Hopanoids are sterol-like membrane lipids widely used as geochemical proxies for bacteria. Currently, the physiological role of hopanoids is not well understood, and this represents one of the major ...limitations in interpreting the significance of their presence in ancient or contemporary sediments. Previous analyses of mutants lacking hopanoids in a range of bacteria have revealed a range of phenotypes under normal growth conditions, but with most having at least an increased sensitivity to toxins and osmotic stress. We employed hopanoid-free strains of Methylobacterium extorquens DM4, uncovering severe growth defects relative to the wild-type under many tested conditions, including normal growth conditions without additional stressors. Mutants overproduce carotenoids-the other major isoprenoid product of this strain-and show an altered fatty acid profile, pronounced flocculation in liquid media, and lower growth yields than for the wild-type strain. The flocculation phenotype can be mitigated by addition of cellulase to the medium, suggesting a link between the function of hopanoids and the secretion of cellulose in M. extorquens DM4. On solid media, colonies of the hopanoid-free mutant strain were smaller than wild-type, and were more sensitive to osmotic or pH stress, as well as to a variety of toxins. The results for M. extorquens DM4 are consistent with the hypothesis that hopanoids are important for membrane fluidity and lipid packing, but also indicate that the specific physiological processes that require hopanoids vary across bacterial lineages. Our work provides further support to emerging observations that the role of hopanoids in membrane robustness and barrier function may be important across lineages, possibly mediated through an interaction with lipid A in the outer membrane.
Combination antimicrobial therapy has been considered a promising strategy to combat the evolution of antimicrobial resistance.
is the causative agent of tularemia and in addition to being found in ...the nature, is recognized as a threat agent that requires vigilance. We investigated the evolutionary outcome of adapting the Live Vaccine Strain (LVS) of
subsp.
to two non-interacting drugs, ciprofloxacin and doxycycline, individually, sequentially, and in combination. Despite their individual efficacies and independence of mechanisms, evolution to the combination arose on a shorter time scale than evolution to the two drugs sequentially. We conducted a longitudinal mutational analysis of the populations evolving to the drug combination, genetically reconstructed the identified evolutionary pathway, and carried out biochemical validation. We discovered that, after the appearance of an initial weak generalist mutation (FupA/B), each successive mutation alternated between adaptation to one drug or the other. In combination, these mutations allowed the population to more efficiently ascend the fitness peak through a series of evolutionary switch-backs. Clonal interference, weak pleiotropy, and positive epistasis also contributed to combinatorial evolution. This finding suggests that the use of this non-interacting drug pair against
may render both drugs ineffective because of mutational switch-backs that accelerate evolution of dual resistance.
Complete genome sequences are now available for many bacterial species that lack sophisticated genetic tools. We describe the development of a broad-host-range cre-lox system that allows antibiotic ...marker recycling in a variety of gram-negative bacteria. This system consists of an allelic exchange vector bearing a kanamycin cassette flanked by loxP sites and a tetracycline-resistant IncP plasmid that provides expression of the Cre recombinase. We demonstrate this system by generating unmarked deletions of genes in two different bacteria, Methylobacterium extorquens AM1 and Burkholderia fungorum LB400. This new antibiotic marker recycling system offers the possibility of creating unmarked mutants in a wide variety of gram-negative bacteria. Furthermore, marker recycling allows the generation of strains bearing multiple genetic manipulations in organisms for which few antibiotic markers are currently available.
Few areas of science have benefited more from the expansion in sequencing capability than the study of microbial communities. Can sequence data, besides providing hypotheses of the functions the ...members possess, detect the evolutionary and ecological processes that are occurring? For example, can we determine if a species is adapting to one niche, or if it is diversifying into multiple specialists that inhabit distinct niches? Fortunately, adaptation of populations in the laboratory can serve as a model to test our ability to make such inferences about evolution and ecology from sequencing. Even adaptation to a single niche can give rise to complex temporal dynamics due to the transient presence of multiple competing lineages. If there are multiple niches, this complexity is augmented by segmentation of the population into multiple specialists that can each continue to evolve within their own niche. For a known example of parallel diversification that occurred in the laboratory, sequencing data gave surprisingly few obvious, unambiguous signs of the ecological complexity present. Whereas experimental systems are open to direct experimentation to test hypotheses of selection or ecological interaction, the difficulty in "seeing ecology" from sequencing for even such a simple system suggests translation to communities like the human microbiome will be quite challenging. This will require both improved empirical methods to enhance the depth and time resolution for the relevant polymorphisms and novel statistical approaches to rigorously examine time-series data for signs of various evolutionary and ecological phenomena within and between species.
The complete and assembled genome sequences were determined for six strains of the alphaproteobacterial genus Methylobacterium, chosen for their key adaptations to different plant-associated niches ...and environmental constraints.
Adaptation under similar selective pressure often leads to comparable phenotypes. A longstanding question is whether such phenotypic repeatability entails similar (parallelism) or different genotypic ...changes (convergence). To better understand this, we characterized mutations that optimized expression of a plasmid-borne metabolic pathway during laboratory evolution of a bacterium. Expressing these pathway genes was essential for growth but came with substantial costs. Starting from overexpression, replicate populations founded by this bacterium all evolved to reduce expression. Despite this phenotypic repetitiveness, the underlying mutational spectrum was highly diverse. Analysis of these plasmid mutations identified three distinct means to modulate gene expression: (1) reducing the gene copy number, (2) lowering transcript stability, and (3) integration of the pathway-bearing plasmid into the host genome. Our study revealed diverse molecular changes beneath convergence to a simple phenotype. This complex genotype-phenotype mapping presents a challenge to inferring genetic evolution based solely on phenotypic changes.
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► Diverse molecular mechanisms underlie parallel changes in gene expression ► Many types of mutational events were beneficial; a minority of these were SNPs ► Although gene expression was optimized, no mutations occurred in the promoter ► Evolutionarily selected mutations optimize expression better than artificially tuning transcription
Phenotypic parallelism is often observed for evolution under similar selection, but to what extent do similar underlying genotypic changes account for this? We examined the genotypic and phenotypic evolution of replicate populations founded by an engineered bacterium expressing an essential but costly metabolic pathway. To balance its cost and benefit, all populations evolved reduced expression of pathway genes toward an intermediate optimum. This phenotypic repeatability, however, involved diverse mutational events that employed at least three distinct molecular mechanisms to alter gene expression.
How do bacteria regulate their cellular physiology in response to starvation? Here, we present a detailed characterization of Escherichia coli growth and starvation over a time-course lasting two ...weeks. We have measured multiple cellular components, including RNA and proteins at deep genomic coverage, as well as lipid modifications and flux through central metabolism. Our study focuses on the physiological response of E. coli in stationary phase as a result of being starved for glucose, not on the genetic adaptation of E. coli to utilize alternative nutrients. In our analysis, we have taken advantage of the temporal correlations within and among RNA and protein abundances to identify systematic trends in gene regulation. Specifically, we have developed a general computational strategy for classifying expression-profile time courses into distinct categories in an unbiased manner. We have also developed, from dynamic models of gene expression, a framework to characterize protein degradation patterns based on the observed temporal relationships between mRNA and protein abundances. By comparing and contrasting our transcriptomic and proteomic data, we have identified several broad physiological trends in the E. coli starvation response. Strikingly, mRNAs are widely down-regulated in response to glucose starvation, presumably as a strategy for reducing new protein synthesis. By contrast, protein abundances display more varied responses. The abundances of many proteins involved in energy-intensive processes mirror the corresponding mRNA profiles while proteins involved in nutrient metabolism remain abundant even though their corresponding mRNAs are down-regulated.
Parafibromin is the 531-amino-acid protein product encoded by HRPT2, a putative tumor suppressor gene recently implicated in the autosomal dominant hyperparathyroidism-jaw tumor familial cancer ...syndrome, sporadic parathyroid cancer, and a minority of families with isolated hyperparathyroidism. Parafibromin contains no identified functional domains but bears sequence homology to Cdc73p, a budding yeast protein component of the RNA polymerase II-associated Paf1 complex. This study addressed the expression and functional properties of human parafibromin. A survey of human and mouse tissues analysed with polyclonal antibodies to parafibromin showed specific immunoreactivity in adrenal and parathyroid glands, kidney, heart, and skeletal muscle. Subcellular fractionation and laser confocal microscopy of normal human parathyroid gland demonstrated expression of parafibromin in both the cytoplasmic and nuclear compartments. Parafibromin was expressed in four parathyroid adenomas but was absent from two parathyroid carcinomas. Transient overexpression of wild-type parafibromin, but not its Leu64Pro missense mutant implicated in parathyroid cancer and familial isolated hyperparathyroidism, inhibited cell proliferation, and blocked expression of cyclin D1, a key cell cycle regulator previously implicated in parathyroid neoplasia. These results demonstrate that human parafibromin is a nucleocytoplasmic protein with functions consistent with its postulated role as a tumor suppressor protein.
The ability of psychrophiles to survive and proliferate at low temperatures implies that they have overcome key barriers inherent to permanently cold environments. These challenges include: reduced ...enzyme activity; decreased membrane fluidity; altered transport of nutrients and waste products; decreased rates of transcription, translation and cell division; protein cold‐denaturation; inappropriate protein folding; and intracellular ice formation. Cold‐adapted organisms have successfully evolved features, genotypic and/or phenotypic, to surmount the negative effects of low temperatures and to enable growth in these extreme environments. In this review, we discuss the current knowledge of these adaptations as gained from extensive biochemical and biophysical studies and also from genomics and proteomics.
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