An oxalate decarboxylase-like cupin domain containing protein is involved in imparting acid stress tolerance in Bacillus amyloliquefaciens MBNC.

We report here the structural and functional properties of an oxalate decarboxylase (OxDC)-like cupin domain-containing protein of Bacillus amyloliquefaciens MBNC and its role in imparting tolerance to acid stress conditions. Quantitative real-time PCR (qPCR) analysis revealed 32-fold and 20-fold upregulation of the target gene [(OxDC')cupin] under acetic acid stress and hydrochloric acid stress, respectively, indicating its association with the acid stress response. Bacterial cells with targeted inactivation of the (OxDC')cupin gene using the pMUTIN4 vector system showed decreased growth and survival rate in acidic pH, with drastically reduced exopolysaccharide production. In Silico protein-protein interaction studies revealed seven genes (viz. glmS, nagA, nagB, tuaF, tuaF, gcvT, and ykgA) related to cell wall biosynthesis and biofilm production to interact with OxDC-like cupin domain containing protein. While all these seven genes were upregulated in B. amyloliquefaciens MBNC after 6 h of exposure to pH 4.5, the mutant cells containing the inactivated (OxDC')cupin gene displayed significantly lower expression (RQ: 0.001-0.02) (compared to the wild-type cells) in both neutral and acidic pH. Our results indicate that the OxDC-like cupin domain containing protein is necessary for cell wall biosynthesis and biofilm production in Bacillus amyloliquefaciens MBNC for survival in acid-stress conditions.

Genomic insights into Bacillus subtilis MBB3B9 mediated aluminium stress mitigation for enhanced rice growth.

Aluminium (Al) toxicity in acid soil ecosystems is a major impediment to crop production as it drastically affects plant root growth, thereby acquisition of nutrients from the soil. Plant growth-promoting bacteria offers an interesting avenue for promoting plant growth under an Al-phytotoxic environment. Here, we report the plant growth-promoting activities of an acid-tolerant isolate of Bacillus subtilis that could ameliorate acid-induced Al-stress in rice (Oryza sativa L.). The whole genome sequence data identified the major genes and genetic pathways in B. subtilis MBB3B9, which contribute to the plant growth promotion in acidic pH. Genetic pathways for organic acid production, denitrification, urea metabolism, indole-3-acetic acid (IAA) production, and cytokinin biosynthesis were identified as major genetic machinery for plant growth promotion and mitigation of Al-stress in plants. The in-vitro analyses revealed the production of siderophores and organic acid production as primary mechanisms for mitigation of Al-toxicity. Other plant growth-promoting properties such as phosphate solubilization, zinc solubilization, and IAA production were also detected in significant levels. Pot experiments involving rice under acidic pH and elevated concentrations of aluminium chloride (AlCl3) suggested that soil treatment with bacterial isolate MBB3B9 could enhance plant growth and productivity compared to untreated plants. A significant increase in plant growth and productivity was recorded in terms of plant height, chlorophyll content, tiller number, panicle number, grain yield, root growth, and root biomass production.

Agricultural Land Use Influences Bacteriophage Community Diversity, Richness, and Heterogeneity.

The last two decades have witnessed a large-scale conversion of crop cultivation areas into small and mid-sized tea plantations in Assam, India. Agricultural land-use pattern positively or negatively influences native hydrology and above- and belowground biodiversity. Very little is known about the effect of agricultural land-use patterns on the soil virus (especially, bacteriophage) community structure and function. This metagenomic-based study evaluated the rhizosphere viral community structure of three interlinked cultivation areas, viz., mixed cropping area (coded as CP1), tea-seed orchard (CP2), and monocropping tea cultivation (CP3). The bacteriophages belonged to four major classes with the dominance of Malgrandaviricetes (CP1: 79.37%; CP2: 64.62%; CP3: 4.85%) followed by Caudoviricetes (CP1: 20.49%; CP2: 35.22%; CP3: 90.29%), Faserviricetes (CP1: 0.03%; CP2: 0.08%; CP3: 3.88%), and Tectiliviricetes (CP1: 0.12%; CP2: 0.07%; CP3: 0.97%). Microviruses dominated the phage population in both CP1 and CP2, representing 79.35% and 64.59% of total bacteriophage abundance. Both CP1 and CP2 had higher bacteriophage richness (species richness, R in CP1: 65; R in CP2: 66) and lower evenness (Pielou's evenness index, J in CP1: 0.531; J in CP2: 0.579) compared to the CP3 (R: 30; J: 0.902). Principal component analysis of edaphic soil factors and bacteriophage community structure showed a reverse-proportional correlation between the levels of Al saturation, and exchangeable Al3+ ions with that of soil pH, and bacteriophage abundance. Our study indicates that monocropping tea cultivation soil bears less viral richness, abundance, and heterogeneity.

Proline confers acid stress tolerance to Bacillus megaterium G18.

Proline plays a multifunctional role in several organisms including bacteria in conferring protection under stress conditions. In this paper we report the role of proline in conferring acid tolerance to Bacillus megaterium G18. An acid susceptible mutant of B. megaterium G18 which required proline for its growth under acid stress condition was generated through Tn5 mutagenesis. Further, targeted inactivation of proC involved in osmo-adaptive proline synthesis in B. megaterium G18 resulted in the loss of ability of the bacterium to grow at low pH (pH 4.5). Exogenous supply of proline (1 mM) to the growth medium restored the ability of the mutant cells to grow at pH 4.5 which was not the same in case of other osmoprotectants tested. Proline was produced and secreted to extracellular medium by B. megaterium G18 when growing in low pH condition as evidenced by the use of Escherichia coli proline auxotrophs and HPLC analysis. Further, pHT01 vector based expression of full length proC gene in the ∆proC mutant cells restored the survival capacity of the mutant cells in acidic pH, suggesting that proline production is an important strategy employed by B. megaterium G18 to survive under acid stress induced osmotic stress.

Identification of Putative Vaccine and Drug Targets against the Methicillin-Resistant Staphylococcus aureus by Reverse Vaccinology and Subtractive Genomics Approaches.

Methicillin-resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen and responsible for causing life-threatening infections. The emergence of hypervirulent and multidrug-resistant (MDR) S. aureus strains led to challenging issues in antibiotic therapy. Consequently, the morbidity and mortality rates caused by S. aureus infections have a substantial impact on health concerns. The current worldwide prevalence of MRSA infections highlights the need for long-lasting preventive measures and strategies. Unfortunately, effective measures are limited. In this study, we focus on the identification of vaccine candidates and drug target proteins against the 16 strains of MRSA using reverse vaccinology and subtractive genomics approaches. Using the reverse vaccinology approach, 4 putative antigenic proteins were identified; among these, PrsA and EssA proteins were found to be more promising vaccine candidates. We applied a molecular docking approach of selected 8 drug target proteins with the drug-like molecules, revealing that the ZINC4235426 as potential drug molecule with favorable interactions with the target active site residues of 5 drug target proteins viz., biotin protein ligase, HPr kinase/phosphorylase, thymidylate kinase, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-L-lysine ligase, and pantothenate synthetase. Thus, the identified proteins can be used for further rational drug or vaccine design to identify novel therapeutic agents for the treatment of multidrug-resistant staphylococcal infection.

Evaluation of Total Phenolic and Flavonoid Contents, Antibacterial and Antibiofilm Activities of Hungarian Propolis Ethanolic Extract against Staphylococcus aureus.

Propolis is a natural bee product that is widely used in folk medicine. This study aimed to evaluate the antimicrobial and antibiofilm activities of ethanolic extract of propolis (EEP) on methicillin-resistant and sensitive Staphylococcus aureus (MRSA and MSSA). Propolis samples were collected from six regions in Hungary. The minimum inhibitory concentrations (MIC) values and the interaction of EEP-antibiotics were evaluated by the broth microdilution and the chequerboard broth microdilution methods, respectively. The effect of EEP on biofilm formation and eradication was estimated by crystal violet assay. Resazurin/propidium iodide dyes were applied for simultaneous quantification of cellular metabolic activities and dead cells in mature biofilms. The EEP1 sample showed the highest phenolic and flavonoid contents. The EEP1 successfully prevented the growth of planktonic cells of S. aureus (MIC value = 50 µg/mL). Synergistic interactions were shown after the co-exposition to EEP1 and vancomycin at 108 CFU/mL. The EEP1 effectively inhibited the biofilm formation and caused significant degradation of mature biofilms (50-200 µg/mL), as a consequence of the considerable decrement of metabolic activity. The EEP acts effectively as an antimicrobial and antibiofilm agent on S. aureus. Moreover, the simultaneous application of EEP and vancomycin could enhance their effect against MRSA infection.

Comparative analysis of prophages carried by human and animal-associated Staphylococcus aureus strains spreading across the European regions.

Staphylococcus aureus is a major human and animal pathogen although the animal-associated S. aureus can be a potential risk of human zoonoses. Acquisition of phage-related genomic islands determines the S. aureus species diversity. This study characterized and compared the genome architecture, distribution nature, and evolutionary relationship of 65 complete prophages carried by human and animal-associated S. aureus strains spreading across the European regions. The analyzed prophage genomes showed mosaic architecture with extensive variation in genome size. The phylogenetic analyses generated seven clades in which prophages of the animal-associated S. aureus scattered in all the clades. The S. aureus strains with the same SCCmec type, and clonal complex favored the harboring of similar prophage sequences and suggested that the frequency of phage-mediated horizontal gene transfer is higher between them. The presence of various virulence factors in prophages of animal-associated S. aureus suggested that these prophages could have more pathogenic potential than prophages of human-associated S. aureus. This study showed that the S. aureus phages are dispersed among the several S. aureus serotypes and around the European regions. Further, understanding the phage functional genomics is necessary for the phage-host interactions and could be used for tracing the S. aureus strains transmission.

Genome-wide comparison of four MRSA clinical isolates from Germany and Hungary.

Staphylococcus aureus is a drug-resistant pathogen, capable of colonizing diverse ecological niches and causing a broad spectrum of infections related to a community and healthcare. In this study, we choose four methicillin-resistant S. aureus (MRSA) clinical isolates from Germany and Hungary based on our previous polyphasic characterization finding. We assumed that the selected strains have a different genetic background in terms of the presence of resistance and virulence genes, prophages, plasmids, and secondary metabolite biosynthesis genes that may play a crucial role in niche adaptation and pathogenesis. To clarify these assumptions, we performed a comparative genome analysis of these strains and observed many differences in their genomic compositions. The Hungarian isolates (SA H27 and SA H32) with ST22-SCCmec type IVa have fewer genes for multiple-drug resistance, virulence, and prophages reported in Germany isolates. Germany isolate, SA G6 acquires aminoglycoside (ant(6)-Ia and aph(3')-III) and nucleoside (sat-4) resistance genes via phage transduction and may determine its pathogenic potential. The comparative genome study allowed the segregation of isolates of geographical origin and differentiation of the clinical isolates from the commensal isolates. This study suggested that Germany and Hungarian isolates are genetically diverse and showing variation among them due to the gain or loss of mobile genetic elements (MGEs). An interesting finding is the addition of SA G6 genome responsible for the drastic decline of the core/pan-genome ratio curve and causing the pan-genome to open wider. Functional characterizations revealed that S. aureus isolates survival are maintained by the amino acids catabolism and favor adaptation to growing in a protein-rich medium. The dispersible and singleton genes content of S. aureus genomes allows us to understand the genetic variation among the CC5 and CC22 groups. The strains with the same genetic background were clustered together, which suggests that these strains are highly alike; however, comparative genome analysis exposed that the acquisition of phage elements, and plasmids through the events of MGEs transfer contribute to differences in their phenotypic characters. This comparative genome analysis would improve the knowledge about the pathogenic S. aureus strain's characterization, and responsible for clinically important phenotypic differences among the S. aureus strains.

Characterization of methicillin-resistant Staphylococcus aureus through genomics approach.

In the present study, a total of 35 S. aureus isolates collected from two different geographical locations viz., Germany and Hungary were tested for their methicillin-resistant phenotype which revealed a high incidence of methicillin-resistant S. aureus. The quantitative test for biofilm production revealed that 73.3% of isolates were biofilm producers. The isolates were further characterized using a set of biochemical and genotypic methods such as amplification and analysis of S. aureus species-specific sequence and mecA gene. The 33 mecA positive isolates were then characterized by the amplification of SCCmec and pvl toxin genes. Further, based on the biofilm-forming phenotype, 15 isolates were selected and characterized through PCR-RFLP of coa gene, polymorphism of spa gene and amplification of biofilm-associated genes. The dendrogram prepared from the results of both biochemical and genotypic analyses of the 15 isolates showed that except for the isolates SA G5 and SA H29, the rest of the isolates grouped themselves according to their locations. Thus, the two isolates were selected for further characterization through whole-genome sequencing. Comparative genome analysis revealed that SA G5 and SA H29 have 97.20% ANI values with 2344 gene clusters (core-genome) of which 16 genes were related to antibiotic resistance genes and 57 genes encode virulence factors. The highest numbers of singleton genes were found in SA H29 that encodes proteins for virulence, resistance, mobile elements, and lanthionine biosynthesis. The high-resolution phylogenetic trees generated based on shared proteins and SNPs revealed a clear difference between the two strains and can be useful in distinguishing closely related genomes. The present study demonstrated that the whole-genome sequence analysis technique is required to get a better insight into the MRSA strains which would be helpful in improving diagnostic investigations in real-time to improve patient care.