The bacterial AAA+ enhancer-binding proteins (EBPs) HrpR and HrpS (HrpRS) of Pseudomonas syringae (Ps) activate σ54-dependent transcription at the hrpL promoter; triggering type-three secretion system-mediated pathogenicity. In contrast with singly acting EBPs, the evolution of the strictly co-operative HrpRS pair raises questions of potential benefits and mechanistic differences this transcription control system offers. Here, we show distinct properties of HrpR and HrpS variants, indicating functional specialization of these non-redundant, tandemly arranged paralogues. Activities of HrpR, HrpS and their control proteins HrpV and HrpG from Ps pv. tomato DC3000 in vitro establish that HrpRS forms a transcriptionally active hetero-hexamer, that there is a direct negative regulatory role for HrpV through specific binding to HrpS and that HrpG suppresses HrpV. The distinct HrpR and HrpS functionalities suggest how partial paralogue degeneration has potentially led to a novel control mechanism for EBPs and indicate subunit-specific roles for EBPs in σ54-RNA polymerase activation.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni 2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

Azospirillum species are plant-associated diazotrophs of the alpha subclass of Proteobacteria. The genomes of five of the six Azospirillum species were analyzed by pulsed-field gel electrophoresis. All strains possessed several megareplicons, some probably linear, and 16S ribosomal DNA hybridization indicated multiple chromosomes in genomes ranging in size from 4.8 to 9.7 Mbp. The nifHDK operon was identified in the largest replicon.

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BackgroundHerbaspirillum seropedicae SmR1 is a nitrogen fixing endophyte associated with important agricultural crops. It produces polyhydroxybutyrate (PHB) which is stored intracellularly as granules. However, PHB metabolism and regulatory control is not yet well studied in this organism.ResultsIn this work we describe the characterization of the PhbF protein from H. seropedicae SmR1 which was purified and characterized after expression in E. coli. The purified PhbF protein was able to bind to eleven putative promoters of genes involved in PHB metabolism in H. seropedicae SmR1. In silico analyses indicated a probable DNA-binding sequence which was shown to be protected in DNA footprinting assays using purified PhbF. Analyses using lacZ fusions showed that PhbF can act as a repressor protein controlling the expression of PHB metabolism-related genes.ConclusionsOur results indicate that H. seropedicae SmR1 PhbF regulates expression of phb-related genes by acting as a transcriptional repressor. The knowledge of the PHB metabolism of this plant-associated bacterium may contribute to the understanding of the plant-colonizing process and the organism's resistance and survival in planta.