Mila Kojadinovic

Legumes symbioses : Absence of Nod genes in photosynthetic bradyrhizobia

Leguminous plants (such as peas and soybeans) and rhizobial soil bacteria are symbiotic partners that communicate through molecular signaling pathways, resulting in the formation of nodules on legume roots and occasionally stems that house nitrogen-fixing bacteria. Nodule formation has been assumed to be exclusively initiated by the binding of bacterial, host-specific lipochito-oligosaccharidic Nod factors, encoded by the nodABC genes, to kinase-like receptors of the plant. Here we show by complete genome sequencing of two symbiotic, photosynthetic, Bradyrhizobium strains, BTAi1 and ORS278, that canonical nodABC genes and typical lipochito-oligosaccharidic Nod factors are not required for symbiosis in some legumes. Mutational analyses indicated that these unique rhizobia use an alternative pathway to initiate symbioses, where a purine derivative may play a key role in triggering nodule formation.

Identifying components of the NF-κB pathway in the beneficial Euprymna scolopes-vibrio fischeri light organ symbiosis

ABSTRACT The Toll/NF-κB pathway is a common, evolutionarily conserved innate immune pathway that modulates the responses of animal cells to microbe-associated molecular patterns (MAMPs). Because MAMPs have been implicated as critical elements in the signaling of symbiont-induced development, an expressed sequence tag library from the juvenile light organ of Euprymna scolopes was used to identify members of the Toll/NF-κB pathway. Full-length transcripts were identified by using 5′ and 3′ RACE PCR. Seven transcripts critical for MAMP-induced triggering of the Toll/NF-κB phosphorylation cascade have been identified, including receptors, signal transducers, and a transcription factor. Further investigations should elucidate the role of the Toll/NF-κB pathway in the initiation of the beneficial symbiosis between E. scolopes and Vibrio fischeri.

Evolution of a bacteriophytochrome from light to redox sensor

Bacteriophytochromes are red/far‐red photoreceptors that bacteria use to mediate sensory responses to their light environment. Here, we show that the photosynthetic bacterium Rhodopseudomonas palustris has two distinct types of bacteriophytochrome‐related protein (RpBphP4) depending upon the strain considered. The first type binds the chromophore biliverdin and acts as a light‐sensitive kinase, thus behaving as a bona fide bacteriophytochrome. However, in most strains, RpBphP4 does not to bind this chromophore. This loss of light sensing is replaced by a redox‐sensing ability coupled to kinase activity. Phylogenetic analysis is consistent with an evolutionary scenario, where a bacteriophytochrome ancestor has adapted from light to redox sensing. Both types of RpBphP4 regulate the synthesis of light harvesting (LH2) complexes according to the light or redox conditions, respectively. They modulate the affinity of a transcription factor binding to the promoter regions of LH2 complex genes by controlling its phosphorylation status. This is the first complete description of a bacteriophytochrome signal transduction pathway involving a two‐component system.

Fitness Is Strongly Influenced by Rare Mutations of Large Effect in a Microbial Mutation Accumulation Experiment

Our understanding of the evolutionary consequences of mutation relies heavily on estimates of the rate and fitness effect of spontaneous mutations generated by mutation accumulation (MA) experiments. We performed a classic MA experiment in which frequent sampling of MA lines was combined with whole genome resequencing to develop a high-resolution picture of the effect of spontaneous mutations in a hypermutator (ΔmutS) strain of the bacterium Pseudomonas aeruginosa. After ∼644 generations of mutation accumulation, MA lines had accumulated an average of 118 mutations, and we found that average fitness across all lines decayed linearly over time. Detailed analyses of the dynamics of fitness change in individual lines revealed that a large fraction of the total decay in fitness (42.3%) was attributable to the fixation of rare, highly deleterious mutations (comprising only 0.5% of fixed mutations). Furthermore, we found that at least 0.64% of mutations were beneficial and probably fixed due to positive selection. The majority of mutations that fixed (82.4%) were base substitutions and we failed to find any signatures of selection on nonsynonymous or intergenic mutations. Short indels made up a much smaller fraction of the mutations that were fixed (17.4%), but we found evidence of strong selection against indels that caused frameshift mutations in coding regions. These results help to quantify the amount of natural selection present in microbial MA experiments and demonstrate that changes in fitness are strongly influenced by rare mutations of large effect.

Testing the role of genetic background in parallel evolution using the comparative experimental evolution of antibiotic resistance

Parallel evolution is the independent evolution of the same phenotype or genotype in response to the same selection pressure. There are examples of parallel molecular evolution across divergent genetic backgrounds, suggesting that genetic background may not play an important role in determining the outcome of adaptation. Here, we measure the influence of genetic background on phenotypic and molecular adaptation by combining experimental evolution with comparative analysis. We selected for resistance to the antibiotic rifampicin in eight strains of bacteria from the genus Pseudomonas using a short term selection experiment. Adaptation occurred by 47 mutations at conserved sites in rpoB, the target of rifampicin, and due to the high diversity of possible mutations the probability of within-strain parallel evolution was low. The probability of between-strain parallel evolution was only marginally lower, because different strains substituted similar rpoB mutations. In contrast, we found that more than 30% of the phenotypic variation in the growth rate of evolved clones was attributable to among-strain differences. Parallel molecular evolution across strains resulted in divergent phenotypic evolution because rpoB mutations had different effects on growth rate in different strains. This study shows that genetic divergence between strains constrains parallel phenotypic evolution, but had little detectable impact on the molecular basis of adaptation in this system.

The SOS response increases bacterial fitness, but not evolvability, under a sublethal dose of antibiotic

Exposure to antibiotics induces the expression of mutagenic bacterial stress–response pathways, but the evolutionary benefits of these responses remain unclear. One possibility is that stress–response pathways provide a short-term advantage by protecting bacteria against the toxic effects of antibiotics. Second, it is possible that stress-induced mutagenesis provides a long-term advantage by accelerating the evolution of resistance. Here, we directly measure the contribution of the Pseudomonas aeruginosa SOS pathway to bacterial fitness and evolvability in the presence of sublethal doses of ciprofloxacin. Using short-term competition experiments, we demonstrate that the SOS pathway increases competitive fitness in the presence of ciprofloxacin. Continued exposure to ciprofloxacin results in the rapid evolution of increased fitness and antibiotic resistance, but we find no evidence that SOS-induced mutagenesis accelerates the rate of adaptation to ciprofloxacin during a 200 generation selection experiment. Intriguingly, we find that the expression of the SOS pathway decreases during adaptation to ciprofloxacin, and this helps to explain why this pathway does not increase long-term evolvability. Furthermore, we argue that the SOS pathway fails to accelerate adaptation to ciprofloxacin because the modest increase in the mutation rate associated with SOS mutagenesis is offset by a decrease in the effective strength of selection for increased resistance at a population level. Our findings suggest that the primary evolutionary benefit of the SOS response is to increase bacterial competitive ability, and that stress-induced mutagenesis is an unwanted side effect, and not a selected attribute, of this pathway.

Response kinetics in the complex chemotaxis signalling pathway of Rhodobacter sphaeroides

Chemotaxis is one of the best-characterized signalling systems in biology. It is the mechanism by which bacteria move towards optimal environments and is implicated in biofilm formation, pathogenesis and symbiosis. The properties of the bacterial chemosensory response have been described in detail for the single chemosensory pathway of Escherichia coli. We have characterized the properties of the chemosensory response of Rhodobacter sphaeroides, an α-proteobacterium with multiple chemotaxis pathways, under two growth conditions allowing the effects of protein expression levels and cell architecture to be investigated. Using tethered cell assays, we measured the responses of the system to step changes in concentration of the attractant propionate and show that, independently of the growth conditions, R. sphaeroides is chemotactic over at least five orders of magnitude and has a sensing profile following Webers Law. Mathematical modelling also shows that, as E. coli, R. sphaeroides is capable of showing fold-change detection (FCD). Our results indicate that general features of bacterial chemotaxis such as the range and sensitivity of detection, adaptation times, adherence to Webers Law and the presence of FCD may be integral features of chemotaxis systems in general, regardless of network complexity, protein expression levels and cellular architecture across different species.

The intriguing CP12‐like tail of adenylate kinase 3 from Chlamydomonas reinhardtii

Adenylate kinases (ADK) are key enzymes that maintain the energetic balance in cellular compartments by catalyzing the reaction: AMP + ATP↔2 ADP. Here, we analyzed the chloroplast ADK 3 from the green alga, Chlamydomonas reinhardtii for the first time. This enzyme bears a C‐terminal extension that is highly similar to the C‐terminal end of the intrinsically disordered protein CP12 that plays a major role in the redox regulation of key enzymes of the Calvin–Benson cycle like glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) and phosphoribulokinase. The only other known example of a CP12‐like extension is found in the GapB isoform of GAPDH, where it is responsible for the autonomous redox regulation of the higher plant A2B2 GAPDH. In this study, we show that the CP12‐like tail is not involved in the redox regulation of ADK 3, but contributes greatly to its stability, and is essential for the post‐translational modification of the Cys221 residue by glutathione. This report highlights the fact that the C‐terminal part of the CP12 protein can act as a moonlighting, intrinsically disordered module conferring additional capabilities to the proteins to which it is added.

New Motion Analysis System for Characterization of the Chemosensory Response Kinetics of Rhodobacter sphaeroides under Different Growth Conditions

ABSTRACT We developed a new set of software tools that enable the speed and response kinetics of large numbers of tethered bacterial cells to be rapidly measured and analyzed. The software provides precision, accuracy, and a good signal-to-noise ratio combined with ease of data handling and processing. The software was tested on the single-cell chemosensory response kinetics of large numbers of Rhodobacter sphaeroides cells grown under either aerobic or photoheterotrophic conditions and either in chemostats or in batch cultures, allowing the effects of growth conditions on responses to be accurately measured. Aerobically and photoheterotrophically grown R. sphaeroides exhibited significantly different chemosensory response kinetics and cell-to-cell variability in their responses to 100 μM propionate. A greater proportion of the population of aerobically grown cells responded to a 100 μM step decrease in propionate; they adapted faster and showed less cell-to-cell variability than photosynthetic populations. Growth in chemostats did not significantly reduce the measured cell to cell variability but did change the adaptation kinetics for photoheterotrophically grown cells.

Complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa

Abstract We report the complete mitochondrial genome sequence of the freshwater diatom Asterionella formosa. The large 61.9 kb circular sequence encodes 34 proteins and 25 tRNAs that are universally conserved in other sequenced diatoms. We fully resolved a unique 24 kb region containing highly conserved repeated sequence units, possibly collocating with an origin of replication.