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Defining the physiological role of a gene product relies on interpreting phenotypes caused by the lack, or alteration, of the respective gene product. Mutations in critical genes often lead to easily recognized phenotypes that can include changes in cellular growth, metabolism, structure etc. However, mutations in many important genes may fail to generate an obvious defect unless additional perturbations are caused by medium or genetic background. The latter scenario is exemplified by RidA proteins. In vitro RidA proteins deaminate numerous imine/enamines, including those generated by serine/threonine dehydratase IlvA (EC:4.3.1.19) from serine or threonine – 2-aminoacrylate (2AA) and 2-aminocrotonate (2AC), respectively. Despite this demonstrable biochemical activity, a lack of RidA has little to no effect on growth of E. coli or S. enterica without the application of additional metabolic perturbation. A cellular role of RidA is to prevent accumulation of 2AA which, if allowed to persist, can irreversibly damage pyridoxal 5’-phosphate (PLP)-dependent enzymes, causing global metabolic stress. Because the phenotypes caused by a lack of RidA are dependent on the unique structure of each metabolic network, the link between RidA function and 2AA stress is difficult to demonstrate in some organisms. The current study used coculture experiments to exacerbate differences in growth caused by the lack of RidA in S. enterica and E. coli. Results described here solidify the established role of RidA in removing 2AA, while also presenting evidence for a role of RidA in enhancing flux towards isoleucine biosynthesis in E. coli. Overall, these data emphasize that metabolic networks can generate distinct responses to perturbation, even when the individual components are conserved.

The distribution of fitness effects of new mutations plays a central role in evolutionary biology. Estimates of the distribution of fitness effect from experimental mutation accumulation lines are compromised by the complete linkage disequilibrium between mutations in different lines. To reduce the linkage disequilibrium, we constructed 2 sets of recombinant inbred lines from a cross of 2 Caenorhabditis elegans mutation accumulation lines. One set of lines ("RIAILs") was intercrossed for 10 generations prior to 10 generations of selfing; the second set of lines ("RILs") omitted the intercrossing. Residual linkage disequilibrium in the RIAILs is much less than in the RILs, which affects the inferred distribution of fitness effect when the sets of lines are analyzed separately. The best-fit model estimated from all lines (RIAILs + RILs) infers a large fraction of mutations with positive effects (∼40%); models that constrain mutations to have negative effects fit much worse. The conclusion is the same using only the RILs. For the RIAILs, however, models that constrain mutations to have negative effects fit nearly as well as models that allow positive effects. When mutations in high linkage disequilibrium are pooled into haplotypes, the inferred distribution of fitness effect becomes increasingly negative-skewed and leptokurtic. We conclude that the conventional wisdom—most mutations have effects near 0, a handful of mutations have effects that are substantially negative, and mutations with positive effects are very rare—is likely correct, and that unless it can be shown otherwise, estimates of the distribution of fitness effect that infer a substantial fraction of mutations with positive effects are likely confounded by linkage disequilibrium. [ABSTRACT FROM AUTHOR]

Severe acute respiratory syndrome coronavirus 2 (SARS‑CoV‑2) variants continue to emerge and cocirculate in humans and wild animals. The factors driving the emergence and replacement of novel variants and recombinants remain incompletely understood. Herein, we comprehensively characterized the competitive fitness of SARS‐CoV‐2 wild type (WT) and three variants of concern (VOCs), Alpha, Beta and Delta, by coinfection and serial passaging assays in different susceptible cells. Deep sequencing analyses revealed cell‐specific competitive fitness: the Beta variant showed enhanced replication fitness during serial passage in Caco‐2 cells, whereas the WT and Alpha variant showed elevated fitness in Vero E6 cells. Interestingly, a high level of neutralizing antibody sped up competition and completely reshaped the fitness advantages of different variants. More importantly, single clone purification identified a significant proportion of homologous recombinants that emerged during the passage history, and immune pressure reduced the frequency of recombination. Interestingly, a recombination hot region located between nucleotide sites 22,995 and 28,866 of the viral genomes could be identified in most of the detected recombinants. Our study not only profiled the variable competitive fitness of SARS‐CoV‐2 under different conditions, but also provided direct experimental evidence of homologous recombination between SARS‐CoV‐2 viruses, as well as a model for investigating SARS‐CoV‐2 recombination. [ABSTRACT FROM AUTHOR]

Competitive fitness assays are widely used in evolutionary biology and typically rely on a reference strain to compare different focal genotypes. This approach implicitly relies on the absence of interaction between the competing genotypes. In other words, the performance of the reference strain must not depend on the competitor. This report scrutinized this assumption by competing diverged Drosophila simulans populations against a common reference strain. We detected strong evidence for interaction between the competing genotypes: (1) Frequency‐dependent selection was common with opposite effects in genetically diverged populations. (2) Temporal heterogeneity of fitness estimates, which can be partially attributed to a competitor‐specific delay in the eclosion of the reference strain. We propose that this inconsistent behavior of the reference strain can be considered a specific case of a genotype × environment interaction. Focal populations could modify the environment of the reference strain, either indirectly by altering the microbiome composition and food availability or directly by genotype‐specific cannibalism. Our results provide new insights into the interaction of diverged genotypes and have important implications for the interpretation of competitive fitness assays. [ABSTRACT FROM AUTHOR]

The surface of fruits is heterogenous in term of its microenvironment hence dictate the kind of microflora that develops during storage. A better understanding of spoilage organisms would lead to better preservation methods. The pomegranate was chosen, since its sturdy and spoils slow at room temperature and is ideal for studying fruit spoilage in-situ. In the current study we isolated organisms from fruit surface and study the spoilage and competition amongst microbial species. Total 17 unique bacterial isolates from pomegranate were identified. The 16S rRNA gene identification placed them in 8 major genera (Acinetobacter, Micrococcus, Pantoea, Microbacterium, Strenotrophomonas, Bacillus, Staphylococcus and Exiguobacterium). Competition assay among isolate suggested that Exiguobacterium is dominant species followed by Micrococcus, Pantoea and Bacillus. The consortium of 3 different combinations (5 bacteria each) of isolated bacteria showed the spoilage phenotype on pomegranate. Except for 3 bacterial isolates, the rest of the isolates produced any one or multiple enzymes associated with the food spoilage (cellulase, amylase, lactase, pectinase and protease). The isolates were checked for the presence of genes associated with antibiotic resistance and 78.9% of the tested micro-organisms were blaTEM positive. Aminoglycoside resistance genes were present in 10% of the tested microbes. This study demonstrated interspecies competition amongst spoilage organisms. This understanding of surface flora of fruit would give better insights to preserve fruits. [ABSTRACT FROM AUTHOR]

CD4+ Foxp3+ T regulatory (Treg) cells are key players in preventing lethal autoimmunity. Tregs undertake differentiation processes and acquire diverse functional properties. However, how Treg's differentiation and functional specification are regulated remains incompletely understood. Here, we report that gradient expression of TCF1 and LEF1 distinguishes Tregs into three distinct subpopulations, particularly highlighting a subset of activated Treg (aTreg) cells. Treg-specific ablation of TCF1 and LEF1 renders the mice susceptible to systemic autoimmunity. TCF1 and LEF1 are dispensable for Treg's suppressive capacity but essential for maintaining a normal aTreg pool and promoting Treg's competitive survival. As a consequence, the development of T follicular regulatory (Tfr) cells, which are a subset of aTreg, is abolished in TCF1/LEF1-conditional knockout mice, leading to unrestrained T follicular helper (Tfh) and germinal center B cell responses. Thus, TCF1 and LEF1 act redundantly to control the maintenance and functional specification of Treg subsets to prevent autoimmunity.

Previous work reported that deletion of the Enzyme IIAB subunits (EIIAB Man and manL ) of the glucose phosphotransferase system (PTS) (glucose-PTS, manLMNO ) in Streptococcus sanguinis impacted carbon catabolite repression and bacterial fitness. Here, a single-nucleotide polymorphism in ManN, ManNA91E, produced the unusual phenotype of increased excretion of organic acids and H 2 O 2 yet elevated PTS activities. To characterize the contributions of each component of the glucose-PTS to bacterial fitness, we performed genetic analyses by deleting from S. sanguinis SK36 the entire operon and each EII Man subunit individually; and genes encoding the catabolite control protein A (Δ ccpA ) and the redox regulator Rex (Δ rex ) for comparison. Deletion of each subunit incurred a growth defect on glucose partly due to elevated excretion of H 2 O 2 ; when supplemented with catalase, this defect was rescued, instead resulting in a significantly higher yield than the parent. All glucose-PTS deletion mutants presented an increased antagonism against the oral pathobiont Streptococcus mutans , a phenotype absent in Δ ccpA despite increased H 2 O 2 output. A shift in the pyruvate node toward mixed acid fermentation and increased arginine deiminase activity enhanced pH homeostasis in glucose-PTS mutants but not Δ ccpA . Despite the purported ability of Rex to regulate central carbon metabolism, deletion of rex had no significant impact on most of the phenotypes discussed here. These findings place glucose-PTS in the pivotal position of controlling central carbon flux in streptococci, with critical outcomes impacting acidogenicity, aciduricity, pH homeostasis, and antagonism, highlighting its potential as a therapeutic target for treating diseases with a dysbiotic microbiome., Importance: Management of carbohydrate metabolism and environmental stress is key to the survival of oral commensal species such as S. sanguinis . Antagonism of oral pathobionts and modulation of the environmental pH and oxidative potential by commensals are crucial to the maintenance of microbial homeostasis and prevention of oral diseases including dental caries. It is therefore vital to understand how these species regulate sugar fermentation, production of acids and ammonia, and stress management in an environment known for a feast-and-famine cycle of carbohydrates and similar fluctuations in pH and oxygen tension. Here, we detail that genetic alterations of the glucose-PTS transporter in S. sanguinis can significantly affect the regulation of factors required for bacterial fitness and homeostatic ability independent of known catabolic regulators. It is then discussed how these changes may impact the survival of streptococcal species and affect caries onset.

Abstract Experimental evolution studies with microorganisms such as bacteria and yeast have been an increasingly important and powerful tool to draw long‐term inferences of how microbes interact. However, while several strains of the same species often exist in natural environments, many ecology and evolution studies in microbes are typically performed with isogenic populations of bacteria or yeast. In the present study, we firstly perform a genotypic and phenotypic characterization of two laboratory and eight natural strains of the yeast Schizosaccharomyces pombe. We then propagated, in a rich resource environment, yeast communities of 2, 3, 4, and 5 strains for hundreds of generations and asked which fitness‐related phenotypes—maximum growth rate or relative competitive fitness—would better predict the outcome of a focal strain during the propagations. While the strain's growth rates would wrongly predict long‐term coexistence, pairwise competitive fitness with a focal strain qualitatively predicted the success or extinction of the focal strain by a simple multigenotype population genetics model, given the initial community composition. Interestingly, we have also measured the competitive fitness of the ancestral and evolved communities by the end of the experiment (≈370 generations) and observed frequent maladaptation to the abiotic environment in communities with more than three members. Overall, our results aid establishing pairwise competitive fitness as good qualitative measurement of long‐term community composition but also reveal a complex adaptive scenario when trying to predict the evolutionary outcome of those communities.

To investigate the phenotypic evolution of West Nile virus (WNV) in California, we competed sixteen isolates made during 2007-08 against COAV997-5nt, a genetically marked clone from the founding 2003 California isolate COAV997-2003. Using in vivo fitness competitions in House Finches (HOFI) and Culex tarsalis mosquitoes, we found that the majority of WNV WN02 and SW03 genotype isolates exhibited elevated replicative fitness in both hosts compared to COAV997-5nt. Increased replicative capacity in HOFIs was not associated with increased mortality, indicating that these isolates had not gained avian virulence. One WN02 isolate from Coachella Valley, a region geographically close to the isolation of COAV997, showed neutral fitness in HOFIs and reduced fitness in Cx. tarsalis. Two isolates from Kern County and Sacramento/Yolo County out-competed COAV997-nt in HOFIs, but were transmitted less efficiently by Cx. tarsalis. Competition demonstrated neutral or increased fitness that appeared independent of both WN02 and SW03 genotypes.

Gametogenesis is an evolutionarily conserved developmental program whereby a diploid progenitor cell undergoes meiosis and cellular remodeling to differentiate into haploid gametes, the precursors for sexual reproduction. Even in the simple eukaryotic organism Saccharomyces cerevisiae, the meiotic transcriptome is very rich and complex, thereby necessitating new tools for functional studies. Here, we report the construction of 5 stage-specific, inducible complementary DNA libraries from meiotic cells that represent over 84% of the genes found in the budding yeast genome. We employed computational strategies to detect endogenous meiotic transcript isoforms as well as library-specific gene truncations. Furthermore, we developed a robust screening pipeline to test the effect of each complementary DNA on competitive fitness. Our multiday proof-of-principle time course revealed 877 complementary DNAs that were detrimental for competitive fitness when overexpressed. The list included mitochondrial proteins that cause dose-dependent disruption of cellular respiration as well as library-specific gene truncations that expose a dominant negative effect on competitive growth. Together, these highquality complementary DNA libraries provide an important tool for systematically identifying meiotic genes, transcript isoforms, and protein domains that are important for a specific biological function. [ABSTRACT FROM AUTHOR]

The filamentous fungus Neurospora crassa, a model microbial eukaryote, has a life cycle with many features that make it suitable for studying experimental evolution. However, it has lacked a general tool for estimating relative fitness of different strains in competition experiments. To remedy this need, we constructed N. crassa strains that contain a modified csr-1 locus and developed an assay for detecting the proportion of the marked strain using a post PCR high resolution melting assay. DNA extraction from spore samples can be performed on 96-well plates, followed by a PCR step, which allows many samples to be processed with ease. Furthermore, we suggest a Bayesian approach for estimating relative fitness from competition experiments that takes into account the uncertainty in measured strain proportions. We show that there is a fitness effect of the mating type locus, as mating type mat a has a higher competitive fitness than mat A. The csr-1* marker also has a small fitness effect, but is still a suitable marker for competition experiments. As a proof of concept, we estimate the fitness effect of the qde-2 mutation, a gene in the RNA interference pathway, and show that its competitive fitness is lower than what would be expected from its mycelial growth rate alone.

ABSTRACT During stationary phase in Escherichia coli, the expression of the ribosome modulation factor (RMF) protein participates in the dimerization of two 70S ribosomes, ultimately creating a 100S particle. 100S ribosomes are commonly thought to function to preserve ribosomes as growth ceases and cells begin to catabolize intracellular components, including proteins, during their transition into stationary phase. Here, we show that the rates of stationary-phase ribosomal degradation are increased in an rmf mutant strain that cannot produce 100S ribosomes, resulting in deficiencies in outgrowth upon reinoculation into fresh medium. Upon coinoculation in LB medium, the mutant exhibits a delay in entry into log phase, differences in growth rates, and an overall reduction in relative fitness during competition. Unexpectedly, the rmf mutant exhibited shorter generation times than wild-type cells during log phase, both in monoculture and during competition. These doubling times of ∼13 min suggest that failure to maintain ribosomal balance affects the control of cell division. Though the timing of entry into and exit from log phase is altered, 100S ribosomes are not essential for long-term viability of the rmf mutant when grown in monoculture. IMPORTANCE Ribosomes are the sole source in any cell for new protein synthesis that is vital to maintain life. While ribosomes are frequently consumed as sources of nutrients under low-nutrient conditions, some ribosomes appear to be preserved for later use. The failure to maintain the availability of these ribosomes can lead to a dire consequence upon the influx of new nutrients, as cells are unable to efficiently replenish their metabolic machinery. It is important to study the repercussions, consequences, and mechanisms of survival in cells that cannot properly maintain the availability of their ribosomes in order to better understand their mechanisms of survival during competition under nutrient-depleted conditions.

Introduction. Such sports as fitness have high demands on the motor system, mainly on the foot arch. Repeated movements can expose the ankle joint and the sole of children practicing fitness sports to high loading. Aim of Study. The aim of this study was to determine the incidence of incorrect body posture, flexibility and flatfoot in children attending competition fitness classes. Material and Methods. The study population was composed of 18 children aged 8.9 to 10.3 years of age. Body posture was assessed using the eye-viewing method according to Klein and Thomas. Backbone flexibility was measured using tests of Schober's and Stibor's symptoms, right and left backbone lateral flexibility, Thomayer's symptom, Otto's inclination and the reclination symptom. Podoscopy was used to assess flatfoot degrees. All indicators were evaluated individually and statistical significance of the relationship between individual dimensions of body posture and flat feet were assessed using the chi-square (X2) test. Results. The highest proportion of incorrect body posture in all tested children was recorded in the dimension of hips and shoulders (78%), backbone curvature (61%) and shoulderblades (55.6%). Flat feet were diagnosed according to Kapandji in 27.8% and 11.2% cases according to Srdečný. When investigating the relationship of flat feet in the test of Kapandji and Srdečný to the dimension of body posture statistical significance was recorded between flat feet and the dimension of hips and shoulders and backbone curvature. In the test of Srdečný a significant relationship was observed between flat feet and the dimensions of head, shoulderblade and abdomen. Conclusions. This study focused on the verification of the impact of the training process in fitness sports practiced by children on their body posture and flatfoot incidence. We recommend observing children for flatfoot symptoms, which can negatively influence the postural function in young athletes. [ABSTRACT FROM AUTHOR]

Experimental evolution studies with microorganisms such as bacteria and yeast have been an increasingly important and powerful tool to draw long‐term inferences of how microbes interact. However, while several strains of the same species often exist in natural environments, many ecology and evolution studies in microbes are typically performed with isogenic populations of bacteria or yeast. In the present study, we firstly perform a genotypic and phenotypic characterization of two laboratory and eight natural strains of the yeast Schizosaccharomyces pombe. We then propagated, in a rich resource environment, yeast communities of 2, 3, 4, and 5 strains for hundreds of generations and asked which fitness‐related phenotypes—maximum growth rate or relative competitive fitness—would better predict the outcome of a focal strain during the propagations. While the strain's growth rates would wrongly predict long‐term coexistence, pairwise competitive fitness with a focal strain qualitatively predicted the success or extinction of the focal strain by a simple multigenotype population genetics model, given the initial community composition. Interestingly, we have also measured the competitive fitness of the ancestral and evolved communities by the end of the experiment (≈370 generations) and observed frequent maladaptation to the abiotic environment in communities with more than three members. Overall, our results aid establishing pairwise competitive fitness as good qualitative measurement of long‐term community composition but also reveal a complex adaptive scenario when trying to predict the evolutionary outcome of those communities. [ABSTRACT FROM AUTHOR]

The potential spread of infectious diseases in response to climate change and rising sea surface temperatures in temperate regions has been a growing concern for the past several decades. Extreme heat waves in the North Atlantic and North Sea regions have been correlated with an increase in human Vibrio infections; of particular concern to human health are Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus. While these species are well-known to cause disease in humans, most environmental strains are not pathogenic. Studying not only the behavior of the pathogenic strains, but that of non-pathogenic environmental isolates, may better elucidate their ecological relationship in their native microbiome and the dispersal of these species in coastal regions. Using red fluorescent protein-tagged and gentamycin-resistant V. cholerae, V. parahaemolyticus, and V. vulnificus strains, we investigated whether increasing temperatures confer greater competitive fitness to these species when incubated within a natural North Sea water sample still containing its microbiome in a small-scale niche investigation. Increased incubation temperatures alone did not confer a competitive advantage to V. cholerae, V. parahaemolyticus, and V. vulnificus. The microbial community could limit Vibrio growth at all temperatures. To the best of our knowledge, we also demonstrate the first (albeit unintentional) genetic modification of multiple species of marine bacteria through the introduction of a genetically modified V. vulnificus strain into a natural water sample in a contained system.

Respiratory chains are crucial for cellular energy conversion and consist of multi‐subunit complexes that can assemble into supercomplexes. These structures have been intensively characterized in various organisms, but their physiological roles remain unclear. Here, we elucidate their function by leveraging a high‐resolution structural model of yeast respiratory supercomplexes that allowed us to inhibit supercomplex formation by mutation of key residues in the interaction interface. Analyses of a mutant defective in supercomplex formation, which still contains fully functional individual complexes, show that the lack of supercomplex assembly delays the diffusion of cytochrome c between the separated complexes, thus reducing electron transfer efficiency. Consequently, competitive cellular fitness is severely reduced in the absence of supercomplex formation and can be restored by overexpression of cytochrome c. In sum, our results establish how respiratory supercomplexes increase the efficiency of cellular energy conversion, thereby providing an evolutionary advantage for aerobic organisms. Synopsis: Mitochondrial respiratory supercomplexes are found in various organisms, however their physiological function remains elusive. This study reveals that supercomplexes enhance mitochondrial electron transfer, resulting in increased competitive fitness. A 3.17 Å cryo‐EM structure of Saccharomyces cerevisiae respiratory supercomplex was generated.Guided by this structure, mutants were created to inhibit supercomplex formation.Mitochondrial electron transfer efficiency is severely impaired in mutants lacking supercomplexes.Disruption of supercomplexes results in decreased competitive fitness. [ABSTRACT FROM AUTHOR]

The misfolding avoidance hypothesis postulates that sequence mutations render proteins cytotoxic and therefore the higher the gene expression, the stronger the operation of selection against substitutions. This translates into prediction that relative toxicity of extant proteins is higher for those evolving faster. In the present experiment, we selected pairs of yeast genes which were paralogous but evolving at different rates. We expressed them artificially to high levels. We expected that toxicity would be higher for ones bearing more mutations, especially that overcrowding should rather exacerbate than reverse the already existing differences in misfolding rates. We did find that the applied mode of overexpression caused a considerable decrease in fitness and that the decrease was proportional to the amount of excessive protein. However, it was not higher for proteins which are normally expressed at lower levels (and have less conserved sequence). This result was obtained consistently, regardless whether the rate of growth or ability to compete in common cultures was used as a proxy for fitness. In additional experiments, we applied factors that reduce accuracy of translation or enhance structural instability of proteins. It did not change a consistent pattern of independence between the fitness cost caused by overexpression of a protein and the rate of its sequence evolution. [ABSTRACT FROM AUTHOR]

Albendazole and ivermectin are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channel (GluCl) genes, but it is unknown whether these genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans , we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required loss of two GluCl genes ( avr-14 and avr-15 ) or loss of three GluCl genes ( avr-14 , avr-15 , and glc-1 ). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance., Competing Interests: COMPETING INTERESTS The authors have declared that no competing interests exist

The 3'-to-5' exoribonuclease in coronavirus (CoV) nonstructural protein 14 (nsp14-ExoN) mediates RNA proofreading during genome replication. ExoN catalytic residues are arranged in three motifs: I (DE), II (E), and III (D). Alanine replacement of the motif I residues (AA-E-D; four nucleotide substitutions) in murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV yields viable mutants with impaired replication and fitness, increased mutation rates, and attenuated virulence in vivo. Despite these impairments, MHV- and SARS-CoV ExoN motif I AA mutants (ExoN-AA) have not reverted at motif I in diverse in vitro and in vivo environments, suggesting that profound fitness barriers prevent motif I reversion. To test this hypothesis, we engineered MHV-ExoN-AA with 1, 2, or 3 nucleotide mutations along genetic pathways to AA-to-DE reversion. We show that engineered intermediate revertants were viable but had no increased replication or competitive fitness compared to that of MHV-ExoN-AA. In contrast, a low-passage-number (passage 10 [P10]) MHVExoN- AA showed increased replication and competitive fitness without reversion of ExoN-AA. Finally, engineered reversion of ExoN-AA to ExoN-DE in the presence of ExoN-AA passage-adaptive mutations resulted in significant fitness loss. These results demonstrate that while reversion is possible, at least one alternative adaptive pathway is more rapidly advantageous than intermediate revertants and may alter the genetic background to render reversion detrimental to fitness. Our results provide an evolutionary rationale for lack of ExoN-AA reversion, illuminate potential multiprotein replicase interactions and coevolution, and support future studies aimed at stabilizing attenuated CoV ExoN-AA mutants. [ABSTRACT FROM AUTHOR]

Chaperone proteins are crucial for proper protein folding and quality control, especially when cells encounter stress caused by non-optimal temperatures. DnaK is one of such essential chaperones in bacteria. Although DnaK has been well characterized, the function of its intrinsically disordered C-terminus has remained enigmatic as the deletion of this region has been shown to either enhance or reduce its protein folding ability. We have shown previously that DnaK interacts with toxin GraT of the GraTA toxin-antitoxin system in Pseudomonas putida. Interestingly, the C-terminal truncation of DnaK was shown to alleviate GraT-caused growth defects. Here, we aim to clarify the importance of DnaK in GraT activity. We show that DnaK increases GraT toxicity, and particularly important is the negatively charged motif in the DnaK C-terminus. Given that GraT has an intrinsically disordered N-terminus, the assistance of DnaK is probably needed for re-modelling the toxin structure. We also demonstrate that the DnaK C-terminal negatively charged motif contributes to the competitive fitness of P. putida at both high and optimal growth temperatures. Thus, our data suggest that the disordered C-terminal end of DnaK enhances the chaperone functionality.

ABSTRACT Cell-cell communication mediated by peptide pheromones (cCF10 [CF]) is essential for high-frequency plasmid transfer in vitro in Enterococcus faecalis. To examine the role of pheromone signaling in vivo, we established either a CF-producing (CF+) recipient or a recipient producing a biologically inactive variant of CF (CF− recipient) in a germfree mouse model 3 days before donor inoculation and determined transfer frequencies of the pheromone-inducible plasmid pCF10. Plasmid transfer was detected in the upper and middle sections of the intestinal tract 5 h after donor inoculation and was highly efficient in the absence of antibiotic selection. The transconjugant/donor ratio reached a maximum level approaching 1 on day 4 in the upper intestinal tract. Plasmid transfer was significantly lower with the CF− recipient. While rescue of the CF− mating defect by coculture with CF+ recipients is easily accomplished in vitro, no extracellular complementation occurred in vivo. This suggests that most pheromone signaling in the gut occurs between recipient and donor cells in very close proximity. Plasmid-bearing cells (donors plus transconjugants) steadily increased in the population from 0.1% after donor inoculation to about 10% at the conclusion of the experiments. This suggests a selective advantage of pCF10 carriage distinct from antibiotic resistance or bacteriocin production. Our results demonstrate that pheromone signaling is required for efficient pCF10 transfer in vivo. In the absence of CF+ recipients, a low level of transfer to CF− recipients occurred in the gut. This may result from low-level host-mediated induction of the donors in the gastrointestinal (GI) tract, similar to that previously observed in serum. IMPORTANCE Horizontal gene transfer is a major factor in the biology of Enterococcus faecalis, an important nosocomial pathogen. Previous studies showing efficient conjugative plasmid transfer in the gastrointestinal (GI) tracts of experimental animals did not examine how the enterococcal sex pheromone response impacts the efficiency of transfer. Our study demonstrates for the first time pheromone-enhanced, high-frequency plasmid transfer of E. faecalis plasmid pCF10 in a mouse model in the absence of antibiotic or bacteriocin selection. Pheromone production by recipients dramatically increased plasmid transfer in germfree mice colonized initially with recipients, followed by donors. The presence of a coresident community of common gut microbes did not significantly reduce in vivo plasmid transfer between enterococcal donors and recipients. In mice colonized with enterococcal recipients, we detected plasmid transfer in the intestinal tract within 5 h of addition of donors, before transconjugants could be cultured from feces. Surprisingly, pCF10 carriage provided a competitive fitness advantage unrelated to antibiotic resistance or bacteriocin production.

Metal resistance has been associated with antibiotic resistance due to co- or cross-resistance mechanisms. Here, metal contaminated mine soil treated with organic wastes was screened for the presence of mobile genetic elements (MGEs). The occurrence of conjugative IncP-1 and mobilizable IncQ plasmids, as well as of class 1 integrons, was confirmed by PCR and Southern blot hybridization, suggesting that bacteria from these soils have gene-mobilizing capacity with implications for the dissemination of resistance factors. Moreover, exogenous isolation of MGEs from the soil bacterial community was attempted under antibiotic selection pressure by using Escherichia coli as recipient. Seventeen putative transconjugants were identified based on increased antibiotic resistance. Metabolic traits and metal resistance of putative transconjugants were investigated, and whole genome sequencing was carried out for two of them. Most putative transconjugants displayed a multi-resistant phenotype for a broad spectrum of antibiotics. They also displayed changes regarding the ability to metabolise different carbon sources, RNA: DNA ratio, growth rate and biofilm formation. Genome sequencing of putative transconjugants failed to detect genes acquired by horizontal gene transfer, but instead revealed a number of nonsense mutations, including in ubiH , whose inactivation was linked to the observed resistance to aminoglycosides. Our results confirm that mine soils contain MGEs encoding antibiotic resistance. Moreover, they point out the role of spontaneous mutations in achieving low-level antibiotic resistance in a short time, which was associated with a trade-off in the capability to metabolise specific carbon sources. [ABSTRACT FROM AUTHOR]

We previously characterized a filamentous lysogenic bacteriophage, ϕRs551, isolated directly from the race 3 biovar 2 phylotype IIB sequevar 1 strain UW551 of Ralstonia solanacearum grown under normal culture conditions. The genome of ϕRs551 was identified with 100% identity in the deposited genomes of 11 race 3 biovar 2 phylotype IIB sequevar 1 strains of R. solanacearum, indicating evolutionary and biological importance, and ORF14 of ϕRs551 was annotated as a putative type-2 repressor. In this study, we determined the effect of the prophage and its ORF14 on the virulence and competitive fitness of its carrier strain UW551 by deleting the orf14 gene only (the UW551 orf14 mutant), and nine of the prophage’s 14 genes including orf14 and six out of seven structural genes (the UW551 prophage mutant), respectively, from the genome of UW551. The two mutants were increased in extracellular polysaccharide production, twitching motility, expression of targeted virulence and virulence regulatory genes (pilT, egl, pehC, hrPB, and phcA), and virulence, suggesting that the virulence of UW551 was negatively regulated by ϕRs551, at least partially through ORF14. Interestingly, we found that the wt ϕRs551-carrying strain UW551 of R. solanacearum significantly outcompeted the wt strain RUN302 which lacks the prophage in tomato plants co-inoculated with the two strains. When each of the two mutant strains was co-inoculated with RUN302, however, the mutants were significantly out-competed by RUN302 for the same colonization site. Our results suggest that ecologically, ϕRs551 may play an important role by regulating the virulence of and offering a competitive fitness advantage to its carrier bacterial strain for persistence of the bacterium in the environment, which in turn prolongs the symbiotic relationship between the phage ϕRs551 and the R. solanacearum strain UW551. Our study is the first toward a better understanding of the co-existence between a lysogenic phage and its carrier plant pathogenic bacterial strain by determining the effect of the prophage Rs551 and its repressor on the virulence and competitive fitness of its carrier strain UW551 of R. solanacearum.

ABSTRACT The coronavirus (CoV) RNA genome is the largest among the single-stranded positive-sense RNA viruses. CoVs encode a proofreading 3′-to-5′ exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is responsible for CoV high-fidelity replication. Alanine substitution of ExoN catalytic residues [ExoN(-)] in severe acute respiratory syndrome-associated coronavirus (SARS-CoV) and murine hepatitis virus (MHV) disrupts ExoN activity, yielding viable mutant viruses with defective replication, up to 20-fold-decreased fidelity, and increased susceptibility to nucleoside analogues. To test the stability of the ExoN(-) genotype and phenotype, we passaged MHV-ExoN(-) 250 times in cultured cells (P250), in parallel with wild-type MHV (WT-MHV). Compared to MHV-ExoN(-) P3, MHV-ExoN(-) P250 demonstrated enhanced replication and increased competitive fitness without reversion at the ExoN(-) active site. Furthermore, MHV-ExoN(-) P250 was less susceptible than MHV-ExoN(-) P3 to multiple nucleoside analogues, suggesting that MHV-ExoN(-) was under selection for increased replication fidelity. We subsequently identified novel amino acid changes within the RNA-dependent RNA polymerase and nsp14 of MHV-ExoN(-) P250 that partially accounted for the reduced susceptibility to nucleoside analogues. Our results suggest that increased replication fidelity is selected in ExoN(-) CoVs and that there may be a significant barrier to ExoN(-) reversion. These results also support the hypothesis that high-fidelity replication is linked to CoV fitness and indicate that multiple replicase proteins could compensate for ExoN functions during replication. IMPORTANCE Uniquely among RNA viruses, CoVs encode a proofreading exoribonuclease (ExoN) in nsp14 that mediates high-fidelity RNA genome replication. Proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] either are nonviable or have significant defects in replication, RNA synthesis, fidelity, fitness, and virulence. In this study, we showed that ExoN(-) murine hepatitis virus can adapt during long-term passage for increased replication and fitness without reverting the ExoN-inactivating mutations. Passage-adapted ExoN(-) mutants also demonstrate increasing resistance to nucleoside analogues that is explained only partially by secondary mutations in nsp12 and nsp14. These data suggest that enhanced resistance to nucleoside analogues is mediated by the interplay of multiple replicase proteins and support the proposed link between CoV fidelity and fitness.

Combined experimental evolutionary and molecular biology approaches have been used to investigate the adaptive radiation of Pseudomonas fluorescens SBW25 in static microcosms leading to the colonisation of the air-liquid interface by biofilm-forming mutants such as the Wrinkly Spreader (WS). In these microcosms, the ecosystem engineering of the early wild-type colonists establishes the niche space for subsequent WS evolution and colonisation. Random WS mutations occurring in the developing population that deregulate diguanylate cyclases and c-di-GMP homeostasis result in cellulose-based biofilms at the air-liquid interface. These structures allow Wrinkly Spreaders to intercept O2 diffusing into the liquid column and limit the growth of competitors lower down. As the biofilm matures, competition increasingly occurs between WS lineages, and niche divergence within the biofilm may support further diversification before system failure when the structure finally sinks. A combination of pleiotropic and epistasis effects, as well as secondary mutations, may explain variations in WS phenotype and fitness. Understanding how mutations subvert regulatory networks to express intrinsic genome potential and key innovations providing a selective advantage in novel environments is key to understanding the versatility of bacteria, and how selection and ecological opportunity can rapidly lead to substantive changes in phenotype and in community structure and function. [ABSTRACT FROM AUTHOR]

The mammalian gastrointestinal (GI) tract is a complex organ system with a twist—a significant portion of its composition is a community of microbial symbionts. The microbiota plays an increasingly appreciated role in many clinically-relevant conditions. It is important to understand the details of biofilm development in the GI tract since bacteria in this state not only use biofilms to improve colonization, biofilm bacteria often exhibit high levels of resistance to common, clinically relevant antibacterial drugs. Here we examine the initial colonization of the germ-free murine GI tract byEnterococcus faecalis—one of the first bacterial colonizers of the naïve mammalian gut. We demonstrate strong morphological similarities to our previousin vitro E. faecalisbiofilm microcolony architecture using 3 complementary imaging techniques: conventional tissue Gram stain, immunofluorescent imaging (IFM) of constitutive fluorescent protein reporter expression, and low-voltage scanning electron microscopy (LV-SEM).E. faecalisbiofilm microcolonies were readily identifiable throughout the entire lower GI tract, from the duodenum to the colon. Notably, biofilm development appeared to occur as discrete microcolonies directly attached to the epithelial surface rather than confluent sheets of cells throughout the GI tract even in the presence of high (>109) fecal bacterial loads. Anin vivocompetition experiment using a pool of 11 selectE. faecalismutant strains containing sequence-defined transposon insertions showed the potential of this model to identify genetic factors involved inE. faecaliscolonization of the murine GI tract. [ABSTRACT FROM AUTHOR]

Antirrhinum majus Rosea1 (Ros1) is an MYB-related transcription factor that induces anthocyanin biosynthesis in plant tissues, and has been shown to be suitable for visual tracking of virus infection in plants. However, activation of anthocyanin biosynthesis has far reaching effects on plant physiology and could consequently have negative effects on viral replication. Therefore, viruses carrying the Ros1 marker might have a low fitness and consequently rapidly lose the marker. To compare the stability of the Ros1 marker, we generated Tobacco etch virus (TEV) based constructs containing either Ros1 or the enhanced green fluorescent protein (eGFP) between the NIb and CP cistrons (TEV-Ros1 and TEV-eGFP, respectively). We measured the within-host competitive fitness of both viruses by direct competitions with a common competitor during infection of Nicotiana tabacum. The fitness of TEV-Ros1 was significantly lower than that of TEV-eGFP, and both recombinant viruses had a significantly lower fitness than the wild-type virus. Nevertheless, after seven weeks of infection in N. tabacum, similar levels of marker gene instability where found for both viruses. Despite lower fitness of the marked virus, Ros1 is therefore a viable alternative marker for tracking viral infection in plants.

Listeria monocytogenes is a foodborne pathogen that is implicated in numerous outbreaks of disease (listeriosis) via fresh produce. The genetic features of L. monocytogenes that allow adherence and growth on produce remain largely uncharacterized. In this study, two non-motile transposon mutants were characterized for attachment, growth, and survival on the surface of cantaloupe rind. One of the mutants, L1E4, harbored a single transposon insertion in a DEAD-box RNA helicase gene (lmo0866 homolog), while the other, M1A5, harbored an insertion in a gene from a flagellum biosynthesis and chemotaxis gene cluster (lmo0694 homolog). When inoculated alone, neither mutant was significantly impaired in growth or survival on the surface of cantaloupe at either 25 or 37 °C. However, when co-inoculated with the wildtype parental strain, the RNA helicase mutant L1E4 had a clear competitive disadvantage, while the relative fitness of M1A5 was not noticeably impacted. Genetic complementation of L1E4 with the intact RNA helicase gene restored relative fitness on cantaloupe. The findings suggest that the DEAD-box RNA helicase encoded by the lmo0866 homolog is critical for relative fitness of L. monocytogenes on cantaloupe. Mutant L1E4 was pleiotropic, being not only non-motile but also cold-sensitive and with reduced hemolytic activity, warranting further studies to elucidate the role of this helicase in the competitive fitness of L. monocytogenes on produce.

Candida albicans is responsible for 400,000 systemic fungal infections annually, with an associated mortality rate of 46 75%. The human gastrointestinal (GI) tract represents the largest natural reservoir of Candida species and is a major source of systemic fungal infections. However, the factors that control GI colonization by Candida species are not completely understood. We hypothesized that the fungal cell wall would play an important role in determining the competitive fitness of Candida species in the mammalian GI tract. To test this hypothesis, we generated a systematic collection of isogenic C. albicans cell wall mutants and measured their fitness in the mouse GI tract via quantitative competition assays. Whereas a large variation in competitive fitness was found among mutants, no correlation was observed between GI fitness and total levels of individual cell wall components. Similar results were obtained in a set of distantly related Candida species, suggesting that total amounts of individual cell wall components do not determine the ability of fungi to colonize the GI tract. We then subjected this collection of Candida strains and species to an extensive quantitative phenotypic profiling in search for features that might be responsible for their differences in GI fitness, but found no association with the ability to grow in GI mimicking and stressful environments or with in vitro and in vivo virulence. The most significant association with GI fitness was found to be the strength of signaling through the Dectin-1 receptor. Using a quantitative assay to measure the amount of exposed ß-glucan on the surface of fungal cells, we found this parameter, unlike total ß-glucan levels, to be strongly predictive of competitive fitness in the mouse GI tract. These data suggest that fungal cell wall architecture, more so than its crude composition, critically determines the ability of fungi to colonize the mammalian GI tract. In particular, recognition of exposed ß-glucan by Dectin-1 receptor appears to severely limit Candida GI fitness and hence represents a promising target to reduce fungal colonization in patients at risks of systemic candidiasis. [ABSTRACT FROM AUTHOR]

Leprosy is a chronic infectious disease caused by Mycobacterium leprae. Fox P3 have been shown to have important implications in various diseases. The present study describes the mechanism of action of Fox P3 in CD4+ CD25+ T cells derived from leprosy patients. Increased molecular interactions of Fox P3 with histone deacetylases 7/9 in the nucleus of CD4+ CD25+ T cells derived from borderline lepromatous leprosy/lepromatous leprosy ( BL/ LL) patients were found to be responsible for Fox P3-driven immune suppression activities during the progression of leprosy. Further, downregulation of CTLA-4 and CD25 genes in si Fox P3-treated PBMCs derived from BL/ LL patients elucidated the transcription-activating nature of Fox P3. This observation was supported by direct binding of Fox P3 to the promoter region of the CTLA-4 and CD25 genes, and Fox P3's molecular interaction with histone acetyl transferases. The study also revealed that the increased expression of mi R155 in CD4+ CD25+ cells from BL/ LL governs the competitive fitness of these cells. Again, reduced Annexin V & propidium iodide staining and Nur77 expression, and concomitantly increased Ki-67 positivity suggested that CD4+ CD25+ cells derived from BL/ LL patients are more competitively fit than those from borderline tuberculoid leprosy/tuberculoid leprosy and healthy controls. Taken together, the study shows the orchestration of Fox P3 leading to competitive fitness of Treg cells in leprosy. [ABSTRACT FROM AUTHOR]

Antirrhinum majus Rosea1 (Ros1) is an MYB-related transcription factor that induces anthocyanin biosynthesis in plant tissues, and has been shown to be suitable for visual tracking of virus infection in plants. However, activation of anthocyanin biosynthesis has far reaching effects on plant physiology and could consequently have negative effects on viral replication. Therefore, viruses carrying the Ros1 marker might have a low fitness and consequently rapidly lose the marker. To compare the stability of the Ros1 marker, we generated Tobacco etch virus (TEV) based constructs containing either Ros1 or the enhanced green fluorescent protein (eGFP) between the NIb and CP cistrons (TEV-Ros1 and TEV-eGFP, respectively). We measured the within-host competitive fitness of both viruses by direct competitions with a common competitor during infection of Nicotiana tabacum. The fitness of TEV-Ros1 was significantly lower than that of TEV-eGFP, and both recombinant viruses had a significantly lower fitness than the wild-type virus. Nevertheless, after seven weeks of infection in N. tabacum, similar levels of marker gene instability where found for both viruses. Despite lower fitness of the marked virus, Ros1 is therefore a viable alternative marker for tracking viral infection in plants. [ABSTRACT FROM AUTHOR]

The fitness of populations adapting to new environments is expected to decline in different environments, but empirical studies often do not lend support for such adaptation costs. We test the idea that the initial fitness of the selected populations in the environment where the cost is estimated is key for interpreting tests of ecological trade-offs. We isolated single clones of the yeast Saccharomyces cerevisiae every ~250 generations from replicate experimental lineages that had been selected during 5000 generations in a glucose-limited environment. We then selected these clones in a galactose-limited environment for ~120 generations. Finally, we estimated single-clone fitness in both environments, before and after selection on galactose. The pleiotropic effects on glucose of selection on galactose evolved from positive to negative as fitness in glucose increased, providing strong support for the importance of initial fitness for determining the sign and magnitude of pleiotropic effects. This demonstrates that the sign of pleiotropic effects for fitness following adaptation to a new environment can change during long-term adaptation to an original environment. We also found no relationship between the size of the fitness changes in galactose and glucose, such that pleiotropic effects in glucose became relatively smaller as the sizes of direct effects on galactose increased. [ABSTRACT FROM AUTHOR]

Many bacteria utilize quorum sensing (QS) to control group behavior in a cell density-dependent manner. Previous studies have demonstrated that Acinetobacter baumannii employs an N-acyl-L-homoserine lactone (AHL)-based QS system to control biological functions and virulence. Here, we report that indole controls biological functions, virulence and AHL signal production in A. baumannii. The biosynthesis of indole is performed by A1S_3160 (AbiS, A cinetobacter b aumannii i ndole s ynthase), which is a novel indole synthase annotated as an alpha / beta hydrolase in A. baumannii. Heterologous expression of AbiS in an Escherichia coli indole-deficient mutant also rescued the production of indole by using a distinct biosynthetic pathway from the tryptophanase TnaA, which produces indole directly from tryptophan in E. coli. Moreover, we revealed that indole from A. baumannii reduced the competitive fitness of Pseudomonas aeruginosa by inhibiting its QS systems and type III secretion system (T3SS). As A. baumannii and P. aeruginosa usually coexist in human lungs, our results suggest the crucial roles of indole in both the bacterial physiology and interspecies communication. IMPORTANCE Acinetobacter baumannii is an important human opportunistic pathogen that usually causes high morbidity and mortality. It employs the N-acyl-L-homoserine lactone (AHL)-type quorum sensing (QS) system, AbaI/AbaR, to regulate biological functions and virulence. In this study, we found that A. baumannii utilizes another QS signal, indole, to modulate biological functions and virulence. It was further revealed that indole positively controls the production of AHL signals by regulating abaI expression at the transcriptional levels. Furthermore, indole represses the QS systems and type III secretion system (T3SS) of P. aeruginosa and enhances the competitive ability of A. baumannii. Together, our work describes a QS signaling network where a pathogen uses to control the bacterial physiology and pathogenesis, and the competitive ability in microbial community.

Abstract: Competition between virus genotypes in insect hosts is a key element of virus fitness, affecting their long-term persistence in agro-ecosystems. Little information is available on virus competition in insect hosts or during serial passages from one cohort of hosts to the next. Here we report on the competition between two genotypes of Spodoptera exigua nucleopolyhedrovirus (SeMNPV), when serially passaged as mixtures in cohorts of 4th instar S. exigua larvae. One of the genotypes was a SeMNPV wild-type isolate, SeUS1, while the other was a SeMNPV recombinant (SeMNPV-XD1) having a greater speed of kill than SeUS1. SeXD1 lacks a suite of genes, including the ecdysteroid UDP-glucosyl transferase (egt) gene. SeXD1 expresses the green fluorescent protein (GFP) gene, enabling the identification of SeXD1 in cell culture and in insects. The relative proportion of SeUS1 and SeXD1 in successive passages of mixed infections in various ratios was determined by plaque assays of budded virus from infected larvae and by polymerase chain reactions and restriction enzyme analyses. The SeUS1 genotype outcompeted recombinant SeXD1 over successive passages. Depending on the initial virus genotype ratio, the recombinant SeXD1 was no longer detected after 6–12 passages. A mathematical model was developed to characterize the competition dynamics. Overall, the ratio SeUS1/XD1 increased by a factor 1.9 per passage. The findings suggest that under the experimental conditions recombinant SeXD1 is displaced by the wild-type strain SeUS1, but further studies are needed to ascertain that this is also the case when the same baculoviruses would be used in agro-ecosystems. [ABSTRACT FROM AUTHOR]

Thermal adaptation to spatially varying environmental conditions occurs in a wide range of species, but what is less clear is the nature of fitness trade-offs associated with this temperature adaptation. Here, populations of the intertidal copepod Tigriopus californicus are examined at both local and latitudinal scales to determine whether these populations have evolved differences in their survival under high temperature stress. A clear pattern of increasing high temperature stress tolerance is seen with decreasing latitude, consistent with temperature adaptation. Additionally, there is also evidence for significant variation in thermal tolerance on a smaller scale. The competitive fitness of pairs of northern and southern copepod populations were also examined under a series of lower, more moderate temperatures. These fitness assays show that the southern populations that have the best survival under extreme high temperatures have lowered competitive fitness at the lower temperatures tested, whereas the fitness of the southern populations exceeded that of the northern populations at the highest temperatures tested. Combined, these results suggest that there may be evolutionary trade-offs between performance at high and stressful temperatures and fitness at moderate temperatures in this species. [ABSTRACT FROM AUTHOR]

Summary: The translation elongation factor G (EFG) is encoded by the fusA gene. Several bacteria possess a second fusA-like locus, fusA2 which encodes EFG2. A comparison of EFG and EFG2 from various bacteria reveals that EFG2 preserves domain organization and maintains significant sequence homology with EFG, suggesting that EFG2 may function as an elongation factor. However, with the single exception of a recent study on Thermus thermophilus EFG2, this class of EFG-like factors has not been investigated. Here, we have characterized EFG2 (MSMEG_6535) from Mycobacterium smegmatis. Expression of EFG2 was detected in stationary phase cultures of M. smegmatis (Msm). Our in vitro studies show that while MsmEFG2 binds guanine nucleotides, it lacks the ribosome-dependent GTPase activity characteristic of EFGs. Furthermore, unlike MsmEFG (MSMEG_1400), MsmEFG2 failed to rescue an E. coli strain harboring a temperature-sensitive allele of EFG, for its growth at the non-permissive temperature. Subsequent experiments showed that the fusA2 gene could be disrupted in M. smegmatis mc2155 with KanR marker. The M. smegmatis fusA2::kan strain was viable and showed growth kinetics similar to that of the parent strain (wild-type for fusA2). However, in the growth competition assays, the disruption of fusA2 was found to confer a fitness disadvantage to M. smegmatis, raising the possibility that EFG2 is of some physiological relevance to mycobacteria. [Copyright &y& Elsevier]