Xanthomonas sontii, and not Xanthomonas sacchari, is the predominant, vertically transmitted core rice seed endophyte.

Seeds endophytes, particularly the abundant, core and vertically transmitted species, are major areas of focus in the host microbiome studies. Apart from being the first members to colonize, they accompany the plant throughout its development stages and also to the next generation. Recently published studies have reported Xanthomonas sacchari as the keystone species, a core endophyte that is vertically transmitted in rice with probiotic properties. Further, the Xanthomonas species was also reported as being involved in the assembly of beneficial bacteria after early inoculation in the rice seeds. However, the strains discussed in these studies were misclassified as Xanthomonas sacchari, a well-known pathogen of sugarcane. By including non-pathogenic Xanthomonas species with plant protective functions reported from rice seeds, we have correctly established the phylogenetic and taxonomic identity of the keystone species as Xanthomonas sontii. This will enable researchers to use the correct reference or lab strain of X. sontii for further systematic and in-depth studies as a model endophyte in plant-microbe interaction apart from its exploitation in seed health.

Taxonomic and Phylogenomic Assessment Identifies Phytopathogenicity Potential of Stenotrophomonas maltophilia Complex.

Stenotrophomonas maltophilia is a versatile bacterium found in plants, water, air, and even hospital settings. Deep taxono phylogenomics studies have revealed that S. maltophilia is a complex of several hidden species that are not differentiated using conventional approaches. In the last two decades, there have been increasing reports of S. maltophilia as a pathogen of diverse plants. Hence, proper taxonogenomic assessment of plant-pathogenic strains and species within the S. maltophilia complex (Smc) is required. In the present study, we formally propose a taxonomic amendment of Pseudomonas hibiscicola and P. beteli, reported as pathogens of Hibiscus rosa-sinensis and Betelvine (Piper betle) plants, respectively, as a misclassified member species of the Smc. Recently, a novel species of the genus, S. cyclobalanopsidis, was reported as a leaf spot pathogen of the oak tree genus Cyclobalanopsis. Interestingly, our investigation also revealed S. cyclobalanopsidis as another plant-pathogenic member species of the Smc lineage. In addition, we provide deep phylo-taxonogenomic evidence that S. maltophilia strain JZL8, reported as a plant pathogen, is a misclassified strain of S. geniculata, making it the fourth member species of the Smc harboring plant-pathogenic strains. Therefore, a proper taxonomic assessment of plant-pathogenic strains and species from the Smc is required for further systematic studies and management.

Insights into the Diversity of Transcription Activator-Like Effectors in Indian Pathotype Strains of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a major rice pathogen, and its genome harbors extensive inter-strain and inter-lineage variations. The emergence of highly virulent pathotypes of Xoo that can overcome major resistance (R) genes deployed in rice breeding programs is a grave threat to rice cultivation. The present study reports on a long-read Oxford nanopore-based complete genomic investigation of Xoo isolates from 11 pathotypes that are reported based on their reaction toward 10 R genes. The investigation revealed remarkable variation in the genome structure in the strains belonging to different pathotypes. Furthermore, transcription activator-like effector (TALE) proteins secreted by the type III secretion system display marked variation in content, genomic location, classes, and DNA-binding domain. We also found the association of tal genes in the vicinity of regions with genome structural variations. Furthermore, in silico analysis of the genome-wide rice targets of TALEs allowed us to understand the emergence of pathotypes compatible with major R genes. Long-read, cost-effective sequencing technologies such as nanopore can be a game changer in the surveillance of major and emerging pathotypes. The resource and findings will be invaluable in the management of Xoo and in appropriate deployment of R genes in rice breeding programs.

Lysobacter arvi sp. nov. Isolated from Farmland Soil.

A bacterial strain designated as UC was isolated from farmland soil. Strain UCT formed a pale yellow colony on nutrient agar. Cell morphology revealed it as the rod-shaped bacterium that stained Gram-negative. The 16S rRNA gene sequence analysis identified strain UCT as a member of the genus Lysobacter that showed high identity with L. soli DCY21T (99.5%), L. panacisoli CJ29T (98.7%), and L. tabacisoli C8-1T (97.9%). It formed a distinct cluster with these strains in the neighbor-joining phylogenetic tree. A similar tree topology was observed in TYGS-based phylogenomic analysis. However, genome sequence analyses of strain UCT showed 87.7% average nucleotide identity and 34.7% digital DNA-DNA hybridization similarity with the phylogenetically closest species, L. soli DCY21T. The similarity was much less with other closely related strains of the genus Lysobacter. The G + C content of strain UCT was 68.1%. Major cellular fatty acids observed were C14:0 iso (13.4%), C15:0 iso (13.6%), and C15:0 anteiso (14.8%). Quinone Q-8 was the major respiratory ubiquinone. Predominant polar lipids were phosphatidylethanolamine, diphosphatidylglycerol, and phosphatidylglycerol. Production of xanthomonadin pigment was observed. Based on phenotypic differences and phylogenomic analysis, strain UCT represents a novel species of the genus Lysobacter, for which the name Lysobacter arvi is proposed. The type strain of the novel species is UCT (= KCTC 92613T = JCM 23757T = MTCC 12824T).

Evaluation of Human Dental Plaque Lactic Acid Bacilli for Probiotic Potential and Functional Analysis in Relevance to Oral Health.

Members of the lactic acid bacillus group are well-known probiotics and primarily isolated from fermented food, dairy products, intestinal and gut environment of human. Since probiotics from the human source are preferred, there exists a huge repertoire of lactobacilli in the human oral cavity which could prove a much better niche to be exploited for these beneficial microorganisms. Therefore, in this study, four lactobacilli strains, including strain DISK7, reported earlier, isolated from dental plaque samples of a healthy humans were evaluated for their probiotic potential. Strains displayed 99.9% of 16S rRNA gene sequence identity with species of the genera Lactobacillus and Limosilactobacillus. All strains showed lactic acid production, tolerance to low pH and antibiotic sensitivity. Variations were observed among strains in their aggregation ability, biofilm formation, bile salt resistance and cholesterol degradation. Further, we analyzed the interaction of strains with other oral commensals and opportunistic pathogens in co-culture experiments. Isolates DISK7 and DISK26 exhibited high co-aggregation (> 70%) with secondary colonizers, Streptococcus pyogenes and Veillonella parvula, respectively, but their aggregation ability was decreased with opportunistic pathogens. Furthermore, strains showed a substantial increase in biofilm in co-culture with other Lactobacillus isolates, indicating their ability to proliferate commensal bacteria in the oral environment. These microbes continually evolve in terms of niche adaptation as evidenced in genome analysis. The highlight of the investigation is the isolation and evaluation of the probiotic lactobacilli from the human oral cavity, which could prove a much better niche to be exploited for the effective commercialization of these beneficial microbes. Taken together, probiotic properties and interaction with commensal bacteria, these isolates exhibit the huge potential to be developed as alternative bioresource agents for maintenance of oral health. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-023-01108-2.

Deep Population Genomics Reveals Systematic and Parallel Evolution at a Lipopolysaccharide Biosynthetic Locus in Xanthomonas Pathogens That Infect Rice and Sugarcane.

The advent of high-throughput sequencing and population genomics has enabled researchers to investigate selection pressure at hypervariable genomic loci encoding pathogen-associated molecular pattern (PAMP) molecules like lipopolysaccharide (LPS). Xanthomonas is a model and a major group of phytopathogenic bacteria that infect hosts in tissue-specific manner. Our in-depth population-based genomic investigation revealed the emergence of major lineages in two Xanthomonas pathogens that infect xylem of rice and sugarcane is associated with the acquisition and later large-scale replacement by distinct type of LPS cassettes. In the population of the rice xylem pathogen, Xanthomonas oryzae pv. oryzae (Xoo) and sugarcane pathogens Xanthomonas sacchari (Xsac) and Xanthomonas vasicola (Xvv), the BXO8 type of LPS cassette is replaced by a BXO1 type of cassette in Xoo and by Xvv type LPS cassette in Xsac and Xvv. These findings suggest a wave of parallel evolution at an LPS locus mediated by horizontal gene transfer (HGT) events during its adaptation and emergence. Aside from xylem pathogens, two closely related lineages of Xoo that infect parenchyma of rice and Leersia hexandra grass have acquired an LPS cassette from Xanthomonas pathogens that infect parenchyma of citrus, walnut, and strawberries, indicating yet another instance of parallel evolution mediated by HGT at an LPS locus. Our targeted and megapopulation-based genome dynamic studies revealed the acquisition and dominance of specific types of LPS cassettes in adaptation and success of a major group of phytopathogenic bacteria. IMPORTANCE Lipopolysaccharide (LPS) is a major microbe associated molecular pattern and hence a major immunomodulator. As a major and outer member component, it is expected that LPS is a frontline defense mechanism to deal with different host responses. Limited studies have indicated that LPS loci are also highly variable at strain and species level in plant-pathogenic bacteria, suggesting strong selection pressure from plants and associated niches. The advent of high-throughput genomics has led to the availability of a large set of genomic resources at taxonomic and population levels. This provides an exciting and important opportunity to carryout megascale targeted and population-based comparative genomic/association studies at important loci like those encoding LPS biosynthesis to understand their role in the evolution of the host, tissue specificity, and also predominant lineages. Such studies will also fill major gap in understanding host and tissue specificity in pathogenic bacteria. Our pioneering study uses the Xanthomonas group of phytopathogens that are known for their characteristic host and tissue specificity. The present deep phylogenomics of diverse Xanthomonas species and its members revealed lineage association and dominance of distinct types of LPS in accordance with their origin, host, tissue specificity, and evolutionary success.

Phylo-Taxonogenomics Supports Revision of Taxonomic Status of 20 Xanthomonas Pathovars to Xanthomonas citri.

Based on phylo-taxonogenomics criteria, we present amended descriptions for 20 pathovars to Xanthomonas citri. Incidentally, 18 were first reported from India. Seven out of twenty are classified as X. axonopodis, 12 out of 20 as X. campestris, and one as X. cissicola. In this study, we have generated genome sequence data of four pathovars, and the genomes of the remaining 16 were used from the published data. Comprehensive genome-based phylogenomic and taxonogenomic analyses reveal that all these pathovars belong to X. citri and need to reconcile their taxonomic status. This proposal will aid in systematic studies of a major species and its constitutent members that infect economically important plants.

Lacrimispora defluvii PI-S10-B5AT sp. nov., an Obligate Anaerobe, Isolated from an Industrial Waste and Reclassification of Hungatella xylanolytica as Lacrimispora xylanolytica and Clostridium indicum as Lacrimispora indica Comb. nov.

A bacterial strain was isolated from the waste slurry of an industrial effluent treatment plant near Patancheru, Hyderabad, India, and designated as PI-S10-B5AT. It was an obligately anaerobic, spore-forming, rod-shaped, motile bacterium that stained Gram-positive. The strain revealed high 16S rRNA gene sequence identity with Hungatella xylanolytica DSM 3808T (99.4%) followed by members of the genus Lacrimispora (98.8-93.3%). However, the average nucleotide identity (ANI) and digital DNA-DNA hybridization of genome sequence exhibited similarity in the range of 94.3-68.7% and 57.4-18.8%, respectively, with all closely related strains. A multi-gene phylogenetic analysis of strain PI-S10-B5AT was performed to investigate the taxonomic affiliation, which revealed formation of a coherent cluster with the members of the genus Lacrimispora. The DNA G + C content was 41.8 mol%. Major polar lipids were glyco- and phospholipids. The fatty acids analysis showed C16:0 to be the major fatty acid. The predominant respiratory quinone was menaquinone-7 (MK-7). Based on phenotypic, chemotaxonomic, and whole-genome phylogenetic analysis, strain PI-S10-B5AT is assigned as a novel species of the genus Lacrimispora, for which the name Lacrimispora defluvii is proposed. The type strain of the novel species is PI-S10-B5AT (= MTCC 12280T; = DSM 24980T) isolated from waste slurry of effluent treatment plant. The genomic analysis of type strains of C. indicum PI-S10-A1BT and H. xylanolytica DSM 3808T showed ANI and AAI values consistent with members of the genus Lacrimispora. Therefore, these strains are ascertained to the genus Lacrimispora and reclassified as Lacrimispora indica and Lacrimispora xylanolytica comb. nov.

Xanthomonas indica sp. nov., a Novel Member of Non-Pathogenic Xanthomonas Community from Healthy Rice Seeds.

Xanthomonas is a major group of pathogenic bacteria infecting staple food crops like rice. Increasingly it is being recognized that non-pathogenic Xanthomonas (NPX) are also important members of a healthy plant microbiome. However, the vast majority of the species described in this genus are of pathogenic nature, and only a few NPX species have been reported till now. Genomic and taxonogenomic analysis of NPX is needed for the management of this important group of bacteria. In this study, two yellow-pigmented bacterial isolates were obtained from healthy rice seeds in Punjab, India. The isolates designated PPL560T and PPL568 were identified as members of the genus Xanthomonas based on biochemical tests and 16S rRNA gene sequence analysis retrieved from the whole-genome sequences. Isolates formed a distinct monophyletic lineage with Xanthomonas sontii and Xanthomonas sacchari as the closest relatives in the phylogenetic tree based on core gene content shared by the representative species of the genus Xanthomonas. Pairwise ortho Average Nucleotide Identity and digital DNA-DNA hybridization values calculated against other species of Xanthomonas were below their respective cut-offs. In planta studies revealed that PPL560T and PPL568 are non-pathogenic to rice plants upon leaf clip inoculation. The absence of type III secretion system-related genes and effectors further supported their non-pathogenic status. Herein, we propose Xanthomonas indica sp. nov. as novel species of the genus Xanthomonas with PPL560T = MTCC 13185 = CFBP 9039 = ICMP 24394 as its type strain and PPL568 as another constituent member.

Deep phylo-taxono genomics reveals Xylella as a variant lineage of plant associated Xanthomonas and supports their taxonomic reunification along with Stenotrophomonas and Pseudoxanthomonas.

Genus Xanthomonas is a group of phytopathogens that is phylogenetically related to Xylella, Stenotrophomonas, and Pseudoxanthomonas, having diverse lifestyles. Xylella is a lethal plant pathogen with a highly reduced genome, atypical GC content and is taxonomically related to these three genera. Deep phylo-taxono genomics reveals that Xylella is a variant Xanthomonas lineage that is sandwiched between Xanthomonas clades. Comparative studies suggest the role of unique pigment and exopolysaccharide gene clusters in the emergence of Xanthomonas and Xylella clades. Pan-genome analysis identified a set of unique genes associated with sub-lineages representing plant-associated Xanthomonas clade and nosocomial origin Stenotrophomonas clade. Overall, our study reveals the importance of reconciling classical phenotypic data and genomic findings in reconstituting the taxonomic status of these four genera. SIGNIFICANCE STATEMENT: Xylella fastidiosa is a devastating pathogen of perennial dicots such as grapes, citrus, coffee, and olives. An insect vector transmits the pathogen to its specific host wherein the infection leads to complete wilting of the plants. The genome of X. fastidiosa is significantly reduced both in terms of size (2 Mb) and GC content (50%) when compared with its relatives such as Xanthomonas, Stenotrophomonas, and Pseudoxanthomonas that have higher GC content (65%) and larger genomes (5 Mb). In this study, using systematic and in-depth genome-based taxonomic and phylogenetic criteria and comparative studies, we assert the need to unify Xanthomonas with its relatives (Xylella, Stenotrophomonas and Pseudoxanthomonas). Interestingly, Xylella revealed itself as a minor variant lineage embedded within two major Xanthomonas lineages comprising member species of different hosts.

Xanthomonas sontii sp. nov., a non-pathogenic bacterium isolated from healthy basmati rice (Oryza sativa) seeds from India.

We report three yellow-pigmented, Gram-negative, aerobic, rod-shaped, motile bacterial isolates designated as PPL1T, PPL2, and PPL3 from healthy basmati rice seeds. Phenotypic and 16S rRNA gene sequence analysis assigned these isolates to the genus Xanthomonas. The 16S rRNA showed a 99.59% similarity with X. sacchari CFBP 4641T, a sugarcane pathogen. Further, biochemical and fatty acid analysis revealed it to be closer to X. sacchari. Still, it differed from other species in general and known rice associated species such as X. oryzae (pathogenic) and X. maliensis (non-pathogenic) in particular. Interestingly, the isolatess in this study were isolated from healthy rice plants but are closely related to species that is pathogenic and isolated from diseased sugarcane. Accordingly, in planta studies revealed that PPL1T, PPL2, and PPL3 are non-pathogenic to rice plants upon leaf inoculation. Taxonogenomic studies based on orthologous average nucleotide identity (OrthoANI) and digital DNA-DNA hybridization (dDDH) values with type strains of Xanthomonas species were below the recommended threshold values for species delineation. Whole genome-based phylogenomic analysis revealed that these isolates formed a distinct monophyletic clade with X. sacchari CFBP 4641T as their closest neighbour. Further, pangenome analysis revealed PPL1T, PPL2, and PPL3 isolates to comprise NRPS cluster along with a large number of unique genes associated with the novel species. Based on polyphasic and genomic approaches, a novel lineage and species associated with healthy rice seeds for which the name Xanthomonas sontii sp. nov. is proposed. The type strain for the X. sontii sp. nov. is PPL1T (JCM 33631T = CFBP 8688T = ICMP 23426T = MTCC 12491T) and PPL2 (JCM 33632 = CFBP 8689 = ICMP 23427 = MTCC 12492) and PPL3 (JCM 33633 = CFBP 8690 = ICMP 23428 = MTCC 12493) as other strains of the species.

Evolutionary insights into adaptation of Staphylococcus haemolyticus to human and non-human niches.

Staphylococcus haemolyticus is a well-known member of human skin microbiome and an emerging opportunistic human pathogen. Presently, evolutionary studies are limited to human isolates even though it is reported from plants with beneficial properties and in environmental settings. In the present study, we report isolation of novel S. haemolyticus strains from surface sterilized rice seeds and compare their genome to other isolates from diverse niches available in public domain. The study showed expanding nature of pan-genome and revealed set of genes with putative functions related to its adaptability. This is seen by presence of type II lanthipeptide cluster in rice isolates, metal homeostasis genes in an isolate from copper coin and gene encoding methicillin resistance in human isolates. The present study on differential genome dynamics and role of horizontal gene transfers has provided novel insights into capability for ecological diversification of a bacterium of significance to human health.

Genomic insights into evolution of extensive drug resistance in Stenotrophomonas maltophilia complex.

We report first complete genomic investigation of extensive drug resistance (XDR) in a nosocomial Stenotrophomonas maltophilia complex strain that is resistant to mainstream drugs (trimethoprim/sulfamethoxazole and levofloxacin). Comprehensive genomic investigation revealed its exclusive fourteen dynamic regions and highly enriched resistome comprising of two sulfonamide resistance genes on two diverse super-integrons of chromosomal origin. In addition, both these integrons harbour array of antibiotic resistance and commonly used disinfectant's resistance genes linked to ISCR elements. Isolation of a novel XDR strain from Indian tertiary care unit belonging to novel ST with diverse array of resistance genes on ISCR linked super-integrons indicates extent and nature of selection pressure in hospitals. Since, repetitive elements have major role in their spread and due to limitations of draft genomes, there is an urgent need to employ complete genome-based investigation for tracking the emergence of XDR at global level and designing strategies of antimicrobial stewardship and disinfection. IMPORTANCE: Hospital settings in India have one of the highest usages of antimicrobials and a heavy patient load. We hereby report a novel clinical isolate of S. maltophilia complex with two super-integrons that harbour array of antimicrobial resistance genes along with biocide and heavy metal resistance genes. Further, the presence of ISCR type of transposable elements on both the integrons indicates their propensity to transfer resistome while their chromosomal origin suggests possibilities for further genomic/phenotypic complexities according to selection pressure. Such complex mobile cassettes in a novel strain is a potential threat to global health care. Hence, to understand the evolution of opportunistic nosocomial pathogen, there is an urgent need to employ cost-effective long read technologies to keep vigilance on novel and XDR pathogens in populous countries. There is also need for surveillance of the usage of disinfectants and other antimicrobials for environmental hygiene and linked/rapid co-evolution of XDR in nosocomial pathogens. Repositories: Complete genome sequence of Stenotrophomonas maltophilia SM866: CP031058.

Bacteriocin isolated from the natural inhabitant of Allium cepa against Staphylococcus aureus.

Extensive usage of antibiotics has led to the emergence of drug-resistant strains of pathogens and hence, there is an urgent need for alternative antimicrobial agents. Antimicrobial Peptides (AMPs) of bacterial origin have shown the potential to replace some conventional antibiotics. In the present study, an AMP was isolated from Bacillus subtilis subsp. spizizenii strain Ba49 present on the Allium cepa, the common onion and named as peptide-Ba49. The isolated AMP was purified and characterized. The purified peptide-Ba49, having a molecular weight of ~ 3.3 kDa as determined using mass spectroscopy, was stable up to 121 °C and in the pH range of 5-10. Its interaction with protein degrading enzymes confirmed the peptide nature of the molecule. The peptide exhibited low minimum inhibitory concentration (MIC) against Staphylococcus aureus and its (Methicillin-resistant Staphylococcus aureus) MRSA strains (MIC, 2-16 µM/mL). Further, time kill kinetic assay was performed and analysis of the results of membrane depolarization and permeabilization assays (TEM, DiBAC4 (3) and PI) suggested peptide-Ba49 to be acting through the change in membrane potential leading to disruption of S. aureus membrane. Additionally, cytotoxicity studies of peptide-Ba49, carried out using three mammalian cell lines viz. HEK 293T, RAW 264.7, and L929, showed limited cytotoxicity on these cell lines at a concentration much higher than its MIC values. All these studies suggested that the AMP isolated from strain Ba49 (peptide-Ba49) has the potential to be an alternative to antibiotics in terms of eradicating the pathogenic as well as drug-resistant microorganisms.

Clostridium indicum sp. nov., a novel anaerobic bacterium isolated from the sludge of an industrial effluent.

A strictly anaerobic bacterial strain, designated as PI-S10-A1B, was isolated from a sludge sample collected from an industrial effluent dump site at Hyderabad, India. Cells stained Gram-positive and contained terminal endospores. Optimal growth was observed at 30 °C and pH 7.0. It showed negative reactions to catalase and oxidase activities. Phylogenetic analysis of the 16S rRNA gene led to strain PI-S10-A1BT being assigned to the genus Clostridium. It displayed high sequence similarity to species of cluster XIVa including Clostridium amygdalinumBR-10T (99.84 %), Clostridium saccharolyticum WM1T (98.93 %) and Clostridium indolis DSM 755T (98.31 %). It formed a coherent cluster with members of cluster XIVa. Despite high 16S rRNA gene sequence similarity, strain PI-S10-A1BT displayed only 25.3 % identity in DNA-DNA hybridization tests with C. amygdalinum BR-10T. A draft genome exhibited low values for average nucleotide identity and in silico DNA-DNA hybridization with strains of cluster XIVa. The DNA G+C content was 42.3 mol%. Major lipids were phosphatidylglycerol and diphosphatidylglycerol, with an abundance of phosphoglycolipids. Further, analysis of the draft genome revealed genomic insights against functional aspects. Considering the phenotypic differences and low genomic identity with phylogenetic relatives, strain PI-S10-A1BT is concluded to represent a new species of the genus Clostridium, for which the name Clostridiumindicum sp. nov. is proposed with type strain PI-S10-A1BT (=MTCC 12282T=DSM 24996T=JCM 32788T).

Ecological and Evolutionary Insights into Xanthomonas citri Pathovar Diversity.

Citrus canker, caused by Xanthomonas citri pv. citri, is a serious disease of citrus plants worldwide. Earlier phylogenetic studies using housekeeping genes revealed that X. citri pv. citri is related to many other pathovars, which can be collectively referred as Xanthomonas citri pathovars (XCPs). From the present study, we report the genome sequences of 18 XCPs and compared them with four XCPs available in the public domain. In a tree based on phylogenomic marker genes, all the XCPs form a monophyletic cluster, suggesting their origin from a common ancestor. Phylogenomic analysis using the type strain further established that all the XCPs belong to one species. Clonal analysis of the core genome revealed the presence of two major lineages within this monophyletic cluster consisting of some clonal variants. Incidentally, the majority of these XCPs were first noticed in India, corroborating their clonal relationship and their common origin. Comparative analysis revealed an open pan-genome and the role of interstrain genomic flux of these XCPs since their diversification from a common ancestor. Even though there are wide variations in type III gene effectomes, we identified three core effectors which can be valuable in resistance-breeding programs. Overall, genomic examination of ecological relatives allowed us to dissect the tremendous genomic potential of X. citri species to rapidly evolve into specialized strains infecting diverse crop plants.IMPORTANCE Host specialization is one of the characteristic features of highly evolved pathogens such as the Xanthomonas group of phytopathogenic bacteria. Since the hosts involve staple crops and economically important fruits such as citrus, detailed understanding of the diversity and evolution of such strains infecting diverse plants is important for quarantine purposes. In the present study, we carried out genomic investigation of members of a phylogenetically and ecologically defined group of Xanthomonas strains pathogenic to diverse plants, including citrus. This group includes the oldest Xanthomonas pathovars and also recently emerged pathovars in a particular country where they are endemic. Our high-throughput genomic study has provided novel insights into the evolution of a unique lineage consisting of serious pathogens and their ecological relatives, suggesting the nature, scope, and pattern of rapid and recent diversification. Further, from the level of species to that of clonal variants, the study revealed interesting genomic patterns in diversification of a Xanthomonas lineage and perhaps will inspire careful study of the host range of the included pathovars.

Characterization of the Antimicrobial Peptide Penisin, a Class Ia Novel Lantibiotic from Paenibacillus sp. Strain A3.

Attempts to isolate novel antimicrobial peptides from microbial sources have been on the rise recently, despite their low efficacy in therapeutic applications. Here, we report identification and characterization of a new efficient antimicrobial peptide from a bacterial strain designated A3 that exhibited highest identity with Paenibacillus ehimensis. Upon purification and subsequent molecular characterization of the antimicrobial peptide, referred to as penisin, we found the peptide to be a bacteriocin-like peptide. Consistent with these results, RAST analysis of the entire genome sequence revealed the presence of a lantibiotic gene cluster containing genes necessary for synthesis and maturation of a lantibiotic. While circular dichroism and one-dimension nuclear magnetic resonance experiments confirmed a random coil structure of the peptide, similar to other known lantibiotics, additional biochemical evidence suggests posttranslational modifications of the core peptide yield six thioether cross-links. The deduced amino acid sequence of the putative biosynthetic gene penA showed approximately 74% similarity with elgicin A and 50% similarity with the lantibiotic paenicidin A. Penisin effectively killed methicillin-resistant Staphylococcus aureus (MRSA) and did not exhibit hemolysis activity. Unlike other lantibiotics, it effectively inhibited the growth of Gram-negative bacteria. Furthermore, 80 mg/kg of body weight of penisin significantly reduced bacterial burden in a mouse thigh infection model and protected BALB/c mice in a bacteremia model entailing infection with Staphylococcus aureus MTCC 96, suggesting that it could be a promising new antimicrobial peptide.

Laterosporulin10: a novel defensin like Class IId bacteriocin from Brevibacillus sp. strain SKDU10 with inhibitory activity against microbial pathogens.

Bacteriocins are antimicrobial peptides (AMPs) produced by bacteria to acquire survival benefits during competitive inter- and intra-species interactions in complex ecosystems. In this study, an AMP-producing soil bacterial strain designated SKDU10 was isolated and identified as a member of the genus Brevibacillus. The AMP produced by strain SKDU10 identified as a class IId bacteriocin with 57.6 % homology to laterosporulin, a defensin-like class IId bacteriocin. However, substantial differences were observed in the antimicrobial activity spectrum of this bacteriocin named laterosporulin10 when compared to laterosporulin. Laterosporulin10 effectively inhibited the growth of Staphylococcus aureus and Mycobacterium tuberculosis (Mtb H37Rv) with LD50 values of 4.0 µM and 0.5 µM, respectively. Furthermore, laterosporulin10 inhibited the growth of Mtb H37Rv strain at about 20 times lower MIC value compared to S. aureus MTCC 1430 or M. smegmatis MC2 155 in vitro and ex vivo. Electron micrographs along with membrane permeabilization studies using FACS analysis revealed that laterosporulin10 is a membrane-permeabilizing peptide. Interestingly, laterosporulin10 was able to efficiently kill Mtb H37Rv strain residing inside the macrophages and did not show haemolysis up to 40 µM concentration.

Genomic insights into the evolutionary origin of Xanthomonas axonopodis pv. citri and its ecological relatives.

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker (CBC) and is a serious problem worldwide. Like CBC, several important diseases in other fruits, such as mango, pomegranate, and grape, are also caused by Xanthomonas pathovars that display remarkable specificity toward their hosts. While citrus and mango diseases were documented more than 100 years ago, the pomegranate and grape diseases have been known only since the 1950s and 1970s, respectively. Interestingly, diseases caused by all these pathovars were noted first in India. Our genome-based phylogenetic studies suggest that these diverse pathogens belong to a single species and these pathovars may be just a group of rapidly evolving strains. Furthermore, the recently reported pathovars, such as those infecting grape and pomegranate, form independent clonal lineages, while the citrus and mango pathovars that have been known for a long time form one clonal lineage. Such an understanding of their phylogenomic relationship has further allowed us to understand major and unique variations in the lineages that give rise to these pathovars. Whole-genome sequencing studies including ecological relatives from their putative country of origin has allowed us to understand the evolutionary history of Xac and other pathovars that infect fruits.

The evolution of host specialization in the vertebrate gut symbiont Lactobacillus reuteri.

Recent research has provided mechanistic insight into the important contributions of the gut microbiota to vertebrate biology, but questions remain about the evolutionary processes that have shaped this symbiosis. In the present study, we showed in experiments with gnotobiotic mice that the evolution of Lactobacillus reuteri with rodents resulted in the emergence of host specialization. To identify genomic events marking adaptations to the murine host, we compared the genome of the rodent isolate L. reuteri 100-23 with that of the human isolate L. reuteri F275, and we identified hundreds of genes that were specific to each strain. In order to differentiate true host-specific genome content from strain-level differences, comparative genome hybridizations were performed to query 57 L. reuteri strains originating from six different vertebrate hosts in combination with genome sequence comparisons of nine strains encompassing five phylogenetic lineages of the species. This approach revealed that rodent strains, although showing a high degree of genomic plasticity, possessed a specific genome inventory that was rare or absent in strains from other vertebrate hosts. The distinct genome content of L. reuteri lineages reflected the niche characteristics in the gastrointestinal tracts of their respective hosts, and inactivation of seven out of eight representative rodent-specific genes in L. reuteri 100-23 resulted in impaired ecological performance in the gut of mice. The comparative genomic analyses suggested fundamentally different trends of genome evolution in rodent and human L. reuteri populations, with the former possessing a large and adaptable pan-genome while the latter being subjected to a process of reductive evolution. In conclusion, this study provided experimental evidence and a molecular basis for the evolution of host specificity in a vertebrate gut symbiont, and it identified genomic events that have shaped this process.