Metagenomic and Metatranscriptomic Responses of Chemical Dispersant Application during a Marine Dilbit Spill

The global increase in marine transportation of dilbit (diluted bitumen) can increase the risk of spills, and the application of chemical dispersants remains a common response practice in spill events. To reliably evaluate dispersant effects on dilbit biodegradation over time, we set large-scale (1500 mL) microcosms without nutrients addition using low dilbit concentration (30 ppm). Shotgun metagenomics and metatranscriptomics were deployed to investigate microbial community responses to naturally and chemically dispersed dilbit. We found that the large-scale microcosms could produce more reproducible community trajectories than small-scale (250 mL) ones based on the 16S rRNA gene amplicon sequencing. In the early-stage large-scale microcosms, multiple genera were involved into the biodegradation of dilbit, while dispersant addition enriched primarily Alteromonas and competed for the utilization of dilbit, causing depressed degradation of aromatics. The metatranscriptomic based Metagenome Assembled Genomes (MAG) further elucidated early-stage microbial antioxidation mechanism, which showed dispersant addition triggered the increased expression of the antioxidation process genes of Alteromonas species. Differently, in the late stage, the microbial communities showed high diversity and richness and similar compositions and metabolic functions regardless of dispersant addition, indicating the biotransformation of remaining compounds can occur within the post-oil communities. These findings can guide future microcosm studies and the application of chemical dispersants for responding to a marine dilbit spill. Importance In this study, we employed microcosms to study the effects of marine dilbit spill and dispersant application on microbial community dynamics over time. We evaluated the impacts of microcosm scale and found that increasing the scale is beneficial for reducing community stochasticity, especially in the late stage of biodegradation. We observed that dispersant application suppressed aromatics biodegradation in the early stage (6 days) whereas exerting insignificant effects in the late stage (50 days), from both substances removal and metagenomic/metatranscriptomic perspectives. We further found that Alteromonas species are vital for the early-stage chemically dispersed oil biodegradation, and clarified their degradation and antioxidation mechanisms. The findings would help to better understand microcosm studies and microbial roles for biodegrading dilbit and chemically dispersed dilbit, and suggest that dispersant evaluation in large-scale systems and even through field trails would be more realistic after marine oil spill response.

Adhesion of enteropathogenic, enterotoxigenic and commensal Escherichia coli to the Major Zymogen Granule Membrane Glycoprotein 2

Pathogenic bacteria, such as enteropathogenic (EPEC) and enterotoxigenic Escherichia coli (ETEC), cause diarrhea in mammals. In particular, E. coli colonize and infect the gastrointestinal tract via type 1 fimbriae (T1F). Here the major zymogen granule membrane glycoprotein 2 (GP2) acts as host cell receptor. GP2 is also secreted by the pancreas and various mucous glands, interacting with luminal type 1 fimbriae-positive E. coli . It is unknown whether GP2 isoforms demonstrate specific E. coli pathotype binding. In this study, we investigated interactions of human, porcine and bovine EPEC, ETEC as well as commensal E. coli isolates with human, porcine and bovine GP2. We first defined pathotype- and host-associated FimH variants. Secondly, we could prove that GP2 isoforms bound to FimH variants to varying degrees. However, the GP2-FimH interactions did not seem to be influenced by the host specificity of E. coli . In contrast, soluble GP2 affected ETEC infection and phagocytosis rates of macrophages. Pre-incubation of ETEC pathotype with GP2 reduced infection of cell lines. Furthermore, pre-incubation of E. coli with GP2 improved the phagocytosis rate of macrophages. Our findings suggest that GP2 plays a role in the defense against E. coli infection and in the corresponding host immune response. IMPORTANCE Infection by pathogenic bacteria such as certain Escherichia coli pathotypes results in diarrhea in mammals. Pathogens, including zoonotic agents, can infect different hosts or show host-specificity. There are Escherichia coli strains which are frequently transmitted between humans and animals, whereas other Escherichia coli strains tend to colonize only one host. This host-specificity is still not fully understood. We show that glycoprotein 2 is a selective receptor for particular Escherichia coli strains or variants of the adhesin FimH but not a selector for a species-specific Escherichia coli group. We demonstrate that GP2 is involved in the regulation of colonization and infection and thus represents a molecule of interest for the prevention or treatment of disease.

Adaptation to an amoeba host leads to Pseudomonas aeruginosa isolates with attenuated virulence

The opportunistic pathogen Pseudomonas aeruginosa , is ubiquitous in the environment, and in humans is capable of causing acute or chronic infections. In the natural environment, predation by bacterivorous protozoa represents a primary threat to bacteria. Here, we determined the impact of long-term exposure of P. aeruginosa to predation pressure. P. aeruginosa persisted when co-incubated with the bacterivorous Acanthamoeba castellanii for extended periods and produced genetic and phenotypic variants. Sequencing of late-stage amoeba-adapted P. aeruginosa isolates demonstrated single nucleotide polymorphisms within genes that encode known virulence factors and this correlated with a reduction in expression of virulence traits. Virulence towards the nematode, Caenorhabditis elegans , was attenuated in late-stage amoeba-adapted P. aeruginosa compared to early-stage amoeba-adapted and non-adapted counterparts. Further, late-stage amoeba-adapted P. aeruginosa showed increased competitive fitness and enhanced survival in amoeba as well as in macrophage and neutrophils. Interestingly, our findings indicate that the selection imposed by amoeba resulted in P. aeruginosa isolates with reduced virulence and enhanced fitness, similar to those recovered from chronic cystic fibrosis infections. Thus, predation by protozoa and long-term colonization of the human host may represent similar environments that select for similar losses of gene function. Importance Pseudomonas aeruginosa is an opportunistic pathogen that causes both acute infections in plants and animals, including humans, and chronic infections in immunocompromised and cystic fibrosis patients. This bacterium is commonly found in soils and water where bacteria are constantly under threat of being consumed by bacterial predators, e.g. protozoa. To escape being killed, bacteria have evolved a suite of mechanisms that protect them from being consumed or digested. Here, we examine the effect of long-term predation on the genotypes and phenotypes expressed by P. aeruginosa . We show that long term co-incubation with protozoa resulted in mutations that resulted in P. aeruginosa becoming less pathogenic. This is particularly interesting as we see similar mutations arise in bacteria associated with chronic infections. Importantly, the genetic and phenotypic traits possessed by late-stage amoeba-adapted P. aeruginosa are similar to what is observed for isolates obtained from chronic cystic fibrosis infections. This notable overlap in adaptation to different host types suggests similar selection pressures amongst host cell types as well as similar adaptation strategies.

High-level Production of Isoleucine and Fusel alcohol by expression of the Feedback Inhibition-insensitive Threonine deaminase in Saccharomyces cerevisiae

A variety of the yeast Saccharomyces cerevisiae with intracellular accumulation of isoleucine (Ile) would be a promising strain for developing a distinct kind of sake, a traditional Japanese alcoholic beverage, because Ile-derived volatile compounds have a great impact on the flavor and taste of fermented foods. In this study, we isolated an Ile-accumulating mutant (strain K9-I48) derived from a diploid sake yeast of S. cerevisiae by conventional mutagenesis. Strain K9-I48 carries a novel mutation in the ILV1 gene encoding the His480Tyr variant of threonine deaminase (TD). Interestingly, the TD activity of the His480Tyr variant was markedly insensitive to feedback inhibition by Ile, but was not upregulated by valine, leading to intracellular accumulation of Ile and extracellular overproduction of 2-methyl-1-butanol, a fusel alcohol derived from Ile, in yeast cells. The present study demonstrated for the first time that the conserved histidine residue located in a linker region between two regulatory domains is involved in allosteric regulation of TD. Moreover, sake brewed with strain K9-I48 contained 2-3 times more 2-methyl-1-butanol and 2-methylbutyl acetate than sake brewed with the parent strain. These findings are valuable for the engineering of TD to increase the productivity of Ile and its derived fusel alcohols. IMPORTANCE Fruit-like flavors of isoleucine-derived volatile compounds, 2-methyl-1-butanol (2MB) and its acetate ester, contribute to a variety of the flavors and tastes of alcoholic beverages. Besides its value as aroma components in foods and cosmetics, 2MB has attracted significant attention as second-generation biofuels. Threonine deaminase (TD) catalyzes the first step in isoleucine biosynthesis and its activity is subject to feedback inhibition by isoleucine. Here, we isolated an isoleucine-accumulating sake yeast mutant and identified a mutant gene encoding a novel variant of TD. The variant TD exhibited much less sensitivity to isoleucine, leading to higher production of 2MB as well as isoleucine than the wild-type TD. Furthermore, sake brewed with a mutant yeast expressing the variant TD contained more 2MB and its acetate ester than that brewed with the parent strain. These findings will contribute to the development of superior industrial yeast strains for high-level production of isoleucine and its related fusel alcohols.

Genomic epidemiology of Salmonella enterica circulating in surface waters used in agriculture and aquaculture in central Mexico

Salmonella enterica (SE) can survive in surface waters (SuWa) and the role of non-host environments in its transmission has acquired increasing relevance. In this study, we conducted comparative genomic analyses of 172 SE isolates collected from SuWa across three months in six states of central Mexico during 2019. SE transmission dynamics were assessed using 87 experimental and 112 public isolates from Mexico collected during 2002-2019. We also studied genetic relatedness between SuWa isolates and human clinical strains collected in North America during 2005-2020. Among experimental isolates, we identified 41 SE serovars and 56 multi-locus sequence types (ST). Predominant serovars were Senftenberg (n=13), Meleagridis, Agona, and Newport (n=12 each), Give (n=10), Anatum (n=8), Adelaide (n=7), and Infantis, Mbandaka, Ohio and Typhimurium (n=6 each). We observed a high genetic diversity in the sample under study, as well as clonal dissemination of strains across distant regions. Some of these strains are epidemiologically important (ST14, ST45, ST118, ST132, ST198, and ST213), and were genotypically close to those involved in clinical cases in North America. Transmission network analysis suggests that SuWa are a relevant source of SE (0.7 source/hub ratio) and contributes to its dissemination as isolates from varied sources and clinical cases have SuWa isolates as common ancestors. Overall, the study shows SuWa act as reservoir of various SE serovars of public health significance. Further research is needed to better understand the mechanisms involved in SuWa contamination by SE, as well as develop interventions to contain its dissemination to food production settings. Study importance Surface waters are heavily used in food production worldwide. Several human pathogens can survive in these waters for long periods and disseminate to food production environments, contaminating our food supply. One of these pathogens is Salmonella enterica , a leading cause of foodborne infections, hospitalizations and deaths in many countries. This research demonstrates the role of surface waters as a vehicle for the transmission of Salmonella along food production chains. It also shows some strains circulating in surface waters are very similar to those implicated in human infections and harbor genes that confer resistance to multiple antibiotics, posing a risk to public health. The study contributes to expand our current knowledge on the ecology and epidemiology of Salmonella in surface waters.

A Micrarchaeon isolate is covered by a proteinaceous S-Layer

In previous publications, it was hypothesized that Micrarchaeota cells are covered by two individual membrane systems. This study proves that at least the recently cultivated “ Candidatus Micrarchaeum harzensis A_DKE” possesses an S-layer covering its cytoplasmic membrane. The potential S-layer protein was found to be among the proteins with the highest abundance in “ Ca. Micrarchaeum harzensis A_DKE” and in silico characterisation of its primary structure indicated homologies to other known S-layer proteins. Homologues of this protein were found in other Micrarchaeota genomes, which raises the question of whether the ability to form an S-layer is a common trait within this phylum. The S-layer protein seems to be glycosylated and the Micrarchaeon expresses genes for N-glycosylation under cultivation conditions, despite not being able to synthesize carbohydrates. Electron micrographs of freeze-etched samples of a previously described co-culture, containing Micrarchaeum A_DKE and a Thermoplasmatales member as its host organism, verified the hypothesis of an S-layer on the surface of “ Ca. Micrarchaeum harzensis A_DKE”. Both organisms are clearly distinguishable by cell size, shape and surface structure. Importance Our knowledge about the DPANN superphylum, which comprises several archaeal phyla with limited metabolic capacities, is mostly based on genomic data derived from cultivation-independent approaches. This study examined the surface structure of a recently cultivated member “ Candidatus Micrarchaeum harzensis A_DKE”, an archaeal symbiont dependent on an interaction with a host organism for growth. The interaction requires direct cell contact between interaction partners, a mechanism which is also described for other DPANN archaea. Investigating the surface structure of “ Ca. Micrarchaeum harzensis A_DKE” is an important step towards understanding the interaction between Micrarchaeota and their host organisms and living with limited metabolic capabilities, a trait shared by several DPANN archaea.

Heat activation and inactivation of bacterial spores. Is there an overlap?

Heat activation at a sublethal temperature is widely applied to promote Bacillus species spore germination. This treatment also has potential to be employed in food processing to eliminate undesired bacterial spores by enhancing their germination, and then inactivating the less heat resistant germinated spores at a milder temperature. However, incorrect heat treatment could also generate heat damage in spores, and lead to more heterogeneous spore germination. Here, the heat activation and heat damage profile of Bacillus subtilis spores was determined by testing spore germination and outgrowth at both population and single spore levels. The heat treatments used were 40-80°C, and for 0-300 min. The results were as follows. 1) Heat activation at 40-70°C promoted L-valine and L-asparagine-glucose-fructose-potassium (AGFK) induced germination in a time dependent manner. 2) The optimal heat activation temperatures for AGFK and L-valine germination via the GerB plus GerK or GerA germinant receptors were 65 and 50-65°C, respectively. 3) Heat inactivation of dormant spores appeared at 70°C, and the heat damage of molecules essential for germination and growth began at 70 and 65°C, respectively. 4) Heat treatment at 75°C resulted in both activation of germination and damage to the germination apparatus, and 80°C treatment caused more pronounced heat damage. 5) For the spores that should withstand adverse environmental temperatures in nature, heat activation seems functional for a subsequent optimal germination process, while heat damage affected both germination and outgrowth. Importance Bacterial spores are thermal resistant structures that can thus survive preservation strategies and revive through the process of spore germination. The more heat resistant spores are the more heterogeneous they germinate upon adding germinants. Upon germination spores can cause food spoilage and cause food intoxication. Here we provide new information on both heat activation and inactivation regimes and their effects on the (heterogeneity of) spore germination.

Rational regulation of reaction specificity of nitrilase for efficient biosynthesis of 2-chloronicotinic acid through a single site mutation

Nitrilase-catalyzed hydrolysis of 2-chloronicotinonitrile (2-CN) is a promising approach for efficient synthesis of 2-chloronicotinic acid (2-CA). Development of nitrilase with ideal catalytic properties is crucial for the biosynthetic route with industrial potentail. Herein, a nitrilase from Rhodococcus zopfii ( Rz NIT), which showed much higher hydration activity than hydrolysis activity, was designed for efficient hydrolysis of 2-CN. Two residues (N165 and W167) significantly affecting the reaction specificity were precisely identified. By tuning these two residues, a single mutation of W167G with abolished hydration activity and 20-fold improved hydrolysis activity was obtained. Molecular dynamics simulation and molecular docking revealed that the mutation generated a larger binding pocket, causing the substrate 2-CN bound more deeply in the pocket and the formation of delocalized π bond between the residues W190 and Y196, which reduced the negative influence of steric hindrance and electron effect caused by chlorine substituent. With mutant W167G as biocatalyst, 100 mM 2-CN was exclusively converted into 2-CA within 16 h. The study provides useful guidance in nitrilase engineering for simultaneous improvement of reaction specificity and catalytic activity, which are highly desirable in value-added carboxylic acids production from nitriles hydrolysis. Importance 2-CA is an important building block for agrochemicals and pharmaceuticals with rapid increase in demand in recent years. It is currently manufactured from 3-cyanopyridine by chemical methods. However, during the final step of 2-CN hydrolysis under high temperature and strong alkaline conditions, by-product 2-CM was generated except for the target product, leading to low yield and tedious separation steps. Nitrilase-mediated hydrolysis is regarded as a promising alternative for 2-CA production, which proceeds under mild conditions. Nevertheless, nitrilase capable of efficient hydrolysis of 2-CN was not reported till now, since the enzymes showed either extremely low activity or surprisingly high hydration activity towards 2-CN. Herein, the reaction specificity of Rz NIT was precisely tuned through a single site mutation. The mutant exhibited remarkably enhanced hydrolysis activity without formation of by-products, providing a robust biocatalyst for 2-CA biosynthesis with industrial potential.

A novel phage indirectly regulates diatom growth by infecting diatom-associated biofilm-forming bacterium

Algae and heterotrophic bacteria have close and intricate interactions, which are regulated by multiple factors in the natural environment. Phages are the major factor determining bacterial mortality. However, their impacts on the algae-associated bacteria and thus on the algae-bacteria interactions are poorly understood. Here, we obtained a diatom-associated bacterium Stappia indica SNL01 that could form biofilm and had an inhibitory effect on the growth of diatom Thalassiosira pseudonana . Meanwhile, the phage SI01 with a double-stranded circular DNA genome (44,247 bp) infecting S. indica SNL01 was isolated. Phylogenetic analysis revealed that phage SI01 represents a novel member of the Podoviridae family. The phage contained multiple lysis genes encoding for cell wall lysing muramidase and spore cortex lysing SleB, as well as depolymerase-like tail spike protein. By lysing the host bacterium and inhibiting the formation of biofilm, this phage could indirectly promote the growth of the diatom. Our results shed new insights into how phages indirectly regulate algal growth by infecting bacteria closely associated with algae or in the phycosphere. IMPORTANCE The impact of phage infection on the algae-bacteria relationship in the ocean is poorly understood. Here, a novel phage infecting the diatom-associated bacterium Stappia indica SNL01 was isolated. This bacterium could form biofilm and had a negative effect on diatom growth. We revealed that this phage contained multiple lysis genes and could inhibit the formation of bacterial biofilm, thus indirectly promoting diatom growth. This study implicates that phages are not only important regulators of bacteria but also have substantial indirect effects on algae as well as the algae-bacteria relationship.