Characterization of novel proteases identified by metagenomic analysis from dairy stabilization ponds.

Cheese whey is the main by-product of dairy industries. It is used as a raw material for other value-added products, like whey protein concentrate. By using enzymes, this product can be further treated to obtain new higher value products, like whey protein hydrolysates. Proteases (EC: 3.4) represent a large segment of industrial enzymes, since they are used in several industries, including food. In this work, we describe three novel enzymes identified using a metagenomic approach. Metagenomic DNA from dairy industry stabilization ponds were sequenced, and the predicted genes were compared against the MEROPS database, focusing on families commercially used to produce whey protein hydrolysates. From a total of 849 candidates, 10 were selected for cloning and expression and three showed activities with both the chromogenic substrate, azocasein, and whey proteins. Particularly, Pr05, an enzyme from the yet uncultured phylum Patescibacteria, showed activity that is comparable to a commercial protease. All these novel enzymes could represent an alternative for dairy industries to produce value-added products from industrial by-products. KEY POINTS: • Over 19,000 proteases were predicted in a sequence-based metagenomic analysis. • Three proteases were successfully expressed and showed activity with whey proteins. • The enzyme Pr05 showed hydrolysis profiles of interest for food industry.

Omicron Waves in Argentina: Dynamics of SARS-CoV-2 Lineages BA.1, BA.2 and the Emerging BA.2.12.1 and BA.4/BA.5.

The COVID-19 pandemic has lately been driven by Omicron. This work aimed to study the dynamics of SARS-CoV-2 Omicron lineages during the third and fourth waves of COVID-19 in Argentina. Molecular surveillance was performed on 3431 samples from Argentina, between EW44/2021 and EW31/2022. Sequencing, phylogenetic and phylodynamic analyses were performed. A differential dynamic between the Omicron waves was found. The third wave was associated with lineage BA.1, characterized by a high number of cases, very fast displacement of Delta, doubling times of 3.3 days and a low level of lineage diversity and clustering. In contrast, the fourth wave was longer but associated with a lower number of cases, initially caused by BA.2, and later by BA.4/BA.5, with doubling times of about 10 days. Several BA.2 and BA.4/BA.5 sublineages and introductions were detected, although very few clusters with a constrained geographical distribution were observed, suggesting limited transmission chains. The differential dynamic could be due to waning immunity and an increase in population gatherings in the BA.1 wave, and a boosted population (for vaccination or recent prior immunity for BA.1 infection) in the wave caused by BA2/BA.4/BA.5, which may have limited the establishment of the new lineages.

Dissecting Light Sensing and Metabolic Pathways on the Millimeter Scale in High-Altitude Modern Stromatolites.

Modern non-lithifying stromatolites on the shore of the volcanic lake Socompa (SST) in the Puna are affected by several extreme conditions. The present study assesses for the first time light utilization and functional metabolic stratification of SST on a millimeter scale through shotgun metagenomics. In addition, a scanning-electron-microscopy approach was used to explore the community. The analysis on SST unveiled the profile of a photosynthetic mat, with cyanobacteria not directly exposed to light, but placed just below a high-UV-resistant community. Calvin-Benson and 3-hydroxypropinate cycles for carbon fixation were abundant in upper, oxic layers, while the Wood-Ljungdahl pathway was dominant in the deeper anoxic strata. The high abundance of genes for UV-screening and oxidant-quenching pigments and CPF (photoreactivation) in the UV-stressed layers could indicate that the zone itself works as a UV shield. There is a remarkable density of sequences associated with photoreceptors in the first two layers. Also, genetic evidence of photosynthesis split in eukaryotic (layer 1) and prokaryotic (layer 2). Photoheterotrophic bacteria, aerobic photoautotrophic bacteria, and anaerobic photoautotrophic bacteria coexist by selectively absorbing different parts of the light spectrum (blue, red, and IR respectively) at different positions of the mat. Genes for oxygen, nitrogen, and sulfur metabolism account for the microelectrode chemical data and pigment measurements performed in previous publications. We also provide here an explanation for the vertical microbial mobility within the SST described previously. Finally, our study points to SST as ideal modern analogues of ancient ST.

Ruminal effects of excessive dietary sulphur in feedlot cattle.

Sulphur (S) dietary excess can limit productive performance and increase polioencephalomalacia (PEM) incidence in feedlot cattle (FC). Sulphur excess ingested is transformed to hydrogen sulphide (H2 S) by sulfo-reducing ruminal bacteria (SRB), being high ruminal H2 S concentration responsible for aforementioned damages. As the ruminal mechanisms involved in H2 S concentrations increase have not been elucidated, this study aimed to evaluate the ruminal environment, and the association between ruminal H2 S and dissimilatory SRB (DSRB) concentration in FC experimentally subjected to S dietary excess. Twelve crossbred steers were randomly assigned to one of two dietary S levels (6 animals per treatment): low (LS, 0.19% S) and high (HS, 0.39% S obtained by sodium sulfate inclusion at 0.86%). The study lasted 38 days, and on days 0, 22 and 38, ruminal gas samples were taken to quantify H2 S concentration, and ruminal fluid to determine total bacteria, DSRB, protozoa, volatile fatty acid and ammonia nitrogen concentration. For ruminal H2 S concentration, S dietary × sampling day interaction was significant (p < 0.001), so that the greater concentration was observed on days 22 and 38 with the HS diet. The remaining ruminal parameters were not affected by dietary S level, and no significant correlation between H2 S and DSRB concentrations was observed. The ruminal adaptation that maximizes H2 S production in FC consuming S excess does not seem to be associated with biological or biochemical alterations, nor DSRB concentration changes. The microbial diversity and ruminal environment were resilient to the S excess evaluated, suggesting that 0.39% of dietary S achieved by 0.86% sodium sulfate addition, could be used without disturbances on digestion nor health of FC.

Phylogenomic analysis for Campylobacter fetus ocurring in Argentina.

BACKGROUND AND AIM: Campylobacter fetus is one of the most important pathogens that severely affects livestock industry worldwide. C. fetus mediated bovine genital campylobacteriosis infection in cattle has been associated with significant economic losses in livestock production in the Pampas region, the most productive area of Argentina. The present study aimed to establish the genomic relationships between C. fetus strains, isolated from the Pampas region, at local and global levels. The study also explored the utility of multi-locus sequence typing (MLST) as a typing technique for C. fetus. MATERIALS AND METHODS: For pangenome and phylogenetic analysis, whole genome sequences for 34 C. fetus strains, isolated from cattle in Argentina were downloaded from GenBank. A local maximum likelihood (ML) tree was constructed and linked to a Microreact project. In silico analysis based on MLST was used to obtain information regarding sequence type (ST) for each strain. For global phylogenetic analysis, a core genome ML-tree was constructed using genomic dataset for 265 C. fetus strains, isolated from various sources obtained from 20 countries. RESULTS: The local core genome phylogenetic tree analysis described the presence of two major clusters (A and B) and one minor cluster (C). The occurrence of 82% of the strains in these three clusters suggested a clonal population structure for C. fetus. The MLST analysis for the local strains revealed that 31 strains were ST4 type and one strain was ST5 type. In addition, a new variant was identified that was assigned a novel ST, ST70. In the present case, ST4 was homogenously distributed across all the regions and clusters. The global analysis showed that most of the local strains clustered in the phylogenetic groups that comprised exclusively of the strains isolated from Argentina. Interestingly, three strains showed a close genetic relationship with bovine strains obtained from Uruguay and Brazil. The ST5 strain grouped in a distant cluster, with strains obtained from different sources from various geographic locations worldwide. Two local strains clustered in a phylogenetic group comprising intercontinental Campylobacter fetus venerealis strains. CONCLUSION: The results of the study suggested active movement of animals, probably due to economic trade between different regions of the country as well as with neighboring countries. MLST results were partially concordant with phylogenetic analysis. Thus, this method did not qualify as a reliable subtyping method to assess C. fetus diversity in Argentina. The present study provided a basic platform to conduct future research on C. fetus, both at local and international levels.

ß-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization.

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. ß-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding ß-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with ß-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-ß-galactoside and lactose. The activity levels of one of these novel ß-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic ß-galactosidases from diary stabilization ponds' metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries' by-products.