Comparison of 26 Sphingomonad Genomes Reveals Diverse Environmental Adaptations and Biodegradative Capabilities

Sphingomonads comprise a physiologically versatile group within the Alphaproteobacteria that includes strains of interest for biotechnology, human health, and environmental nutrient cycling. In this study, we compared 26 sphingomonad genome sequences to gain insight into their ecology, metabolic versatility, and environmental adaptations. Our multilocus phylogenetic and average amino acid identity (AAI) analyses confirm that Sphingomonas, Sphingobium, Sphingopyxis, and Novosphingobium are well-resolved monophyletic groups with the exception of Sphingomonas sp. strain SKA58, which we propose belongs to the genus Sphingobium. Our pan-genomic analysis of sphingomonads reveals numerous species-specific open reading frames (ORFs) but few signatures of genus-specific cores. The organization and coding potential of the sphingomonad genomes appear to be highly variable, and plasmid-mediated gene transfer and chromosome-plasmid recombination, together with prophage- and transposon-mediated rearrangements, appear to play prominent roles in the genome evolution of this group. We find that many of the sphingomonad genomes encode numerous oxygenases and glycoside hydrolases, which are likely responsible for their ability to degrade various recalcitrant aromatic compounds and polysaccharides, respectively. Many of these enzymes are encoded on megaplasmids, suggesting that they may be readily transferred between species. We also identified enzymes putatively used for the catabolism of sulfonate and nitroaromatic compounds in many of the genomes, suggesting that plant-based compounds or chemical contaminants may be sources of nitrogen and sulfur. Many of these sphingomonads appear to be adapted to oligotrophic environments, but several contain genomic features indicative of host associations. Our work provides a basis for understanding the ecological strategies employed by sphingomonads and their role in environmental nutrient cycling.     

X-ray sterilization of biopharmaceutical manufacturing equipment—Extractables profile of a film material and copolyester Tritan™ compared to gamma irradiation

The biopharmaceutical industry gains enormous flexibility in production processes by using sterilized preassembled single-use devices. Gamma irradiation is an established sterilization technology that may be restricted in the future by the availability of ⁶⁰Co as irradiation source and irradiation capacities. X-ray technology is considered an alternative type of radiation for sterilizing SU equipment. In the context of extractables and leachables—one concern connected with the use of single-use process equipment—the effect of X-ray irradiation on the extractables profile of the materials needs to be compared to established gamma irradiation to qualify this alternative technology. An approach is presented to obtain robust and comprehensive extractables data for materials used in SU devices after sterilization either using X-ray or gamma irradiation. A careful selection of the test items and the test design allows a one-to-one comparison of data obtained from a combination of orthogonal analytical techniques. The extractables of a modern SU film material and the copolyester Tritan™ are evaluated. The data presented allow a risk evaluation on the safety of this new sterilization modality for biopharmaceutical applications. It is demonstrated that the extractables profile of a polymer is not affected by the type of irradiation used for sterilization.     

Infection and transmission of SARS‐CoV‐2 depend on heparan sulfate proteoglycans

The current pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) and outbreaks of new variants highlight the need for preventive treatments. Here, we identified heparan sulfate proteoglycans as attachment receptors for SARS‐CoV‐2. Notably, neutralizing antibodies against SARS‐CoV‐2 isolated from COVID‐19 patients interfered with SARS‐CoV‐2 binding to heparan sulfate proteoglycans, which might be an additional mechanism of antibodies to neutralize infection. SARS‐CoV‐2 binding to and infection of epithelial cells was blocked by low molecular weight heparins (LMWH). Although dendritic cells (DCs) and mucosal Langerhans cells (LCs) were not infected by SARS‐CoV‐2, both DC subsets efficiently captured SARS‐CoV‐2 via heparan sulfate proteoglycans and transmitted the virus to ACE2‐positive cells. Notably, human primary nasal cells were infected by SARS‐CoV‐2, and infection was blocked by pre‐treatment with LMWH. These data strongly suggest that heparan sulfate proteoglycans are important attachment receptors facilitating infection and transmission, and support the use of LMWH as prophylaxis against SARS‐CoV‐2 infection.     

The interaction of beta-amyloid protein with cellular membranes stimulates its own production

Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ_(1-40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ_(1-40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.     

Genome Survey and Characterization of Endophytic Bacteria Exhibiting a Beneficial Effect on Growth and Development of Poplar Trees

The association of endophytic bacteria with their plant hosts has a beneficial effect for many different plant species. Our goal is to identify endophytic bacteria that improve the biomass production and the carbon sequestration potential of poplar trees (Populus spp.) when grown in marginal soil and to gain an insight in the mechanisms underlying plant growth promotion. Members of the Gammaproteobacteria dominated a collection of 78 bacterial endophytes isolated from poplar and willow trees. As representatives for the dominant genera of endophytic gammaproteobacteria, we selected Enterobacter sp. strain 638, Stenotrophomonas maltophilia R551-3, Pseudomonas putida W619, and Serratia proteamaculans 568 for genome sequencing and analysis of their plant growth-promoting effects, including root development. Derivatives of these endophytes, labeled with gfp, were also used to study the colonization of their poplar hosts. In greenhouse studies, poplar cuttings (Populus deltoides × Populus nigra DN-34) inoculated with Enterobacter sp. strain 638 repeatedly showed the highest increase in biomass production compared to cuttings of noninoculated control plants. Sequence data combined with the analysis of their metabolic properties resulted in the identification of many putative mechanisms, including carbon source utilization, that help these endophytes to thrive within a plant environment and to potentially affect the growth and development of their plant hosts. Understanding the interactions between endophytic bacteria and their host plants should ultimately result in the design of strategies for improved poplar biomass production on marginal soils as a feedstock for biofuels.Endophytic bacteria are bacteria that reside within the living tissue of their host plants without substantively harming it (19, 26). They are ubiquitous in most plant species, latently residing or actively colonizing the tissues. The diversity of cultivable bacterial endophytes is exhibited not only in the variety of plant species colonized but also in the many taxa involved, with most being members of common soil bacterial genera such as Enterobacter, Pseudomonas, Burkholderia, Bacillus, and Azospirillum (21, 23). Endophytic bacteria have several mechanisms by which they can promote plant growth and health. These mechanisms are of prime importance for the use of plants as feedstocks for biofuels and for carbon sequestration through biomass production. This is vital when considering the aim of improving biomass production of marginal soils, thus avoiding competition for agricultural resources, which is one of the critical socioeconomic issues of the increased use of biofuels.Like rhizosphere bacteria, endophytic bacteria have been shown to have plant growth-promoting activity that can be due to the production of phytohormones, enzymes involved in growth regulator metabolism, such as ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, auxins, indole-3-acetic acid (IAA), acetoin, 2,3-butanediol, cytokinins (3, 13-15, 20, 30), or combinations thereof. They can also improve plant growth via the fixation of nitrogen (diazotrophy) (9, 38).Typical examples of marginal soils include soils that have deteriorated due to the presence of heavy metals or organic contaminants. These are often soils with a history of industrial, military, or mining activities. Endophytic bacteria can assist their host plants in overcoming phytotoxic effects caused by environmental contamination (5, 11, 12, 36), which is of direct relevance for waste management and pollution control via phytoremediation technologies. When nonsterile poplar cuttings (Populus trichocarpa × deltoides cv. Hoogvorst) were inoculated with the endophyte Burkholderia cepacia VM1468, a derivative of B. cepacia Bu72 which possesses the pTOM-Bu61 plasmid coding for a constitutively expressed toluene degradation pathway, it was observed that in addition to decreasing the phytotoxicity and releasing toluene, strain VM1468 also considerably improved the growth of poplar trees in the absence of toluene (36). This observation, which was the first of its kind for poplar trees, prompted us to further study the poplar tree-associated beneficial endophytic bacteria in order to improve the overall performance of poplar trees, as it can enhance multiple applications, including biomass production, carbon sequestration, and phytoremediation. This was done by screening endophytic bacteria for their plant growth-promoting capabilities toward poplar trees by performing colonization studies with gfp-labeled strains, by examining their metabolic properties, and by initiating the genome sequencing of several strains.     

Complete genome sequence of Ignisphaera aggregans type strain (AQ1.S1)

Ignisphaera aggregans Niederberger et al. 2006 is the type and sole species of genus Ignisphaera. This archaeal species is characterized by a coccoid-shape and is strictly anaerobic, moderately acidophilic, heterotrophic hyperthermophilic and fermentative. The type strain AQ1.S1(T) was isolated from a near neutral, boiling spring in Kuirau Park, Rotorua, New Zealand. This is the first completed genome sequence of the genus Ignisphaera and the fifth genome (fourth type strain) sequence in the family Desulfurococcaceae. The 1,875,953 bp long genome with its 2,009 protein-coding and 52 RNA genes is a part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Selective release and function of one of the two FMN groups in the cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha.

The soluble, cytoplasmic NAD+-reducing [NiFe]-hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H2-NAD+ reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN-b) firmly bound, but it lost its physiological activity--the reduction of NAD+ by H2. This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H2 was not dependent on the presence of FMN-a. Enzyme devoid of FMN-a could not be activated by NADH. As NADH-dehydrogenase activity was not dependent on the presence of FMN-a, and because FMN-b did not dissociate from the reduced enzyme, we conclude that FMN-b is functional in the NADH-dehydrogenase activity catalyzed by the HoxFU dimer. The possible function of FMN-a as a hydride acceptor in the hydrogenase reaction catalyzed by the HoxHY dimer is discussed.     

Complete genome sequence of the halophilic and highly halotolerant Chromohalobacter salexigens type strain (1H11(T))

Chromohalobacter salexigens is one of nine currently known species of the genus Chromohalobacter in the family Halomonadaceae. It is the most halotolerant of the so-called 'moderately halophilic bacteria' currently known and, due to its strong euryhaline phenotype, it is an established model organism for prokaryotic osmoadaptation. C. salexigens strain 1H11(T) and Halomonas elongata are the first and the second members of the family Halomonadaceae with a completely sequenced genome. The 3,696,649 bp long chromosome with a total of 3,319 protein-coding and 93 RNA genes was sequenced as part of the DOE Joint Genome Institute Program DOEM 2004.