial variation in resistance to L. lactis, but not P. aeruginosa, infection. Specifically, hosts exposed to species-rich bacterial communities were more likely to survive the infection. 5. We conclude that biotic characteristics of the host environment, specifically the number of species of bacteria hosts encounter, shape host resistance to bacterial infection in nature. We discuss our results in the context of what is known about the evolutionary ecology of resistance in invertebrate systems.     

Local biotic environment shapes the spatial scale of bacteriophage adaptation to bacteria

The ecological, epidemiological, and evolutionary consequences of host-parasite interactions are critically shaped by the spatial scale at which parasites adapt to hosts. The scale of interaction between hyperparasites and their parasites is likely to be influenced by the host of the parasite and potentially likely to differ among within-host environments. Here we examine the scale at which bacteriophages adapt to their host bacteria by studying natural isolates from the surface or interior of horse chestnut leaves. We find that phages are more infective to bacteria from the same tree relative to those from other trees but do not differ in infectivity to bacteria from different leaves within the same tree. The results suggest that phages target common bacterial species, including an important plant pathogen, within plant host tissues; this result has important implications for therapeutic phage epidemiology. Furthermore, we show that phages from the leaf interior are more infective to their local hosts than phages from the leaf surface are to theirs, suggesting either increased resistance of bacteria on the leaf surface or increased phage adaptation within the leaf. These results highlight that biotic environment can play a key role in shaping the spatial scale of parasite adaptation and influencing the outcome of coevolutionary interactions.     

The bacterial and fungal nest microbiomes in populations of the social spider Stegodyphus dumicola

Social spiders of the species Stegodyphus dumicola live in communal nests with hundreds of individuals and are characterized by extremely low species-wide genetic diversity. The lack of genetic diversity in combination with group living imposes a potential threat for infection by pathogens. We therefore proposed that specific microbial symbionts inhabiting the spider nests may provide antimicrobial defense. To compare the bacterial and fungal diversity in 17 nests from three different locations in Namibia, we used 16S rRNA gene and internal transcribed spacer (ITS2) sequencing. The nest microbiomes differed between geographically distinct spider populations and appeared largely determined by the local environment. Nevertheless, we identified a core microbiome consisting of four bacterial genera (Curtobacterium, Modestobacter, Sphingomonas, Massilia) and four fungal genera (Aureobasidium, Didymella, Alternaria, Ascochyta), which likely are selected from surrounding soil and plants by the nest environment. We did not find indications for a strain- or species-specific symbiosis in the nests. Isolation of bacteria and fungi from nest material retrieved a few bacterial strains with antimicrobial activity but a number of antimicrobial fungi, including members of the fungal core microbiome. The significance of antimicrobial taxa in the nest microbiome for host protection remains to be shown.     

At Least Two Origins of Fungicide Resistance in Grapevine Downy Mildew Populations

Quinone outside inhibiting (QoI) fungicides represent one of the most widely used groups of fungicides used to control agriculturally important fungal pathogens. They inhibit the cytochrome bc₁ complex of mitochondrial respiration. Soon after their introduction onto the market in 1996, QoI fungicide-resistant isolates were detected in field plant pathogen populations of a large range of species. However, there is still little understanding of the processes driving the development of QoI fungicide resistance in plant pathogens. In particular, it is unknown whether fungicide resistance occurs independently in isolated populations or if it appears once and then spreads globally by migration. Here, we provide the first case study of the evolutionary processes that lead to the emergence of QoI fungicide resistance in the plant pathogen Plasmopara viticola. Sequence analysis of the complete cytochrome b gene showed that all resistant isolates carried a mutation resulting in the replacement of glycine by alanine at codon 143 (G143A). Phylogenetic analysis of a large mitochondrial DNA fragment including the cytochrome b gene (2,281 bp) across a wide range of European P. viticola isolates allowed the detection of four major haplotypes belonging to two distinct clades, each of which contains a different QoI fungicide resistance allele. This is the first demonstration that a selected substitution conferring resistance to a fungicide has occurred several times in a plant-pathogen system. Finally, a high population structure was found when the frequency of QoI fungicide resistance haplotypes was assessed in 17 French vineyards, indicating that pathogen populations might be under strong directional selection for local adaptation to fungicide pressure.     

Pre‐adapting parasitic phages to a pathogen leads to increased pathogen clearance and lowered resistance evolution with Pseudomonas aeruginosa cystic fibrosis bacterial isolates

Recent years have seen renewed interest in phage therapy – the use of viruses to specifically kill disease‐causing bacteria – because of the alarming rise in antibiotic resistance. However, a major limitation of phage therapy is the ease at with bacteria can evolve resistance to phages. Here, we determined whether in vitro experimental coevolution can increase the efficiency of phage therapy by limiting the resistance evolution of intermittent and chronic cystic fibrosis Pseudomonas aeruginosa lung isolates to four different phages. We first pre‐adapted all phage strains against all bacterial strains and then compared the efficacy of pre‐adapted and nonadapted phages against ancestral bacterial strains. We found that evolved phages were more efficient in reducing bacterial densities than ancestral phages. This was primarily because only 50% of bacterial strains were able to evolve resistance to evolved phages, whereas all bacteria were able to evolve some level of resistance to ancestral phages. Although the rate of resistance evolution did not differ between intermittent and chronic isolates, it incurred a relatively higher growth cost for chronic isolates when measured in the absence of phages. This is likely to explain why evolved phages were more effective in reducing the densities of chronic isolates. Our data show that pathogen genotypes respond differently to phage pre‐adaptation, and as a result, phage therapies might need to be individually adjusted for different patients.