Pseudomonas putida KT2440 is naturally endowed to withstand industrial-scale stress conditions

Pseudomonas putida is recognized as a very promising strain for industrial application due to its high redox capacity and frequently observed tolerance towards organic solvents. In this research, we studied the metabolic and transcriptional response of P. putida KT2440 exposed to large-scale heterogeneous mixing conditions in the form of repeated glucose shortage. Cellular responses were mimicked in an experimental setup comprising a stirred tank reactor and a connected plug flow reactor. We deciphered that a stringent response-like transcriptional regulation programme is frequently induced, which seems to be linked to the intracellular pool of 3-hydroxyalkanoates (3-HA) that are known to serve as precursors for polyhydroxyalkanoates (PHA). To be precise, P. putida is endowed with a survival strategy likely to access cellular PHA, amino acids and glycogen in few seconds under glucose starvation to obtain ATP from respiration, thereby replenishing the reduced ATP levels and the adenylate energy charge. Notably, cells only need 0.4% of glucose uptake to build those 3-HA-based energy buffers. Concomitantly, genes that are related to amino acid catabolism and β-oxidation are upregulated during the transient absence of glucose. Furthermore, we provide a detailed list of transcriptional short- and long-term responses that increase the cellular maintenance by about 17% under the industrial-like conditions tested.publishersversionpublishe

Hot Start PCR with heat-activatable primers: a novel approach for improved PCR performance

The polymerase chain reaction (PCR) is widely used for applications which require a high level of specificity and reliability, such as genetic testing, clinical diagnostics, blood screening, forensics and biodefense. Great improvements to PCR performance have been achieved by the use of Hot Start activation strategies that aim to prevent DNA polymerase extension until more stringent, higher temperatures are reached. Herein we present a novel Hot Start activation approach in PCR where primers contain one or two thermolabile, 4-oxo-1-pentyl (OXP) phosphotriester (PTE) modification groups at 3′-terminal and 3′-penultimate internucleotide linkages. Studies demonstrated that the presence of one or more OXP PTE modifications impaired DNA polymerase primer extension at the lower temperatures that exist prior to PCR amplification. Furthermore, incubation of the OXP-modified primers at elevated temperatures was found to produce the corresponding unmodified phosphodiester (PDE) primer, which was then a suitable DNA polymerase substrate. The OXP-modified primers were tested in conventional PCR with endpoint detection, in one-step reverse transcription (RT)–PCR and in real-time PCR with SYBR Green I dye and Taqman® probe detection. When OXP-modified primers were used as substitutes for unmodified PDE primers in PCR, significant improvement was observed in the specificity and efficiency of nucleic acid target amplification

Fructose-Bisphophate Aldolase Exhibits Functional Roles between Carbon Metabolism and the hrp System in Rice Pathogen Xanthomonas oryzae pv. oryzicola

Fructose-bisphophate aldolase (FbaB), is an enzyme in glycolysis and gluconeogenesis in living organisms. The mutagenesis in a unique fbaB gene of Xanthomonas oryzae pv. oryzicola, the causal agent of rice bacterial leaf streak, led the pathogen not only unable to use pyruvate and malate for growth and delayed its growth when fructose was used as the sole carbon source, but also reduced extracellular polysaccharide (EPS) production and impaired bacterial virulence and growth in rice. Intriguingly, the fbaB promoter contains an imperfect PIP-box (plant-inducible promoter) (TTCGT-N9-TTCGT). The expression of fbaB was negatively regulated by a key hrp regulatory HrpG and HrpX cascade. Base substitution in the PIP-box altered the regulation of fbaB with the cascade. Furthermore, the expression of fbaB in X. oryzae pv. oryzicola RS105 strain was inducible in planta rather than in a nutrient-rich medium. Except other hrp-hrc-hpa genes, the expression of hrpG and hrpX was repressed and the transcripts of hrcC, hrpE and hpa3 were enhanced when fbaB was deleted. The mutation in hrcC, hrpE or hpa3 reduced the ability of the pathogen to acquire pyruvate and malate. In addition, bacterial virulence and growth in planta and EPS production in RΔfbaB mutant were completely restored to the wild-type level by the presence of fbaB in trans. This is the first report to demonstrate that carbohydrates, assimilated by X. oryzae pv. oryzicola, play critical roles in coordinating hrp gene expression through a yet unknown regulator

Actin: its cumbersome pilgrimage through cellular compartments

In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin

Comparative genome analysis and genome-guided physiological analysis of Roseobacter litoralis

<p>Abstract</p> <p>Background</p> <p><it>Roseobacter litoralis </it>OCh149, the type species of the genus, and <it>Roseobacter denitrificans </it>OCh114 were the first described organisms of the <it>Roseobacter </it>clade, an ecologically important group of marine bacteria. Both species were isolated from seaweed and are able to perform aerobic anoxygenic photosynthesis.</p> <p>Results</p> <p>The genome of <it>R. litoralis </it>OCh149 contains one circular chromosome of 4,505,211 bp and three plasmids of 93,578 bp (pRLO149_94), 83,129 bp (pRLO149_83) and 63,532 bp (pRLO149_63). Of the 4537 genes predicted for <it>R. litoralis</it>, 1122 (24.7%) are not present in the genome of <it>R. denitrificans</it>. Many of the unique genes of <it>R. litoralis </it>are located in genomic islands and on plasmids. On pRLO149_83 several potential heavy metal resistance genes are encoded which are not present in the genome of <it>R. denitrificans</it>. The comparison of the heavy metal tolerance of the two organisms showed an increased zinc tolerance of <it>R. litoralis</it>. In contrast to <it>R. denitrificans</it>, the photosynthesis genes of <it>R. litoralis </it>are plasmid encoded. The activity of the photosynthetic apparatus was confirmed by respiration rate measurements, indicating a growth-phase dependent response to light. Comparative genomics with other members of the <it>Roseobacter </it>clade revealed several genomic regions that were only conserved in the two <it>Roseobacter </it>species. One of those regions encodes a variety of genes that might play a role in host association of the organisms. The catabolism of different carbon and nitrogen sources was predicted from the genome and combined with experimental data. In several cases, e.g. the degradation of some algal osmolytes and sugars, the genome-derived predictions of the metabolic pathways in <it>R. litoralis </it>differed from the phenotype.</p> <p>Conclusions</p> <p>The genomic differences between the two <it>Roseobacter </it>species are mainly due to lateral gene transfer and genomic rearrangements. Plasmid pRLO149_83 contains predominantly recently acquired genetic material whereas pRLO149_94 was probably translocated from the chromosome. Plasmid pRLO149_63 and one plasmid of <it>R. denitrifcans </it>(pTB2) seem to have a common ancestor and are important for cell envelope biosynthesis. Several new mechanisms of substrate degradation were indicated from the combination of experimental and genomic data. The photosynthetic activity of <it>R. litoralis </it>is probably regulated by nutrient availability.</p

Retrotransfer in Escherichia coli conjugation: bidirectional exchange or de novo mating?

DNA can be transferred among eubacteria and to plants and fungi by related, plasmid-mediated processes collectively referred to as bacterial conjugation. Conjugation occurs between cells in contact with one another and results in the unidirectional delivery of DNA from a bacterial donor to a recipient. Recent experiments that have reexamined the directionality of DNA flow during conjugation have come to different conclusions, some suggesting that genetic material also flows from recipient cells into the donor and that this process, termed retrotransfer, is likewise directed by donor-encoded functions. Given that bacteria are perhaps united with all living creatures by conjugation, the possibility of gene flow into donor bacteria during conjugation raises interesting evolutionary and biocontainment issues. Here we report that plasmid transmission from bacterial recipients to donors is not a donor-mediated event. Movement of genetic material from recipients to donors was inhibited by streptomycin, which does not inhibit the conjugative donor, indicating that retrotransfer requires gene expression in recipients. Furthermore, retrotransfer was reduced in matings mediated by plasmids that encode strong entry exclusion, to a similar degree as matings between two donors. Therefore we suggest that retrotransfer is in fact newly initiated conjugation between transconjugants and donors