In-silico functional analysis of hypothetical proteins from Lactiplantibacillus plantarum plasmids reveals enrichment of cell envelope proteins.

Lactiplantibacillus plantarum is one of the important species of lactic acid bacterium (LAB) found in diverse environments, with many strains exhibiting probiotic properties. In our previous study, 41.6% of protein families (PFs) encoded by 395 plasmids from several L. plantarum strains were found to be hypothetical proteins with no predicted function. This study aimed at predicting the functions of these 647 hypothetical proteins using 21 different bioinformatics methods. As a result, 160 PFs could be newly annotated. A lower proportion of plasmid-specific functions was annotated as compared to the functions shared between plasmids and chromosomes. Also, hypothetical proteins were less conserved than the annotated proteins across L.plantarum plasmids. Based on the subcellular localization, cell envelope proteins represented the biggest category in the newly annotated proteins. Transporters (112 PFs) which was a part of cell envelop proteins represented the largest functional group. Additionally, 40 and 25 other PFs were predicted to contain signal peptides and transmembrane helices, respectively. We speculate that such hypothetical proteins might be involved in the transport of various chemicals and environmental interactions in L. plantarum. In the future, functional characterization of these proteins through wet-lab experimental approach can provide novel insights into their contribution to the physiology, probiotic properties, and industrial utility of these bacteria.

Non-redundant nature of Lactiplantibacillus plantarum plasmidome revealed by comparative genomic analysis of 105 strains.

Lactiplantibacillus plantarum is a homofermentative lactic acid bacterium (LAB) most often found in fermented foods with many strains displaying probiotic properties. Strains belonging to L. plantarum are more stress tolerant and metabolically flexible than other lactobacilli and display larger genomes and higher plasmid abundance. This study aimed at understanding whether plasmids play a particular role in L. plantarum as compared to chromosomes by comparative genomic analysis. Assessment of chromosomes and 395 plasmids of 105 strains with publicly available complete genome sequences revealed that the majority of the plasmids encoded protein families (PFs) (57.6%) were not encoded by the chromosomes. The most abundant PFs unique to plasmids contained hypothetical proteins while others were involved in exopolysaccharides biosynthesis, biofilm formation, stress tolerance, and carbohydrate metabolism. The sequences of common plasmid-encoded and chromosome-encoded PFs differed from each other, suggesting that they might exhibit different biochemical properties. Common PF genes were predominantly present on larger plasmids pointing to another possible way to reduce redundancy by encoding shared PFs by low copy number plasmids. Overall, this study demonstrates the unique contributions of the plasmids to the versatility, survival, and evolutionary success of L. plantarum while also highlighting a need to functionally characterize hypothetical proteins encoded by them.

Benchmarking of 16S rRNA gene databases using known strain sequences.

16S rRNA gene analysis is the most convenient and robust method for microbiome studies. Inaccurate taxonomic assignment of bacterial strains could have deleterious effects as all downstream analyses rely heavily on the accurate assessment of microbial taxonomy. The use of mock communities to check the reliability of the results has been suggested. However, often the mock communities used in most of the studies represent only a small fraction of taxa and are used mostly as validation of sequencing run to estimate sequencing artifacts. Moreover, a large number of databases and tools available for classification and taxonomic assignment of the 16S rRNA gene make it challenging to select the best-suited method for a particular dataset. In the present study, we used authentic and validly published 16S rRNA gene type strain sequences (full length, V3-V4 region) and analyzed them using a widely used QIIME pipeline along with different parameters of OTU clustering and QIIME compatible databases. Data Analysis Measures (DAM) revealed a high discrepancy in ratifying the taxonomy at different taxonomic hierarchies. Beta diversity analysis showed clear segregation of different DAMs. Limited differences were observed in reference data set analysis using partial (V3-V4) and full-length 16S rRNA gene sequences, which signify the reliability of partial 16S rRNA gene sequences in microbiome studies. Our analysis also highlights common discrepancies observed at various taxonomic levels using various methods and databases.

Plasmids encode niche-specific traits in Lactobacillaceae.

Species belonging to the family Lactobacillaceae are found in highly diverse environments and play an important role in fermented foods and probiotic products. Many of these species have been individually reported to harbour plasmids that encode important genes. In this study, we performed comparative genomic analysis of publicly available data for 512 plasmids from 282 strains represented by 51 species of this family and correlated the genomic features of plasmids with the ecological niches in which these species are found. Two-thirds of the species had at least one plasmid-harbouring strain. Plasmid abundance and GC content were significantly lower in vertebrate-adapted species as compared to nomadic and free-living species. Hierarchical clustering highlighted the distinct nature of plasmids from the nomadic and free-living species than those from the vertebrate-adapted species. EggNOG-assisted functional annotation revealed that genes associated with transposition, conjugation, DNA repair and recombination, exopolysaccharide production, metal ion transport, toxin-antitoxin system, and stress tolerance were significantly enriched on the plasmids of the nomadic and in some cases nomadic and free-living species. On the other hand, genes related to anaerobic metabolism, ABC transporters and the major facilitator superfamily were overrepresented on the plasmids of the vertebrate-adapted species. These genomic signatures correlate with the comparatively nutrient-depleted, stressful and dynamic environments of nomadic and free-living species and nutrient-rich and anaerobic environments of vertebrate-adapted species. Thus, these results indicate the contribution of the plasmids in the adaptation of lactobacilli to their respective habitats. This study also underlines the potential application of these plasmids in improving the technological and probiotic properties of lactic acid bacteria.

A Diverse Repertoire of Exopolysaccharide Biosynthesis Gene Clusters in Lactobacillus Revealed by Comparative Analysis in 106 Sequenced Genomes.

Production of exopolysaccharides (EPS) is one of the unique features of Lactobacillus genus. EPS not only have many physiological roles such as in stress tolerance, quorum sensing and biofilm formation, but also have numerous applications in the food and pharmaceutical industries. In this study, we identified and compared EPS biosynthesis gene clusters in 106 sequenced Lactobacillus genomes representing 27 species. Of the 146 identified clusters, only 41 showed the typical generic organization of genes as reported earlier. Hierarchical clustering showed highly varied nature of the clusters in terms of the gene composition; nonetheless, habitat-wise grouping was observed for the gene clusters from host-adapted and nomadic strains. Of the core genes required for EPS biosynthesis, epsA, B, C, D and E showed higher conservation, whereas gt, wzx and wzy showed high variability in terms of the number and composition of the protein families. Analysis of the distribution pattern of the protein families indicated a higher proportion of mutually exclusive families in clusters from host-adapted and nomadic strains, whereas those from the free-living group had very few unique families. Taken together, this analysis highlights high variability in the EPS gene clusters amongst Lactobacillus with some of their properties correlated to the habitats.

Genomic and physiological analyses of an indigenous strain, Enterococcus faecium 17OM39.

The human gut microbiome plays a crucial role in human health and efforts need to be done for cultivation and characterisation of bacteria with potential health benefits. Here, we isolated a bacterium from a healthy Indian adult faeces and investigated its potential as probiotic. The cultured bacterial strain 17OM39 was identified as Enterococcus faecium by 16S rRNA gene sequencing. The strain 17OM39 exhibited tolerance to acidic pH, showed antimicrobial activity and displayed strong cell surface traits such as hydrophobicity and autoaggregation capacity. The strain was able to tolerate bile salts and showed bile salt hydrolytic (BSH) activity, exopolysaccharide production and adherence to human HT-29 cell line. Importantly, partial haemolytic activity was detected and the strain was susceptible to the human serum. Genomics investigation of strain 17OM39 revealed the presence of diverse genes encoding for proteolytic enzymes, stress response systems and the ability to produce essential amino acids, vitamins and antimicrobial compound Bacteriocin-A. No virulence factors and plasmids were found in this genome of the strain 17OM39. Collectively, these physiological and genomic features of 17OM39 confirm the potential of this strain as a candidate probiotic.