Lineage-specific evolution of Aquibium, a close relative of Mesorhizobium, during habitat adaptation.

The novel genus Aquibium that lacks nitrogenase was recently reclassified from the Mesorhizobium genus. The genomes of Aquibium species isolated from water were smaller and had higher GC contents than those of Mesorhizobium species. Six Mesorhizobium species lacking nitrogenase were found to exhibit low similarity in the average nucleotide identity values to the other 24 Mesorhizobium species. Therefore, they were classified as the non-N2-fixing Mesorhizobium lineage (N-ML), an evolutionary intermediate species. The results of our phylogenomic analyses and the loss of Rhizobiales-specific fur/mur indicated that Mesorhizobium species may have evolved from Aquibium species through an ecological transition. Halotolerant and alkali-resistant Aquibium and Mesorhizobium microcysteis belonging to N-ML possessed many tripartite ATP-independent periplasmic transporter and sodium/proton antiporter subunits composed of seven genes (mrpABCDEFG). These genes were not present in the N2-fixing Mesorhizobium lineage (ML), suggesting that genes acquired for adaptation to highly saline and alkaline environments were lost during the evolution of ML as the habitat changed to soil. Land-to-water habitat changes in Aquibium species, close relatives of Mesorhizobium species, could have influenced their genomic evolution by the gain and loss of genes. Our study indicated that lineage-specific evolution could have played a significant role in shaping their genome architecture and conferring their ability to thrive in different habitats.IMPORTANCEPhylogenetic analyses revealed that the Aquibium lineage (AL) and non-N2-fixing Mesorhizobium lineage (N-ML) were monophyletically grouped into distinct clusters separate from the N2-fixing Mesorhizobium lineage (ML). The N-ML, an evolutionary intermediate species having characteristics of both ancestral and descendant species, could provide a genomic snapshot of the genetic changes that occur during adaptation. Genomic analyses of AL, N-ML, and ML revealed that changes in the levels of genes related to transporters, chemotaxis, and nitrogen fixation likely reflect adaptations to different environmental conditions. Our study sheds light on the complex and dynamic nature of the evolution of rhizobia in response to changes in their environment and highlights the crucial role of genomic analysis in understanding these processes.

TRAP transporter TakP: a key player in the resistance against selenite-induced oxidative stress in Rhodobacter sphaeroides.

Almost one-third of all proteins require metal ions as an essential component in key biological processes and approximately half of all enzymes are associated with one or more metal ions. The naturally occurring selenium is very toxic at higher levels, but few bacteria can reduce it into the less toxic insoluble elemental selenium. Selenium is required for the synthesis of selenocysteine, an essential residue involved in the active sites of various enzymes. The purple non-sulphur bacteria, Rhodobacter sphaeroidesis demonstrated for its selenite reduction capacity. The exact mechanism of selenite toxicity is unknown but it reacts with glutathione to form selenodiglutathione, producing the highly toxic compounds namely, H2O2and O2-. A R. sphaeroidesstrain with mutated takP gene, a member of the TRAP (tripartite ATP-independent periplasmic) family of transporter, was reported to be showing more resistance towards selenite in the growth medium but the reason for the resistance is unknown. TRAP transporters are the best-studied family of substrate-binding protein and in our previous study it was confirmed that the gene takP in R. sphaeroides is down-regulated by a small non-coding RNA SorY, providing more resistance to the bacterium against the oxidative stress. By comparative growth analysis and sensitivity assays in the presence of 2 mM selenite, it was observed that the SorY knockout strain is more sensitive to selenite while overexpression of the sRNA conferred more resistance to the bacterium like the takP mutant strain. TakP is involved in the import of malate into the cell, which under oxidative stress needs to be down-regulated to limit malate flux into the cell. Limited malate flux leads to metabolic rearrangements in the cell to avoid excessive generation of prooxidant NADH and facilitate constant generation of antioxidant NADPH. In the presence and absence of selenite, a drastic increase in the NADPH and decrease in the NADH levels are reported respectively. Accumulation of metallic selenium in the cytoplasm was detected via atomic absorption spectrophotometer and our analysis clearly demonstrated the presence of more selenium in the electron micrographs of the SorY knockout strain compared to the takP mutant grown under dark semi-aerobic growth conditions in the presence of selenite. Hence based on our analysis, it is confirmed that lack of TakP transporter led to reduced selenite influx into the cytoplasm, relieving cells with limited generation of ROS, eventually exhibiting more resistance against selenite-induced oxidative stress.

Selective Nutrient Transport in Bacteria: Multicomponent Transporter Systems Reign Supreme.

Multicomponent transporters are used by bacteria to transport a wide range of nutrients. These systems use a substrate-binding protein to bind the nutrient with high affinity and then deliver it to a membrane-bound transporter for uptake. Nutrient uptake pathways are linked to the colonisation potential and pathogenicity of bacteria in humans and may be candidates for antimicrobial targeting. Here we review current research into bacterial multicomponent transport systems, with an emphasis on the interaction at the membrane, as well as new perspectives on the role of lipids and higher oligomers in these complex systems.

Crystal structure of the sugar acid-binding protein CxaP from a TRAP transporter in Advenella mimigardefordensis strain DPN7T.

Recently, CxaP, a sugar acid substrate binding protein (SBP) from Advenella mimigardefordensis strain DPN7T , was identified as part of a novel sugar uptake strategy. In the present study, the protein was successfully crystallized. Although several SBP structures of tripartite ATP-independent periplasmic transporters have already been solved, this is the first structure of an SBP accepting multiple sugar acid ligands. Protein crystals were obtained with bound d-xylonic acid, d-fuconic acid d-galactonic and d-gluconic acid, respectively. The protein shows the typical structure of an SBP of a tripartite ATP-independent periplasmic transporter consisting of two domains linked by a hinge and spanned by a long α-helix. By analysis of the structure, the substrate binding site of the protein was identified. The carboxylic group of the sugar acids interacts with Arg175, whereas the coordination of the hydroxylic groups at positions C2 and C3 is most probably realized by Arg154 and Asn151. Furthermore, it was observed that 2-keto-3-deoxy-d-gluconic acid is bound in protein crystals that were crystallized without the addition of any ligand, indicating that this molecule is prebound to the protein and is displaced by the other ligands if they are available. DATABASE: Structural data of CxaP complexes are available in the worldwide Protein Data Bank (https://www.rcsb.org) under the accession codes 7BBR (2-keto-3-deoxy-d-gluconic acid), 7BCR (d-galactonic acid), 7BCN (d-xylonic acid), 7BCO (d-fuconic acid) and 7BCP (d-gluconic acid).

The periplasmic component of the DctPQM TRAP-transporter is part of the DctS/DctR sensory pathway in Rhodobacter sphaeroides.

Rhodobacter sphaeroides can use C4-dicarboxylic acids to grow heterotrophically or photoheterotropically, and it was previously demonstrated in Rhodobacter capsulatus that the DctPQM transporter system is essential to support growth using these organic acids under heterotrophic but not under photoheterotrophic conditions. In this work we show that in R. sphaeroides this transporter system is essential for photoheterotrophic and heterotrophic growth, when C4-dicarboxylic acids are used as a carbon source. We also found that over-expression of dctPQM is detrimental for photoheterotrophic growth in the presence of succinic acid in the culture medium. In agreement with this, we observed a reduction of the dctPQM promoter activity in cells growing under these conditions, indicating that the amount of DctPQM needs to be reduced under photoheterotrophic growth. It has been reported that the two-component system DctS and DctR activates the expression of dctPQM. Our results demonstrate that in the absence of DctR, dctPQM is still expressed albeit at a low level. In this work, we have found that the periplasmic component of the transporter system, DctP, has a role in both transport and in signalling the DctS/DctR two-component system.

The Salmonella enterica serovar Typhimurium virulence factor STM3169 is a hexuronic acid binding protein component of a TRAP transporter.

The intracellular pathogen S. Typhimurium is a leading cause of foodborne illness across the world and is known to rely on a range of virulence factors to colonize the human host and cause disease. The gene coding for one such factor, stm3169, was determined to be upregulated upon macrophage entry and its disruption reduces survival in the macrophage. In this study we characterize the STM3169 protein, which forms the substrate binding protein (SBP) of an uncharacterized tripartite ATP-independent periplasmic (TRAP) transporter. Genome context analysis of the genes encoding this system in related bacteria suggests a function in sugar acid transport. We demonstrate that purified STM3169 binds d-glucuronic acid with high affinity and specificity. S. Typhimurium LT2 can use this sugar acid as a sole carbon source and the genes for a probable catabolic pathway are present in the genome. As this gene was previously implicated in macrophage survival, it suggests a role for d-glucuronate as an important carbon source for S. Typhimurium in this environment.

"Just a spoonful of sugar...": import of sialic acid across bacterial cell membranes.

Eukaryotic cell surfaces are decorated with a complex array of glycoconjugates that are usually capped with sialic acids, a large family of over 50 structurally distinct nine-carbon amino sugars, the most common member of which is N-acetylneuraminic acid. Once made available through the action of neuraminidases, bacterial pathogens and commensals utilise host-derived sialic acid by degrading it for energy or repurposing the sialic acid onto their own cell surface to camouflage the bacterium from the immune system. A functional sialic acid transporter has been shown to be essential for the uptake of sialic acid in a range of human bacterial pathogens and important for host colonisation and persistence. Here, we review the state-of-play in the field with respect to the molecular mechanisms by which these bio-nanomachines transport sialic acids across bacterial cell membranes.

Sialic acid acquisition in bacteria-one substrate, many transporters.

The sialic acids are a family of 9-carbon sugar acids found predominantly on the cell-surface glycans of humans and other animals within the Deuterostomes and are also used in the biology of a wide range of bacteria that often live in association with these animals. For many bacteria sialic acids are simply a convenient source of food, whereas for some pathogens they are also used in immune evasion strategies. Many bacteria that use sialic acids derive them from the environment and so are dependent on sialic acid uptake. In this mini-review I will describe the discovery and characterization of bacterial sialic acids transporters, revealing that they have evolved multiple times across multiple diverse families of transporters, including the ATP-binding cassette (ABC), tripartite ATP-independent periplasmic (TRAP), major facilitator superfamily (MFS) and sodium solute symporter (SSS) transporter families. In addition there is evidence for protein-mediated transport of sialic acids across the outer membrane of Gram negative bacteria, which can be coupled to periplasmic processing of different sialic acids to the most common form, ß-D-N-acetylneuraminic acid (Neu5Ac) that is most frequently taken up into the cell.

Transport and catabolism of the sialic acids N-glycolylneuraminic acid and 3-keto-3-deoxy-D-glycero-D-galactonononic acid by Escherichia coli K-12.

Escherichia coli can transport and catabolize the common sialic acid, N-acetylneuraminic acid (Neu5Ac), as a sole source of carbon and nitrogen, which is an important mucus-derived carbon source in the mammalian gut. Herein we demonstrate that E. coli can also grow efficiently on the related sialic acids, N-glycolylneuraminic acid (Neu5Gc) and 3-keto-3-deoxy-D-glycero-D-galactonononic acid (KDN), which are transported via the sialic acid transporter NanT and catabolized using the sialic acid aldolase NanA. Catabolism of Neu5Gc uses the same pathway as Neu5Ac, likely producing glycolate instead and acetate during its breakdown and catabolism of KDN requires NanA activity, while other components of the Neu5Ac catabolism pathway are non-essential. We also demonstrate that these two sialic acids can support growth of an E. coli ∆nanT strain expressing sialic acid transporters from two bacterial pathogens, namely the tripartite ATP-independent periplasmic transporter SiaPQM from Haemophilus influenzae and the sodium solute symport transporter STM1128 from Salmonella enterica ssp. Typhimurium, suggesting that the ability to use Neu5Gc and KDN in addition to Neu5Ac is present in a number of human pathogens.