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In this work, the role of the rkpU and rkpJ genes in the production of the K-antigen polysacchandes (KPS) and in the symbiotic capacity of Sinorhizobium fredii HH103, a broad host-range rhizobial strain able to nodulate soybean and many other legumes, was studied. The rkpJ-and rkpU-encoded products are orthologous to Escherichia coil proteins involved in capsule export. S. fredii HH103 mutant derivatives were contructed in both genes. To our knowledge, this is the first time that the role of rkpU in KPS production has been studied in rhizobia. Both rkpJ and rkpU mutants were unable to produce KPS. The rkpU derivative also showed alterations in its lipopolysaccharide (LPS). Neither KPS production nor rkpJ and rkpU expression was affected by the presence of the flavonoid genistein. Soybean (Glycine max) plants inoculated with the S. fredii HH103 rkpU and rkpJ mutants showed reduced nodulation and clear symptoms of nitrogen starvation. However, neither the rkpJ nor the rkpU mutants were significantly impaired in their symbiotic interaction with cowpea (Vigna unguiculata). Thus, we demonstrate for the first time to our knowledge the involvement of the rkpU gene in rhizobial KPS production and also show that the symbiotic relevance of the S. fredii HH103 KPS depends on the specific bacterium-legume interaction. DOI 10.1099/mic.0.042499-0

Abstract Background [NiFe] hydrogenases are enzymes that catalyze the oxidation of hydrogen into protons and electrons, to use H2 as energy source, or the production of hydrogen through proton reduction, as an escape valve for the excess of reduction equivalents in anaerobic metabolism. Biosynthesis of [NiFe] hydrogenases is a complex process that occurs in the cytoplasm, where a number of auxiliary proteins are required to synthesize and insert the metal cofactors into the enzyme structural units. The endosymbiotic bacterium Rhizobium leguminosarum requires the products of eighteen genes (hupSLCDEFGHIJKhypABFCDEX) to synthesize an active hydrogenase. hupF and hupK genes are found only in hydrogenase clusters from bacteria expressing hydrogenase in the presence of oxygen. Results HupF is a HypC paralogue with a similar predicted structure, except for the C-terminal domain present only in HupF. Deletion of hupF results in the inability to process the hydrogenase large subunit HupL, and also in reduced stability of this subunit when cells are exposed to high oxygen tensions. A ΔhupF mutant was fully complemented for hydrogenase activity by a C-terminal deletion derivative under symbiotic, ultra low-oxygen tensions, but only partial complementation was observed in free living cells under higher oxygen tensions (1% or 3%). Co-purification experiments using StrepTag-labelled HupF derivatives and mass spectrometry analysis indicate the existence of a major complex involving HupL and HupF, and a less abundant HupF-HupK complex. Conclusions The results indicate that HupF has a dual role during hydrogenase biosynthesis: it is required for hydrogenase large subunit processing and it also acts as a chaperone to stabilize HupL when hydrogenase is synthesized in the presence of oxygen.

Inthe intricate maturation process of [NiFe]-hydrogenases, theFe(CN)2CO cofactor is first assembled in a HypCD complexwith iron coordinated by cysteines from both proteins and CO is addedafter ligation of cyanides. The small accessory protein HypC is knownto play a role in delivering the cofactor needed for assembling thehydrogenase active site. However, the chemical nature of the Fe(CN)2CO moiety and the stability of the cofactor–HypC complexare open questions. In this work, we address geometries, properties,and the nature of bonding of all chemical species involved in formationand binding of the cofactor by means of quantum calculations. We alsostudy the influence of environmental effects and binding to cysteineson vibrational frequencies of stretching modes of CO and CN used todetect the presence of Fe(CN)2CO. Carbon monoxide is foundto be much more sensitive to sulfur binding and the polarity of themedium than cyanides. The stability of the HypC–cofactor complexis analyzed by means of molecular dynamics simulation of cofactor-freeand cofactor-bound forms of HypC. The results show that HypC is stableenough to carry the cofactor, but since its binding cysteine is locatedat the N-terminal unstructured tail, it presents large motions insolution, which suggests the need for a guiding interaction to achievedelivery of the cofactor. [ABSTRACT FROM AUTHOR]

The Type VI secretion systems (T6SSs) allow bacteria to translocate effector proteins to other bacteria or to eukaryotic cells. However, little is known about the role of T6SS in endosymbiotic bacteria. In this work we describe the T6SS of Rhizobium etli Mim1, a bacteria able to effectively nodulate common beans. Structural genes and those encoding possible effectors have been identified in a 28-gene DNA region of R. etli Mim1 pRetMIM1f plasmid. Immunodetection of Hcp protein, a conserved key structural component of T6SS systems, indicates that this secretion system is active at high cell densities, in the presence of root exudates, and in bean nodules. Rhizobium etli mutants affected in T6SS structural genes produced plants with lower dry weight and smaller nodules than the wild-type strain, indicating for the first time that the T6SS plays a positive role in Rhizobium –legume symbiosis. [ABSTRACT FROM AUTHOR]

Rhizobium leguminosarum bv. viciae ( Rlv ) UPM791 effectively nodulates pea and lentil, but bacteroids contain a number of proteins differentially expressed depending on the host. One of these host-dependent proteins (C189) is similar to a d i a mino b utyr a te-2-oxoglutarate a mino t ransferase (DABA-AT). DABA-AT activity was demonstrated with cell extracts and with purified protein, so C189 was renamed as Dat. The dat gene was strongly induced in the central, active area of pea nodules, but not in lentil. Mutants defective in dat were impaired in symbiotic performance with pea plants, exhibiting reduced shoot dry weight, smaller nodules, and a lower competitiveness for nodulation. In contrast, there were no significant differences between mutant and wild-type in symbiosis with lentil plants. A comparative metabolomic approach using cell-free extracts from bacteroids induced in pea and lentil showed significant differences among the strains in pea bacteroids whereas no significant differences were found in lentil. Targeted metabolomic analysis revealed that the dat mutation abolished the presence of 2,4-diaminobutyrate (DABA) in pea nodules, indicating that DABA-AT reaction is oriented toward the production of DABA from L-aspartate semialdehyde. This analysis also showed the presence of L-homoserine, a likely source of aspartate semialdehyde, in pea bacteroids but not in those induced in lentil. The dat mutant showed impaired growth when cells were grown with L-homoserine as nitrogen source. Inclusion of DABA or L-homoserine as N source suppressed pantothenate auxotropy in Rlv UPM791, suggesting DABA as source of the pantothenate precursor β-alanine. These data indicate that Rlv UPM791 Dat enzyme is part of an adaptation mechanism of this bacterium to a homoserine-rich environment such as pea nodule and rhizosphere., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ballesteros-Gutiérrez, Albareda, Barbas, López-Gonzálvez, Dunn and Palacios.)