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The type IV secretion system (T4SS) of the plant intracellular symbiont Sinorhizobium meliloti 1021 is required for conjugal transfer of DNA. However, it is not required for host invasion and persistence, unlike the T4SSs of closely related mammalian intracellular pathogens. A comparison of the requirement for a bacterial T4SS in plant versus animal host invasion suggests an important difference in the intracellular niches occupied by these bacteria.

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with [beta]-1,3, [beta]-1,4, [beta]-1,6, [alpha]-1,3, and [alpha]-l,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGR[OMEGA]exoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGR[OMEGA]exoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal[(Glc).sub.5][(GlcA).sub.2] PvGal. When used as inoculants, both the exo-deficient mutants and NGR[OMEGA]exoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, ~50 [micro]g per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-[beta]-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes.

The article discusses a study which demonstrated that increased exopolysaccharide succinoglycan production is not detrimental to symbiotic development but enhances the symbiotic productivity of Sinorhizobium meliloti 1021 with the host plant Medicago truncatula cv. ]emalong A17. The study showed that increased succinoglycan production was engineered by overexpression of the exoYgene. It also proposed several possible roles for succinoglycan in infection thread initiation and development.

The patB gene product is required for growth and survival of the filamentous cyanobacterium Anabaena sp. strain PCC 7120 in the absence of combined nitrogen. A patB::gfp fusion demonstrated that this gene is expressed exclusively in heterocysts.patB mutants have a normal initial pattern of heterocyst spacing along the filament but differentiate excess heterocysts after several days in the absence of combined nitrogen. Expression of hetR and patS, two critical regulators of the heterocyst development cascade, are normal for patB mutants, indicating that patB acts downstream of them in the differentiation pathway. A patB deletion mutant suffers an almost complete cessation of growth and nitrogen fixation within 24 h of combined nitrogen removal. In contrast, a new PatB mutant that is defective in its N-terminal ferredoxin domain, or a previously described mutant that has a frameshift removing its C-terminal helix-turn-helix domain, grows very slowly and differentiates multiple contiguous heterocysts under nitrogen-deficient conditions. [ABSTRACT FROM AUTHOR]

Identifies cytochrome c oxidase operon in the nitrogen-fixing cyanobacterium Anabaena specie strain PCC 7120. Correlation between heterocysts and nitrogenases functions; Differentiation of heterocysts by Anabaena strain; Expression of operons in the proheterocysts.

Liberibacter crescens is the only cultured member of its genus, which includes the devastating plant pathogen "Candidatus Liberibacter asiaticus," associated with citrus greening/Huanglongbing (HLB). L. crescens has a larger genome and greater metabolic flexibility than "Ca. Liberibacter asiaticus" and the other uncultured plant-pathogenic Liberibacter species, and it is currently the best model organism available for these pathogens. L. crescens grows slowly and dies rapidly under current culture protocols and this extreme fastidiousness makes it challenging to study. We have determined that a major cause of rapid death of L. crescens in batch culture is its alkalinization of the medium (to pH 8.5 by the end of logarithmic phase). The majority of this alkalinization is due to consumption of alpha-ketoglutaric acid as its primary carbon source, with a smaller proportion of the pH rise due to NH3 production. Controlling the pH rise with higher buffering capacity and lower starting pH improved recoverability of cells from 10-day cultures by >1,000-fold. We have also performed a detailed analysis of L. crescens growth with total cell numbers calibrated to the optical density and the percentage of live and recoverable bacteria determined over 10- day time courses. We modified L. crescens culture conditions to greatly enhance survival and increase maximum culture density. The similarities between L. crescens and the pathogenic liberibacters make this work relevant to efforts to culture the latter organisms. Our results also suggest that growth-dependent pH alteration that overcomes medium buffering should always be considered when growing fastidious bacteria. [ABSTRACT FROM AUTHOR]

Relatively little is known about the phages that infect agriculturally important nitrogen-fixing rhizobial bacteria. Here we report the genome and cryo-electron microscopy structure of the Sinorhizobium meliloti-infecting T4 superfamily phage φM9. This phage and its close relative Rhizobium phage vB_RleM_P10VF define a new group of T4 superfamily phages. These phages are distinctly different from the recently characterized cyanophage-like S. meliloti phages of the φM12 group. Structurally, φM9 has a T=16 capsid formed from repeating units of an extended gp23-like subunit that assemble through interactions between one subunit and the adjacent E-loop insertion domain. Though genetically very distant from the cyanophages, the φM9 capsid closely resembles that of the T4 superfamily cyanophage Syn9. φM9 also has the same T=16 capsid architecture as the very distant phage SPO1 and the herpesviruses. Despite their overall lack of similarity at the genomic and structural levels, φM9 and S. meliloti phage φM12 have a small number of open reading frames in common that appear to encode structural proteins involved in interaction with the host and which may have been acquired by horizontal transfer. These proteins are predicted to encode tail baseplate proteins, tail fibers, tail fiber assembly proteins, and glycanases that cleave host exopolysaccharide. [ABSTRACT FROM AUTHOR]

Although the role that lipopolysaccharide (LPS) plays in the symbiosis between Sinorhizobium meliloti and alfalfa has been studied for over a decade, its function in this process remains controversial and poorly understood. This is largely due to a lack of mutants affected by its synthesis. In one of the definitive studies concerning this issue, Clover et al. (R. H. Clover, J. Kieber, and E. R. Signer, J. Bacteriol. 171:3961-3967, 1989) identified a series of mutants with putative LPS defects, judged them to be symbiotically proficient on Medicago sativa, and concluded that LPS might not have a symbiotic function in S. meliloti. The mutations in these strains were never characterized at the molecular level nor was the LPS from most of them analyzed. We have transduced these mutations from the Rm2011 background from which they were originally isolated into the sequenced strain Rml021 and have characterized the resulting strains in greater detail. We found the LPS from these mutants to display a striking complexity of phenotypes on polyacrylamide electrophoresis gels, including additional rough LPS bands and alterations in the molecular weight distribution of the smooth LPS. We found that some of the mutants contain insertions in genes that are predicted to be involved in the synthesis of carbohydrate components of LPS, including ddhB, lpsB, lpsC, and lpsE. The majority, however, code for proteins predicted to be involved in a wide variety of functions not previously recognized to play a role in LPS synthesis, including a possible transcription elongation factor (GreA), a possible queuine synthesis protein, and a possible chemotaxis protein. Furthermore, using more extensive assays, we have found that most of these strains have symbiotic deficiencies. These results support more recent findings that alterations in LPS structure can affect the ability of S. meliloti to form an effective symbiosis. [ABSTRACT FROM AUTHOR]

Rhizobia are nitrogen-fixing bacteria that establish endosymbiotic associations with legumes. Nodule formation depends on various bacterial carbohydrates, including lipopolysaccharides, K-antigens, and exopolysaccharides (EPS). An acidic EPS from Rhizobium sp. strain NGR234 consists of glucosyl (Glc), galactosyl (Gal), glucuronosyl (GlcA), and 4,6-pyruvylated galactosyl (PvGal) residues with β-1,3, β-1,4, β-1,6, α-1,3, and α-1,4 glycoside linkages. Here we examined the role of NGR234 genes in the synthesis of EPS. Deletions within the exoF, exoL, exoP, exoQ, and exoY genes suppressed accumulation of EPS in bacterial supernatants, a finding that was confirmed by chemical analyses. The data suggest that the repeating subunits of EPS are assembled by an ExoQ/ExoP/ExoF-dependent mechanism, which is related to the Wzy polymerization system of group 1 capsular polysaccharides in Escherichia coli. Mutation of exoK (NGR Ω exoK), which encodes a putative glycanase, resulted in the absence of low-molecular-weight forms of EPS. Analysis of the extracellular carbohydrates revealed that NGR Ω exoK is unable to accumulate exo-oligosaccharides (EOSs), which are O-acetylated nonasaccharide subunits of EPS having the formula Gal(Glc)5(GlcA)2PvGal. When used as inoculants, both the exo-deficient mutants and NGR Ω exoK were unable to form nitrogen-fixing nodules on some hosts (e.g., Albizia lebbeck and Leucaena leucocephala), but they were able to form nitrogen-fixing nodules on other hosts (e.g., Vigna unguiculata). EOSs of the parent strain were biologically active at very low levels (yield in culture supernatants, ∼50 µg per liter). Thus, NGR234 produces symbiotically active EOSs by enzymatic degradation of EPS, using the extracellular endo-β-1,4-glycanase encoded by exoK (glycoside hydrolase family 16). We propose that the derived EOSs (and not EPS) are bacterial components that play a crucial role in nodule formation in various legumes. [ABSTRACT FROM AUTHOR]

The prairie vole ( Microtus ochrogaster ) is an important model for the study of social monogamy and dual parental care of offspring. Characterization of specific host species-microbe strain interactions is critical for understanding the effects of the microbiota on mood and behavior. The five metagenome-assembled genome sequences reported here represent an important step in defining the prairie vole microbiome., (Copyright © 2020 Donovan et al.)

Sinorhizobium phage ΦM6 infects the nitrogen-fixing rhizobial bacterium Sinorhizobium meliloti. ΦM6 most closely resembles marine phages, such as Puniceispirillum phage HMO-2011, rather than previously sequenced rhizobial phages. The 68,176-bp genome is predicted to encode 121 open reading frames, only 10 of which have similarity to those of otherwise-unrelated Sinorhizobium phages.

Chagas disease, caused by infection with the protozoan parasite Trypanosoma cruzi , is a leading cause of heart disease ("chagasic cardiomyopathy") in Latin America, disproportionately affecting people in resource-poor areas. The efficacy of currently approved pharmaceutical treatments is limited mainly to acute infection, and there are no effective treatments for the chronic phase of the disease. Preclinical models of Chagas disease have demonstrated that antigen-specific CD8 + gamma interferon (IFN-γ)-positive T-cell responses are essential for reducing parasite burdens, increasing survival, and decreasing cardiac pathology in both the acute and chronic phases of Chagas disease. In the present study, we developed a genetically adjuvanted, dendritic cell-based immunotherapeutic for acute Chagas disease in an attempt to delay or prevent the cardiac complications that eventually result from chronic T. cruzi infection. Dendritic cells transduced with the adjuvant, an adenoviral vector encoding a dominant negative isoform of Src homology region 2 domain-containing tyrosine phosphatase 1 (SHP-1) along with the T. cruzi Tc24 antigen and trans -sialidase antigen 1 (TSA1), induced significant numbers of antigen-specific CD8 + IFN-γ-positive cells following injection into BALB/c mice. A vaccine platform transduced with the adenoviral vector and loaded in tandem with the recombinant protein reduced parasite burdens by 76% to >99% in comparison to a variety of different controls and significantly reduced cardiac pathology in a BALB/c mouse model of live Chagas disease. Although no statistical differences in overall survival rates among cohorts were observed, the data suggest that immunotherapeutic strategies for the treatment of acute Chagas disease are feasible and that this approach may warrant further study., (Copyright © 2017 American Society for Microbiology.)

Unlabelled: The acidic polysaccharide succinoglycan produced by the rhizobial symbiont Sinorhizobium meliloti 1021 is required for this bacterium to invade the host plant Medicago truncatula and establish a nitrogen-fixing symbiosis. S. meliloti mutants that cannot make succinoglycan cannot initiate invasion structures called infection threads in plant root hairs. S. meliloti exoH mutants that cannot succinylate succinoglycan are also unable to form infection threads, despite the fact that they make large quantities of succinoglycan. Succinoglycan produced by exoH mutants is refractory to cleavage by the glycanases encoded by exoK and exsH, and thus succinoglycan produced by exoH mutants is made only in the high-molecular-weight (HMW) form. One interpretation of the symbiotic defect of exoH mutants is that the low-molecular-weight (LMW) form of succinoglycan is required for infection thread formation. However, our data demonstrate that production of the HMW form of succinoglycan by S. meliloti 1021 is sufficient for invasion of the host M. truncatula and that the LMW form is not required. Here, we show that S. meliloti strains deficient in the exoK- and exsH-encoded glycanases invade M. truncatula and form a productive symbiosis, although they do this with somewhat less efficiency than the wild type. We have also characterized the polysaccharides produced by these double glycanase mutants and determined that they consist of only HMW succinoglycan and no detectable LMW succinoglycan. This demonstrates that LMW succinoglycan is not required for host invasion. These results suggest succinoglycan function is not dependent upon the presence of a small, readily diffusible form., Importance: Sinorhizobium meliloti is a bacterium that forms a beneficial symbiosis with legume host plants. S. meliloti and other rhizobia convert atmospheric nitrogen to ammonia, a nutrient source for the host plant. To establish the symbiosis, rhizobia must invade plant roots, supplying the proper signals to prevent a plant immune response during invasion. A polysaccharide, succinoglycan, produced by S. meliloti is required for successful invasion. Here, we show that the critical feature of succinoglycan that allows infection to proceed is the attachment of a "succinyl" chemical group and that the chain length of succinoglycan is much less important for its function. We also show that none of the short-chain versions of succinoglycan is produced in the absence of two chain-cleaving enzymes., (Copyright © 2016 Mendis et al.)