Bacillus atrophaeus HAB-5 is a plant growth-promoting rhizobacterium (PGPR) that exhibits several biotechnological traits, such as enhancing plant growth, colonizing the rhizosphere, and engaging in biocontrol activities. In this study, we conducted whole-genome sequencing of B. atrophaeus HAB-5 using the single-molecule real-time (SMRT) sequencing platform by Pacific Biosciences (PacBio; United States), which has a circular chromosome with a total length of 4,083,597 bp and a G + C content of 44.21%. The comparative genomic analysis of B. atrophaeus HAB-5 with other strains, Bacillus amyloliquefaciens DSM7, B. atrophaeus SRCM101359, Bacillus velezensis FZB42, B. velezensis HAB-2, and Bacillus subtilis 168, revealed that these strains share 2,465 CDSs, while 599 CDSs are exclusive to the B. atrophaeus HAB-5 strain. Many gene clusters in the B. atrophaeus HAB-5 genome are associated with the production of antimicrobial lipopeptides and polypeptides. These gene clusters comprise distinct enzymes that encode three NRPs, two Transat-Pks, one terpene, one lanthipeptide, one T3PKS, one Ripp, and one thiopeptide. In addition to the likely IAA-producing genes (trpA, trpB, trpC, trpD, trpE, trpS, ywkB, miaA, and nadE), there are probable genes that produce volatile chemicals (acoA, acoB, acoR, acuB, and acuC). Moreover, HAB-5 contained genes linked to iron transportation (fbpA, fetB, feuC, feuB, feuA, and fecD), sulfur metabolism (cysC, sat, cysK, cysS, and sulP), phosphorus solubilization (ispH, pstA, pstC, pstS, pstB, gltP, and phoH), and nitrogen fixation (nif3-like, gltP, gltX, glnR, glnA, nadR, nirB, nirD, nasD, narl, narH, narJ, and nark). In conclusion, this study provides a comprehensive genomic analysis of B. atrophaeus HAB-5, pinpointing the genes and genomic regions linked to the antimicrobial properties of the strain. These findings advance our knowledge of the genetic basis of the antimicrobial properties of B. atrophaeus and imply that HAB-5 may employ a variety of commercial biopesticides and biofertilizers as a substitute strategy to increase agricultural output and manage a variety of plant diseases.

Cronobacter species are potential pathogens that can contaminate powdered infant formula. C. sakazakii and C. malonaticus are the most common species of Cronobacter associated with infections. This study mined new molecular targets for the detection of C. sakazakii and C. malonaticus by using comparative genome approaches. Specific target genes mngB and ompR were obtained and used to detect C. sakazakii and C. malonaticus, respectively. A novel detection method, termed ladder-shape melting temperature isothermal amplification (LMTIA), was developed and evaluated. The detection limit for pure C. sakazakii DNA was 1 pg per reaction and 1 fg per reaction for C. malonaticus. The C. sakazakii, C. malonaticus, and the reference stains were all correctly identified. The amplicons can be successfully visualized and identified by naked eyes when hydroxy naphthol blue dye (HNB dye) was used in the reaction. Therefore, the LMTIA assays developed in this study showed potential application for microorganism identification and detection.

Grapevine (Vitis vinifera) is one of the major economic fruit crops but suffers many diseases, causing damage to the quality of grapes. Strain G166 was isolated from the rhizosphere of grapevine and was found to exhibited broad-spectrum antagonistic activities against fungal pathogens on grapes in vitro, such as Coniella diplodiella, Botrytis cinerea, and Colletotrichum gloeosporioides. Whole-genome sequencing revealed that G166 contained a 6,613,582 bp circular chromosome with 5749 predicted coding DNA sequences and an average GC content of 60.57%. TYGS analysis revealed that G166 belongs to Pseudomonas viciae. Phenotype analysis indicated that P. viciae G166 remarkably reduced the severity of grape white rot disease in the grapevine. After inoculation with C. diplodiella, more H2O2 and MDA accumulated in the leaves and resulted in decreases in the Pn and chlorophyll content. Conversely, G166-treated grapevine displayed less oxidative damage with lower H2O2 levels and MDA contents under the pathogen treatments. Subsequently, G166-treated grapevine could sustain a normal Pn and chlorophyll content. Moreover, the application of P. viciae G166 inhibited the growth of mycelia on detached leaves and berries, while more disease symptoms occurred in non-bacterized leaves and berries. Therefore, P. viciae G166 served as a powerful bioagent against grape white rot disease. Using antiSMASH prediction and genome comparisons, a relationship between non-ribosomal peptide synthase clusters and antifungal activity was found in the genome of P. viciae G166. Taken together, P. viciae G166 shows promising antifungal potential to improve fruit quality and yield in ecological agriculture.

This study focused on L. paracasei strains isolated from fermented palm sap in southern Thailand that exhibit potential probiotic characteristics, including antibiotic susceptibility, resistance to gastrointestinal stresses, and antimicrobial activity against various pathogens. However, a thorough investigation of the whole genome sequences of L. paracasei isolates is required to ensure their safety and probiotic properties for human applications. This study aimed to sequence the genome of L. paracasei isolated from fermented palm sap, to assess its safety profile, and to conduct a comprehensive comparative genomic analysis with other Lacticaseibacillus species. The genome sizes of the seven L. paracasei strains ranged from 3,070,747 bp to 3,131,129 bp, with a GC content between 46.11% and 46.17% supporting their classification as nomadic lactobacilli. In addition, the minimal presence of cloud genes and a significant number of core genes suggest a high degree of relatedness among the strains. Meanwhile, phylogenetic analysis of core genes revealed that the strains possessed distinct genes and were grouped into two distinct clades. Genomic analysis revealed key genes associated with probiotic functions, such as those involved in gastrointestinal, oxidative stress resistance, vitamin synthesis, and biofilm disruption. This study is consistent with previous studies that used whole-genome sequencing and bioinformatics to assess the safety and potential benefits of probiotics in various food fermentation processes. Our findings provide valuable insights into the potential use of seven L. paracasei strains isolated from fermented palm sap as probiotic and postbiotic candidates in functional foods and pharmaceuticals.

Brucellosis is a notifiable disease induced by a facultative intracellular Brucella pathogen. In this study, eight Brucella abortus and eighteen Brucella melitensis strains from Egypt were annotated and compared with RB51 and REV1 vaccines respectively. RAST toolkit in the BV-BRC server was used for annotation, revealing genome length of 3,250,377 bp and 3,285,803 bp, 3289 and 3323 CDS, 48 and 49 tRNA genes, the same number of rRNA (3) genes, 583 and 586 hypothetical proteins, 2697 and 2726 functional proteins for B. abortus and B. melitensis respectively. B. abortus strains exhibit a similar number of candidate genes, while B. melitensis strains showed some differences, especially in the SRR19520422 Faiyum strain. Also, B. melitensis clarified differences in antimicrobial resistance genes (KatG, FabL, MtrA, MtrB, OxyR, and VanO-type) in SRR19520319 Faiyum and (Erm C and Tet K) in SRR19520422 Faiyum strain. Additionally, the whole genome phylogeny analysis proved that all B. abortus strains were related to vaccinated animals and all B. melitensis strains of Menoufia clustered together and closely related to Gharbia, Dameitta, and Kafr Elshiek. The Bowtie2 tool identified 338 (eight B. abortus) and 4271 (eighteen B. melitensis) single nucleotide polymorphisms (SNPs) along the genomes. These variants had been annotated according to type and impact. Moreover, thirty candidate genes were predicted and submitted at GenBank (24 in B. abortus) and (6 in B. melitensis). This study contributes significant insights into genetic variation, virulence factors, and vaccine-related associations of Brucella pathogens, enhancing our knowledge of brucellosis epidemiology and evolution in Egypt.

In our previous microbiome profiling analysis, Lactobacillus (L.) johnsonii was suggested to contribute to resistance against chronic heat stress-induced diarrhea in weaned piglets. Forty-nine L. johnsonii strains were isolated from these heat stress-resistant piglets, and their probiotic properties were assessed. Strains N5 and N7 exhibited a high survival rate in acidic and bile environments, along with an antagonistic effect against Salmonella. To identify genes potentially involved in these observed probiotic properties, the complete genome sequences of N5 and N7 were determined using a combination of Illumina and nanopore sequencing. The genomes of strains N5 and N7 were found to be highly conserved, with two N5-specific and four N7-specific genes identified. Multiple genes involved in gastrointestinal environment adaptation and probiotic properties, including acidic and bile stress tolerance, anti-inflammation, CAZymes, and utilization and biosynthesis of carbohydrate compounds, were identified in both genomes. Comparative genome analysis of the two genomes and 17 available complete L. johnsonii genomes revealed 101 genes specifically harbored by strains N5 and N7, several of which were implicated in potential probiotic properties. Overall, this study provides novel insights into the genetic basis of niche adaptation and probiotic properties, as well as the genome diversity of L. johnsonii.

Lacticaseibacillus rhamnosus is a group of probiotic strains that have gained popularity for their potential health benefits such as promoting digestive health, boosting the immune system, improving lactose digestion, preventing and treating antibiotic-associated diarrhea, reducing the severity and duration of certain infections, and preventing the formation of dental plaque. In particular, L. rhamnosus strains SD4 and SD11 have promising human and animal health applications due to their ability to inhibit the growth of harmful pathogens. This study presents an in silico genomic analysis of L. rhamnosus strains SD4 and SD11. We analyzed draft genomes and conducted comparative genome analyses against several other probiotic strains, aiming to gain insights into the genomes of the two strains and to compare them to related strains isolated from other sources. We also aimed to clarify the functional mechanisms and adaptation of these strains to specific environments. Comprehensive insights into the genomes of L. rhamnosus SD4 and SD11 could enhance our understanding of their capacity to colonize, adapt, and exhibit probiotic properties after administration. This study holds significance in advancing our understanding of the potential health benefits associated with these strains and in elucidating the underlying mechanisms responsible for their effectiveness in humans and animals.

Sisomicin is a broad-spectrum aminoglycoside antibiotic and is the precursor of netilmicin and plazomicin. However, the fermentation level of sisomicin is still low compared with other antibiotics, which restricts the application of sisomicin and its derivatives. In this study, to improve sisomicin production, breeding of high-yielding sisomicin strains was conducted with chemical mutagenesis using Micromonospora inyoensis OG-1 (titer, 1042 U·mL-1) as the starting strain. Protoplast preparation was conducted under optimal conditions, and protoplast mutagenesis was performed with a suitable concentration of diethyl sulfate. Subsequently, a high-yielding and genetically stable strain (H6-32) was obtained by screening, with a sisomicin titer of 1486 U·mL-1 (an increase of 42.6%). Finally, carbon and nitrogen sources were optimized to further improve sisomicin production, and a sisomicin titer of 1780 U·mL-1 was ultimately obtained by controlling the dissolved oxygen level at 30% in a 5-L fermenter, which to the best of our knowledge is the highest reported titer ever achieved by fermentation. Comparative genome analysis showed that a total of 13 genes in the genome of the mutant strain H6-32 were mutated compared to the original strain. This study not only provides a reference for further breeding of high-yielding strains and fermentation optimization, but also enhances our understanding of sisomicin production.

Extreme thermophiles Calditerricola satsumensis DD2 and D3 were isolated from mesothermal municipal sludge, a material used for hyperthermal composting. To understand the ecologically anomalous findings, their behavior at various temperatures, membrane fatty acid composition, and draft genome sequences were compared with those of C. satsumensis YMO81T and Calditerricola yamamurae YMO722T, already isolated from hyperthermal compost. All four strains grew between 56 and 83 °C. However, strains DD2 and D3 were stable for ≥48 h at a wide range of temperatures (20-75 °C), while strains YMO81T and YMO722T were highly labile at lower temperatures. The former strains maintained their colony-forming ability for >180 days at 20 °C, while the latter strains lost it within 1 d. All four strains showed similar composition of membrane fatty acid, which were not affected by 20 °C treatment. Comparative draft genome analyses showed that 13 candidate genes were present only in strains DD2 and D3, and the specific expression of six gene homologs was confirmed. A DNA chaperone, site-specific recombinase XerD homolog, had tetra adenine sequence at its upper gene region, and was up-regulated by 20 °C treatment in DD2 and D3, suggesting a possible role in the cold tolerance of sludge-derived strains. In addition, the lack of another possible DNA chaperone, a homolog of the ATP-dependent DNA helicase, in the compost-derived strains may accelerate their sensitivity to cold shock. In conclusion, we speculate that the specific phenotypic and genotypic characteristics of sludge-derived strains are responsible for their unusual ecological distribution at ambient temperatures.

In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis.
In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant.
This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.