L. plantarum HA9 culture conditions

Lactic acid bacteria (LAB) were isolated from classic artisanal milk cheeses. Isolation was done using, with minor modifications, a previously published approach [19]. In short, we plated each sample on MRS agar after diluting and homogenizing 1 gram of each sample in MRS broth. Then incubated the plates anaerobically at 37 °C for 48 hours and selected colonies with different morphologies. The colonies were purified by sub-culturing and confirmed them as Lactobacillus spp. based on their catalase-negative, gram-positive, rod-shaped appearance. The bacterial isolates stored in MRS broth with 20% glycerol.

Extraction of genomic DNA from L. plantarum HA9

Using the bacterial DNA extraction kit from GENEAID, Korea, genomic DNA was isolated from strain HA9 [20]. Thermo Fisher Scientific’s NanoDrop 2000 spectrophotometer and Life Technologies’ Qubit 3.0 were used for DNA quantification and concentration measurements, respectively, and 0.8% (w/v) agarose gel electrophoresis was used to assess the purity of the DNA. Then, the highly conserved section of ribosomal RNA was amplified with universal primers 27F and 1492R, the PCR product was sequenced with these primers [21], and the results were confirmed by a blast analysis of the NCBI database [22].

Whole-genome sequence analysis of L. plantarum HA9

The genome of L. plantarum HA9 was sequenced using the Illumina and PacBio Sequel II technologies (HN00194138; Psomagen/USA). After passing quality control (QC), the DNA from the samples was randomly fragmented and ligated with 5' and 3' adapters to produce a library. The generated NGS library will then be sequenced. Following the completion of the sequencing, the raw data was analyzed for parameters such as GC (percentage), total bases, and total reads. To reduce analytical biases, quick QC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and quality filtering were utilized. We used the phred quality score (Q20(%) and Q30(%) to assess data quality at the end of each cycle [23].

Genome annotation of L. plantarum HA9

Coding genes were predicted using Prodigal (Prokaryotic Dynamic Programming Gene-finding Algorithm) software [24] Filtering for proteins with the highest sequence similarity using annotations with e 1e-5, the Diamond software [25] was employed to contrast expected coding sequences in order to provide functional annotation information. The HMMER software [26] was utilized for comparing these sequences with protein group models in order to identify the families that scored the highest. The predicted code sequence regions were compared against the KEGG (Kyoto Encyclopedia of Genes and Genomes), COG (Cluster of Orthologous Groups of proteins), GO (Gene Ontology), CAZy (Carbohydrate-Active Enzymes Database), and NR (non-redundant) databases in order to complete the foundational functional prediction and annotation. Using the Circus (v0.69) program [27] many data sets were shown in one single genomic circular map.

Genome analysis

PATRICS, a sophisticated genome analysis service, constructed L. plantarum genome [28]. The L. plantarum reference genome mapped sequencing reads. Functional genes in subsystem categories were annotated in the genome, mapping results were compared, conserved and unique sequencing properties were identified, and high-quality maps were created to verify and display the described features. Bioinformatics techniques removed duplicates, found variants, and mapped filtered data reads to the reference genome [29-31].

The quality of the HA9 DNA

To guarantee the concentration and quality of DNA, the bioinformatics tools provided by PATRIC were employed. The genome isolates of strain HA9 displayed a coarse consistency score of 97.8, was complemented by a fine consistency score of 96.1, and achieved a checkM completeness rating of 100. As a result, this genome exemplified exceptional overall quality.

Genomic characteristics of HA9

The cutting-edge PacBio Sequel II sequencing technology was employed to unveil the entire genome sequence of L. plantarum strain HA9. This breakthrough resulted in NCBI receiving the HA9 genome sequence (accession ID: JAUTDL000000000.1). Impressively, the HA9 genome spans 3,321,986 base pairs, consisting of a singular circular chromosome with a GC content of 44.40%, and notably, no plasmids were detected. Table 1 encapsulates the anticipated 3,138 genes, along with 2 rRNAs (5S, 16S, and 23S) and 59 tRNAs present in this comprehensive genome. Meanwhile, the strain HA9 whole-genome circle map is shown in Figure 1, meticulously delineating key genomic features such as homologous genes, GC content, genomic islands, gene distribution across forward and antisense strands, and COG functional classification of genes, and more intricate details. Noteworthy is the genome's high quality, substantiated by annotation statistics and comparison with PATRIC genomes of the same species. Following meticulous read filtering, the phred quality scores of bases exceeded Q20 and Q30 at an impressive rate of 97.8% and 94.0%, respectively. Furthermore, Comprehensive Genome Analysis unveiled 81 contigs spanning 3,314,413 base pairs, housing 1,420 coding proteins in this meticulously assembled genome, as detailed in Table 2. The genome's GC content averages 44.3%, with Figure 1 providing a schematic representation of GC content and GC skew analysis. Each protein subsystem within the genome contributes to specific biological activities or structural complexes, with a striking 278 genome-specific subsystems found across annotation. Figure 2 offers a comprehensive overview of the genome's subsystem organization, highlighting its intricate biological functionality and structural complexity.

Phylogenetic Analysis

For phylogenetic research, PATRIC offers representative and reference genomes. Based on 16S rRNA sequences, Figure 3 shows a neighbor-joining phylogenetic tree; Figure 4 shows the nearest reference and typical genotypes found during the research.

Figures & Tables

Discussion

16S rRNA analysis was used to identify the HA9 strain and classify it as L. plantarum. This taxonomy corresponds to the species commonly present in sauerkraut [32].

The size of the HA9 genome and the proportion of its GC content were identical to those previously reported for L. plantarum strains. Within the lactobacilli group, the genome of the L. plantarum species is among the largest genomes overall, ranging in size from 2.91 to 3.7 Mb. The whole genome of L. plantarum has a median length of 3.27272 Mb, a median protein counts of 2997, and a GC content of 44.5%, according to the NCBI database. It is considered that the large genomes of L. plantarum are directly related to the environmental flexibility and extraordinary adaptability it possesses to a wide variety of settings [33].

The HA9 strain of L. plantarum occupies a distinct position on the evolutionary tree when compared to other strains of the same species. Its closest counterparts include strains such as L. plantarum WCFS1, which is among the most extensively researched lactobacilli. Notably, the genome of L. plantarum WCFS1 was unveiled many years ago. This particular strain of L. plantarum demonstrates remarkable resilience and a relatively high survival rate when passing through the digestive system, and it can be cultivated to high densities. Additionally, it is easily modifiable genetically [34]. Furthermore, the genome of L. plantarum WCFS1 has not been found to include any transferable antibiotic resistance genes [35]. To understand the distinctions between HA9 and WCFS1 better, further comprehensive comparative genomic investigations are required.

A viable technique to enrich fermented meals and boost the health-promoting impact of probiotics is to make use of starter cultures that produce vitamins or probiotics [36]. On the other hand, future investigations both in silico and in vitro should shed light on the manner in which several biochemical biosynthesis processes are linked to the genetic ability to biosynthesize vitamins. We also evaluated genetic data linked to safety evaluation, like genes that encode toxins and virulence factors, however we were unable to find any risk-associated sequences in the genome of L. plantarum HA9.

Acknowledgments

Authors acknowledge Medical and science colleges/Misan University and Science college for their support. 

Conflict of Interest

The authors declare that there is no conflict of interest.

Author Contributions

Al-kaabi H. Q. and Chelab R. L.  designed the study and performed the experiments. In addition, both authors analyzed the data and wrote the manuscript.

References