Phenotypic variation of Pseudomonas brassicacearum as a plant root-colonization strategy

Pseudomonas brassicacearum was isolated as a major root-colonizing population from Arabidopsis thaliana. The strain NFM421 of P. brassicacearum undergoes phenotypic variation during A. thaliana and Brassica napus root colonization in vitro as well as in soil, resulting in different colony appearance on agar surfaces. Bacteria forming translucent colonies (phase II cells) essentially were localized at the surface of young roots and root tips, whereas wild-type cells (phase I cells) were localized at the basal part of roots. The ability of phase II cells to spread and colonize new sites on root surface correlates with over-production of flagellin as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of surface proteins and microsequencing. Moreover, phase II cells showed a higher ability to swim and to swarm on semisolid agar medium. Phase I and phase II cells of P. brassicacearum NFM421 were tagged genetically with green fluorescent protein and red fluorescent protein. Confocal scanning laser microscopy was used to localize phase II cells on secondary roots and root tips of A. thaliana, whereas phase I cells essentially were localized at the basal part of roots. These experiments were conducted in vitro and in soil. Phenotypic variation on plant roots is likely to be a colonization strategy that may explain the high colonization power of P. brassicacearum.     

荧光假单胞杆菌2P24中gacA、dsbA和phoP/phoQ基因对小RNA因子RsmZ的转录调控

Pseudomonas fluorescens 2P24是分离自麦田的植物病害生物防治菌株,产生抗生素2, 4-二乙酰基间苯三酚（2,4-diacetylphloroglucinol;2,4-DAPG）是其主要防病机制。菌株2P24中小RNA基因rsmZ正调控抗生素2,4-DAPG的产量。【目的】本文研究上游调控因子对RsmZ转录表达的影响,以进一步理解抗生素产生机制。【方法】构建了rsmZ: : lacZ的转录融合结构,将含有该结构的报告载体转入2P24的多个调控基因缺失突变体中,检测相应的缺失基因对rsmZ转录水平的调控作用。【结果】结果表明,反应调控因子GacA对rsmZ基因的转录具有正调控作用,二硫键合成蛋白DsbA对其负调控;双因子调控系统PhoP/PhoQ突变后,rsmZ基因的转录明显滞后。【结论】小RNA基因rsmZ在菌株2P24中受到多个基因的调控,并在信号传递网络中起到重要作用。     

Complete genome sequence of a beneficial plant root-associated bacterium, Pseudomonas brassicacearum

To shed light on the genetic equipment of the beneficial plant-associated bacterium Pseudomonas brassicacearum, we sequenced the whole genome of the strain NFM421. Its genome consists of one chromosome equipped with a repertoire of factors beneficial for plant growth. In addition, a complete type III secretion system and two complete type VI secretion systems were identified. We report here the first genome sequence of this species.     

Translation Enhancing ACA Motifs and Their Silencing by a Bacterial Small Regulatory RNA

GcvB is an archetypal multi-target small RNA regulator of genes involved in amino acid uptake or metabolism in enteric bacteria. Included in the GcvB regulon is the yifK locus, encoding a conserved putative amino acid transporter. GcvB inhibits yifK mRNA translation by pairing with a sequence immediately upstream from the Shine-Dalgarno motif. Surprisingly, we found that some target sequence mutations that disrupt pairing, and thus were expected to relieve repression, actually lower yifK expression and cause it not to respond to GcvB variants carrying the corresponding compensatory changes. Work prompted by these observations revealed that the GcvB target sequence in yifK mRNA includes elements that stimulate translation initiation. Replacing each base of an ACA trinucleotide near the center of the target sequence, by any other base, caused yifK expression to decrease. Effects were additive, with some triple replacements causing up to a 90% reduction. The enhancer activity did not require the ACA motif to be strictly positioned relative to the Shine-Dalgarno sequence, nor did it depend on a particular spacing between the latter and the initiating AUG. The dppA mRNA, another GcvB target, contains four ACA motifs at the target site. Quite strikingly, replacement of all four ACAs by random trinucleotide sequences yielded variants showing over 100-fold reduction in expression, virtually inactivating the gene. Altogether, these data identify the ACA motif as a translation-enhancing module and show that GcvB''s ability to antagonize the enhancer function in target mRNAs is quintessential to the regulatory effectiveness of this sRNA.     

A Single Vegetative Actin Isovariant Overexpressed under the Control of Multiple Regulatory Sequences Is Sufficient for Normal Arabidopsis Development

The relative significance of gene regulation and protein isovariant differences remains unexplored for most gene families, particularly those participating in multicellular development. Arabidopsis thaliana encodes three vegetative actins, ACT2, ACT7, and ACT8, in two ancient and highly divergent subclasses. Mutations in any of these differentially expressed actins revealed only mild phenotypes. However, double mutants were extremely dwarfed, with altered cell and organ morphology and an aberrant F-actin cytoskeleton (e.g., act2-1 act7-4 and act8-2 act7-4) or totally root-hairless (e.g., act2-1 act8-2). Our studies suggest that the three vegetative actin genes and protein isovariants play distinct subclass-specific roles during plant morphogenesis. For example, during root development, ACT7 was involved in root growth, epidermal cell specification, cell division, and root architecture, and ACT2 and ACT8 were essential for root hair tip growth. Also, genetic complementation revealed that the ACT2 and ACT8 isovariants, but not ACT7, fully rescued the root hair growth defects of single and double mutants. Moreover, we synthesized fully normal plants overexpressing the ACT8 isovariant from multiple actin regulatory sequences as the only vegetative actin in the act2-1 act7-4 background. In summary, it is evident that differences in vegetative actin gene regulation and the diversity in actin isovariant sequences are essential for normal plant development.     

HAlign 3: Fast Multiple Alignment of Ultra-Large Numbers of Similar DNA/RNA Sequences

HAlign is a cross-platform program that performs multiple sequence alignments based on the center star strategy. Here we present two major updates of HAlign 3, which helped improve the time efficiency and the alignment quality, and made HAlign 3 a specialized program to process ultra-large numbers of similar DNA/RNA sequences, such as closely related viral or prokaryotic genomes. HAlign 3 can be easily installed via the Anaconda and Java release package on macOS, Linux, Windows subsystem for Linux, and Windows systems, and the source code is available on GitHub (https://github.com/malabz/HAlign-3).     

Sequences of the 5S rRNAs of Azotobacter vinelandii, Pseudomonas aeruginosa and Pseudomonas fluorescens with some notes on 5S RNA secondary structure

Recently published alignments of available 5 S rRNA sequences have shown that a rigid base pairing pattern, pointing to the existence of a universal five-helix secondary structure for all 5 S RNAs, can be superimposed on such alignments. For a few species, the alignment and the base pairing pattern show distortions with respect to the large majority of sequences. Their 5 S RNAs may form exceptional secondary structures, or there may just be errors in the published sequences. We have examined such a case, Pseudomonas fluorescens, and found the sequence to be in error. The corrected sequence, as well as those of the related species Azotobacter vinelandii and Pseudomonas aeruginosa, fit perfectly in the 5 S RNA sequence alignment and in the five-helix secondary structure model. There exists comparative evidence for the frequent presence of non-standard base pairs at several points of the 5 S RNA secondary structure.     

Algorithm of Regulatory Signal Recognition in DNA Sequences

An algorithm is proposed for extracting regulatory signals from DNA sequences. The algorithm complexity is nearly quadratic. The results of testing the algorithm on artificial and natural sequences are presented.