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Abstract Background When heterologous recombinant proteins are produced in Escherichia coli, they often precipitate to form insoluble aggregates of unfolded polypeptides called inclusion bodies. These structures are associated with chaperones like IbpA. However, there are reported cases of "non-classical" inclusion bodies in which proteins are soluble, folded and active. Results We report that the Brucella abortus PdhS histidine kinase fused to the mCherry fluorescent protein forms intermediate aggregates resembling "non-classical" inclusion bodies when overproduced in E. coli, before forming "classical" inclusion bodies. The intermediate aggregates of PdhS-mCherry are characterized by the solubility of PdhS-mCherry, its ability to specifically recruit known partners fused to YFP, suggesting that PdhS is folded in these conditions, and the quick elimination (in less than 10 min) of these structures when bacterial cells are placed on fresh rich medium. Moreover, soluble PdhS-mCherry foci do not systematically colocalize with IpbA-YFP, a marker of inclusion bodies. Instead, time-lapse experiments show that IbpA-YFP exhibits rapid pole-to-pole shuttling, until it partially colocalizes with PdhS-mCherry aggregates. Conclusion The data reported here suggest that, in E. coli, recombinant proteins like PdhS-mCherry may transit through a soluble and folded state, resembling previously reported "non-classical" inclusion bodies, before forming "classical" inclusion bodies. The dynamic localization of IbpA-YFP foci suggests that the IbpA chaperone could scan the E. coli cell to find its substrates.

The bacterial pathogen Brucella abortus was recently demonstrated to recruit the essential cytoplasmic histidine kinase PdhS to its old pole. Here, we report identification of the fumarase FumC as a specific partner for the N-terminal 'sensing' domain of PdhS, using an ORFeome-based yeast two-hybrid screen. We observed that FumC and PdhS colocalize at the old pole of B. abortus, while the other fumarase FumA is not polarly localized. FumC is not required for PdhS localization, and polar FumC localization is not FumA dependent. FumC homologs are not polarly localized in Sinorhizobium meliloti and Caulobacter crescentus, suggesting that polar recruitment of FumC by PdhS is evolutionarily recent. doi: 10.1128/JB.00066-10

The cell envelope of Gram-negative bacteria is an essential organelle that is important for cell shape and protection from toxic compounds. Proteins involved in envelope biogenesis are therefore attractive targets for the design of new antibacterial agents. In a search for new envelope assembly factors, we screened a collection of Escherichia coli deletion mutants for sensitivity to detergents and hydrophobic antibiotics, a phenotype indicative of defects in the cell envelope. Strains lacking yciM were among the most sensitive strains of the mutant collection. Further characterization of yciM mutants revealed that they display a thermo sensitive growth defect on low-osmolarity medium and that they have a significantly altered cellmorphology. At elevated temperatures, yciM mutants form bulges containing cytoplasmic material and subsequently lyse. We also discovered that yciM genetically interacts with envC, a gene encoding a regulator of the activity of peptidoglycan amidases. Altogether, these results indicate that YciM is required for envelope integrity. Biochemical characterization of the protein showed that YciM is anchored to the inner membrane via its N terminus, the rest of the protein being exposed to the cytoplasm. Two CXXC motifs are present at the C terminus of YciM and serve to coordinate a redox-sensitive iron center of the rubredoxin type. Both the N-terminal membrane anchor and the C-terminal iron center of YciM are important for function. [ABSTRACT FROM AUTHOR]

Acinetobacter baumannii is one of the most problematic nosocomial pathogens that can efficiently thrive within hospital settings, mainly due to resistances toward antibiotics, desiccation, disinfectants, human serum and oxidative stress. Recently, increased resistance against last-resort antibiotics earns this bacterium the highest priority concern classified by the Centers for Disease Control and Prevention and the World Health Organization. An obvious hallmark of this bacterium is the high heterogeneity observed among A. baumannii isolates, with a limited core genome. This feature complexifies the study of A. baumannii bacteria as an entity, subsequently reflected in a diversity of phenotypes of not only antimicrobial and environmental resistance but also virulence. A high degree of genome plasticity, along with the use of a limited subset of established strains, can lead to strain-specific observations, decreasing the global understanding of this pathogenic agent. Phenotypic variability of A. baumannii strains is easily observable such as with the macrocolony morphologies, in vitro and in vivo virulence, natural competence level, production of different capsular polysaccharide structures and cellular densities. Some strains encode an extensive amount of virulence factors, while others, including the established strains, lack several key ones. The lack/excess of genes or specific physiological processes might interfere with in vivo and in vitro experiments, thus providing a limited impact on the global understanding of Acinetobacter bacteria. As an answer to the high heterogeneity among A. baumannii strains, we propose a first comprehensive database that includes the bacterial strains and the associated phenotypic and genetic data. This new repository, freely accessible to the entire scientific community, allows selecting the best bacterial isolate(s) related to any biological question, using an efficient and fast exchange platform. Database URL: https://acinetobase.vib.be/., (© The Author(s) 2022. Published by Oxford University Press.)