International Journal of Systematic and Evolutionary Microbiology

The objective of this study was to re-analyse the molecular phylogeny and/or the morphology of all species, which have been attributed to the so-far mono-generic fungal family Ambisporaceae. The genus Ambispora has been well-known for its spore bi-morphy described even from single spore clusters. Triple-walled spores are differentiated on sporiferous saccules, while mono-walled spores are formed on simple subtending hyphae. New phylogenetic analyses reveal dissimilarities of [≥]10% in partial nrDNA gene of three different stable phylogenetic clades and thus suggest the division of Ambispora into three genera, which simultaneously request for advanced morphological separations. These advances are primarily based on the more diverse spore wall composition of the ambisporoid-acaulosporoid morph rather than on the rather simple-glomoid morph. While all known species of the triple-walled morph have an evanescent to semi-permanent outer spore wall, i) Am. fennica, Am. brasiliensis, Am. gerdemannii and Am. nicolsonii have a smooth, permanent central spore wall (Am. fennica clade, A), ii) the central wall of Am. appendicula, Am. callosa, Am. leptoticha and Am. jimgerdemannii is alveolate (Am. appendicula clade, B), and iii) the central wall of Am. granatensis is smooth, but easily degraded, thus rather short-lived and not permanent but evanescent (Am. granatensis clade, C). In conclusion, species of the Am. fennica clade represent the genus Ambispora, while species of the Am. appendicula clade represent the new genus Appendiculaspora, and the mono-specific Am. granatensis clade represents the new genus Ephemerapareta. Species of an additional morph, with triple-walled spores, but apparently formed on subtending hyphae, and having a diagnostic reticulate, football-like middle wall, are here separated from the revised genus Ambispora based solely on morphological analyses, since molecular identification analyses so far failed and remained merely unknown. This later morph and genus is based on the type species Pelotaspora reticulata comb. nov, and on P. austrolatina sp. nov. Concomitant molecular phylogenetic and morphological analyses are needed to attribute not only Pelotaspora spp., but also those species, for which hitherto only the ambisporoid-glomoid morph has been observed correctly within the family Ambisporaceae. Without molecular analyses, such species with glomoid but unknown ambisporoid-acaulosporoid morph have to be retained within Ambispora.

Three novel bioluminescent bacterial strains, VNB-15T, VNB-16 and SChm4, were isolated from water of the Black Sea (Russia) and intestines of the Black Sea horse mackerel. Cells of the isolated strains are motile Gram negative slightly curved rods with single polar flagellum. The temperature range for growth was 10-35{degrees}C, the optimum being 20-25{degrees}C. The pH range for growth was 6.0-9.0, the optimum being 7.0-8.0. The bacteria were able to grow in the presence of 0.5 to 5.0% NaCl (w/v), the optimum being 1.0-4.0% (w/v). Phylogenetic analysis based on comparison of 16S rRNA sequences shows these strains to have kinship with the species Vibrio jasicida, Vibrio hyugaensis, Vibrio alginolyticus, Vibrio campbelli, Vibrio rotiferianus, Vibrio harveyi and Vibrio owensii with sequence similarity from 99.6 to 98.0%. Phylogenetic analysis based on comparison of the sequences of genes gyrB, recA, pyrH, gapA, rpoA, mreB, ftsZ, topA shows that the strains VNB-15T, VNB-16 and SChm4 to form a cluster within the V. harveyi clade and belong to a new species of the Vibrio genus. Comparison of the complete genomic sequence of VNB-15T with typical strains of nearby species also indicates that VNB-15T belongs to a separate species (maximum similarity 98% with V. hyugaensis and 96% with V. jasicida). VNB-15T differs from closely related species by its ability to utilize glucose, mannitol, inositol, sorbitol, rhamnose and sucrose, and to form lysine decarboxylase, ornithine decarboxylase, lipase, acid phosphatase, -glucosidase, {beta}-glucosidase and N-acetyl-{beta}-D-glucosaminidase enzymes. Based on phylogenetic analysis and phenotypic characteristics, Vibrio aquamarinus sp. nov. is proposed. The type strain is VNB-15T (= VKPM B-11245T = DSM 26054 T). RepositoriesThe GenBank accession numbers for the gapA, 16S rRNA, gyrB, pyrH, rpoA, recA, mreB, ftsZ, topA genes sequences of strain VNB-15T are JQ319116-JQ319121, KX242381, KX 242384, KX242387, respectively. The GenBank accession numbers for the gapA, gyrB, pyrH, recA, rpoA,16S rRNA, mreB, ftsZ, topA genes sequences of strain VNB-16 are KP221561-KP221566, KX242382, KX 242385, KX242388 respectively. The GenBank accession numbers for the 16S rRNA, gapA gyrB, rpoA, recA, pyrH, mreB, ftsZ, topA genes sequences of strain SChm4 are KX242375-KX242380, KX242383, KX 242386, KX242389, respectively. The GenBank/EMBL/DDBJ accession numbers for the housekeeping gene sequences used in this study are detailed in supplementary Table S1, Figures S1-S8. The genome of Vibrio aquamarinus sp. nov. VNB-15T, comprising two chromosomes and a plasmid, has been assembled and deposited in the NCBI database under the submission number SUB14585067

Non-pathogenic Xanthomonas (NPX) from a diverse plant hosts are being reported on an increasing basis. There are also reports of multiple species forming communities on a single host plant, such as rice, and, given their role as core endophytes in protecting plants from pathogens, it is essential to isolate and characterization of more NPX species from diverse host plants. Using phylogenomic analysis of publicly available Xanthomonas genome sequences, we identified a novel clade comprising NPX strains from diverse hosts. One of the strains previously reported from our lab is from healthy rice seeds and was reported to be non-pathogenic, with bio-protection function against the bacterial leaf blight pathogen. Genomic investigation confirmed the lack of type III secretion system and its effectors, consistent with their non-pathogenic nature. These strains also harbour core and unique biosynthetic loci identified in other non-pathogenic Xanthomonas (NPX) strains. Further investigation using multiple genomic-based taxonomic indices indicates that these strains represent a potential new species. Hence, we propose Xanthomonas imtechensis sp. nov. as a new species of the genus Xanthomonas, with the type strain being PPL568 = MTCC 13186 = CFBP 9040 = ICMP 24395.

The genus Gardnerella comprises a group of fastidious bacteria associated with the female urogenital tract and has undergone extensive taxonomic revision in recent years. In this study, a bacterial strain, designated CCPDSM, was isolated from the female urinary microbiome and subjected to a comprehensive polyphasic taxonomic characterization. The 16S rRNA gene sequence confirmed that this strain is a member of the genus Gardnerella, and phylogenetic analyses based on cpn60 sequences, together with phylogenomic reconstruction placed strain CCPDSM within the genus Gardnerella as a distinct and well-supported lineage. Genome-based relatedness indices (ANIb, ANIm, TETRA and dDDH), demonstrated clear separation of CCPDSM from all validly published Gardnerella species. In contrast, comparisons with two publicly available closely related genomes yielded values above accepted species delineation thresholds, supporting their assignment to the same taxon. Phenotypic characterization, together with genome-based functional predictions, revealed a fastidious, fermentative metabolic profile that further differentiated CCPDSM from its closest relatives, while remaining consistent with traits characteristic of the genus. On the basis of combined phylogenetic, genomic and phenotypic evidence, strain CCPDSM is proposed as representing a novel species within the genus Gardnerella, for which the name Gardnerella fastidiominuta sp. nov. is proposed, with strain CCPDSM (=CECT 31324=CCP 588) designated as the type strain. This study expands the recognized diversity of Gardnerella and highlights the female urinary tract as a reservoir of previously uncharacterized species within this genus.

Currently, the fungal class Archaeosporomycetes consists of one order, Archaeosporales with four families: Archaeosporaceae, Ambisporaceae, Geosiphonaceae, and Polonosporaceae. In the present study, the objective was to re-analyze the phylogeny and morphology of the Archaeosporomycetes from order to genus level. The different ecological strategies and, consequently, distinct evolutionary patterns of these taxa, as well as their morphological characters and other data updated here, suggest the need to divide Archaeosporales into four orders: (i) the type order Archaeosporales, (ii) Ambisporales ord. nov., both with four genera, (iii) Geosiphonales and (iv) Polonosporales ord. nov., both with single families and genera. Remarkably, the order Geosiphonales was described in the past, but was not considered in the Archaeosporomycetes until now. Phylogenetically, the four main clades (orders here proposed) of Archaeosporomycetes are well supported, with bootstrap values higher than 95% in all analyses, except Ambisporales/Ambisporaceae for RAxML-NG FBP analysis in the SSU tree (75%). Ecologically, this class includes three orders of arbuscular mycorrhizal fungi (AMF) forming symbiotic associations with plants, while Geosiphonales form an endocytobiosis with the cyanobacterium Nostoc. Morphologically, there are at least two AMF orders with spore bimorphism, which has not (yet) been described for Polonosporales. The only known species of Polonosporales, Polonospora polonica, forms spores directly on the neck of sporiferous saccules and the spores can morphologically be differentiated from all other taxa in Archaeosporomycetes by the formation of three permanent, rather thick spore walls, of which two form de novo during spore formation. The outer spore wall of Archaeosporales and Ambisporales are semi-permanent, evanescent or even short-lived, or show multiple fissures during aging, when it is more resistant. Ambisporales can easily be differentiated from Archaeosporales for instance by larger spores of the acaulosporoid morph and thicker spore walls. Our phylogenetic analyses suggested that Archaeosporales can be divided into two families: Antiquisporaceae that was described to form intraradical hyphae, vesicles and spores, staining darkly in Trypan blue, and Archaeosporaceae whose hyphae generally do not or only faintly stain in this reagent, and vesicles and intraradical spores have been rarely, if ever reported.

ASS1 was isolated as a motile Stenotrophomonas strain from crude oil-contaminated soils in Tabasco, Mexico. We characterized this strain using physiological and biochemical traits. ASS1 grew at temperature 25-37 (optimally at 37 {degrees} C) and at pH 6 to 8 (optimally at pH 7 to 8). The assembled genome has a total length of 4.56MB with a G + C content of 66.6%. The 16S rRNA gene sequence analysis confirmed that this strain belongs to the genus Stenotrophomonas. Based on the 16S rRNA analysis, Stenotrophomonas geniculata ATCC 19374 is the closest species, and it shares 99.86% similarity with ASS1. Similarly, a phylogenomic tree based on core genome sequence revealed that the closest species to ASS1 is Stenotrophomonas geniculata ATCC 19374. The major fatty acids in ASS1 are C16:0, antesio C15:0, iso C12:0, iso C15:0, iso C17:0 and C18:0. The genome of ASS1 consists of 4,373,402 bp. The Average Nucleotide Identity (ANI) values for ASS1 which it shared with its closest phylogenetic neighbors, are Stenotrophomonas geniculata ATCC 19374 = JCM 13324 [T] 92.66 %, Stenotrophomonas maltophilia 13637[T] 92.15%, Stenotrophomonas maltophilia K279a 92.13% Stenotrophomonas maltophilia R551-3 92.15% Stenotrophomonas maltophilia MTCC 434 [T] 92.08% and Pseudomonas hibisicicola ATCC [T] 91.66%. ASS1 possesses genes that are essential for the degradation of Polycyclic Aromatic hydrocarbon. Genes such as 1, 2 dihydroxyl 1, 2 dihydronaphthalene dehydrogenase; MG068 17425, homologous to 2 hydroxyl chromene 2 carboxylate isomerases; MG 18055, homologous to salicylaldehyde dehydrogenase and MG068 20095, homologous to naphthalene 1, 2 dioxygenases were identified in ASS1. The dDDH value between ASS1 and its closest neighbor Stenotrophomonas geniculata ATCC 19374 = JCM 13324 [T] is 50%, which is the highest for all the typed species and as such we proposed that ASS1 is a novel species with the name Stenotrophomonas oleivorans sp. nov. sp. nov. and ASS1T as the typed strain

The fastidious basidiomycete Rhizoctonia (Ceratobasidium) theobromae is a biotrophic pathogen that causes Vascular-Streak Dieback (VSD) of Theobroma cacao (cocoa). The fungus has also been identified as the cause of an emergent disease known as Cassava Witches Broom Disease (CWBD) raising concerns that the pathogen is spreading to alternative hosts and to new regions. Interestingly, while VSD of cocoa and CWBD are reported as co-present in several countries, there is currently no evidence for cross-infection between species. The fungus is difficult to culture in vitro due its slow growth and Kochs postulates have not been definitive on either host. The complete fungus life cycle therefore remains enigmatic, though studies have progressed knowledge on pathology within the both the cocoa and cassava hosts. We have conducted limited field trials and sequenced mating (MAT) and ITS loci of isolates from various infected hosts and regions. We hypothesize that (i) genetic variation at MAT loci correlates with region or host (ii) long amplicon ITS sequences between isolates are more definitive for polymorphisms (iii) life-cycle traits of R. theobromae may be inferred from MAT loci (iv) cassava grown under VSD infected cocoa will be infected and develop symptoms of CWBD. We did not find any cross-infection in field trials, and we show that the pathogen is highly homozygous, despite undergoing meiosis, indicating a predominantly homothallic life cycle. Our data indicate that the pathogen is likely host specific and regionally divergent and suggests that host specificity on cocoa and cassava evolved by selection from a common ancestor rather than a host jump.

The strain LEGE 10371, isolated from the surface of a marine sponge at Praia da Memoria, Portugal, was characterized as a new Thalassoporum species (Pseudanabaenales) using a polyphasic approach that included 16S rRNA gene phylogenetic analysis (Maximum Likelihood and Bayesian Inference), 16S-23S ITS secondary structures, p-distance calculations, MALDI-TOF MS profiling, and morphological analysis by optical and scanning electron microscopy, as well as ecological and biochemical characterization. Phylogenetically, LEGE 10371 clustered within the Thalassoporum clade, however distant from the other existent species of the genus. The p-distance analysis revealed low sequence identity with other Thalassoporum species, with a maximum value of 97.2% to Th. komareki. The MALDI-TOF profile displayed high-intensity peaks at approximately 3,000, 4,000, 6,000 and 8,000 m/z, representing strong candidates for diagnostic markers of the new species. Morphologically, the new species differ from the other species of the genus by presenting trichomes with more than 10 cells and lack of aerotopes. Biocompatibility of the fractions was evaluated in HaCaT keratinocytes, showing no cytotoxic effects at most tested concentrations. PCR screening targeting mcyE, sxtG, anaC, and cyrA confirmed the absence of the genetic potential for the production of major cyanotoxins. Chemical characterization revealed a pigment-rich profile dominated by chlorophyll-a and carotenoids, including {beta}-carotene, zeaxanthin, lutein, and mixoxanthophyll. Bioactivity assays showed superoxide anion radical scavenging by the aqueous fraction (IC2 {approx} 0.042-0.045 mg mL-{superscript 1}), strong nitric oxide radical scavenging by the acetonic fraction (IC = 0.045 mg mL-{superscript 1}), and lipoxygenase inhibition ([~]41%, for a fraction concentration of 0.25 mg mL-), suggesting a potential contribution of these fractions to modulate inflammation-related pathways. Additionally to this results, the polyphasic analysis permitted to confirm previous data that Pseudanabaena and Limnothrix represent the same generic entity. Both genera clustered together, presented high 16S rRNA gene identity (up to 99.9%) and share the same morphological and ecological features. Consequently, we formally proposed the synonimization of Limnothrix into Pseudanabaena.

Diversisporales comprises species with worldwide distribution that produce glomoid, otosporoid, or tricisporoid spores. The recent reorganization of the order by Oehl et al. (2016) recognizes two families, Diversisporaceae and Corymbiglomeraceae, comprising one and five genera, respectively. Several Glomeromycotan specimens collected in northern and southeastern Mexico and in French Polynesian atolls were characterized using both morphological and molecular analyses. Phylogenetic inference revealed that they represent new members in the Diversisporales, supporting the reorganization of the genus Redeckera into three independent lineages: Albocarpum gen. nov., with A. arenaceum sp. nov., A. leptohyphum sp. nov., and A. fulvum comb. nov., Pulvinocarpum pulvinatum gen. et comb. nov., and Redeckera, which retains five species, including R. varelae sp. nov. In addition, we described Melanocarpum mexicanum gen. et sp. nov. and Diversispora papillosa sp. nov. A broader phylogeny, based on eDNA sequences and representative of Diversisporales species, including the newly described taxa, further supported the split of Redeckera and suggested three additional clades likely corresponding to a new family and two new genera, awaiting the discovery of representative morphospecies to be formally described. Using eDNA sequences metadata, the occurrences of the newly described taxa were mapped, allowing to recognize distribution patterns, mostly in the pantropical zone, distinguish widespread and rare species, and suggest possible endemisms. Finally, the coexistence of species forming large sporocarps (A. fulvum and A. leptohyphum) alongside species forming spores in loose aggregates (A. arenaceum), prompted us to propose a possible sporulation dimorphism in Albocarpum, an argument previously raised to explain the nested placement of Corymbiglomus and Paracorymbiglomus within the Redeckera clade.

The genus Exiguobacterium comprises Gram-positive, non-spore-forming, facultative anaerobic bacteria known for their remarkable adaptability to extreme environments, including soils, hot springs, glaciers, and the gastrointestinal tracts of certain organisms. Despite their unique adaptations for surviving in extreme environments, their pathogenicity is well documented. Here, we analyzed the phenotypical traits of two Mexican strains of Exiguobacterium--JVH47, isolated from contaminated urban sediments in Mexico City, and P4526, from the less human-impacted Cuatro Cienegas Basin. Furthermore, strains were related via comparative genomics using publicly available genomes. Phenotypic characterization demonstrated that both strains thrive across a wide range of temperatures (20-50 {degrees}C), pH (7-11), and salinity (up to 7% NaCl). Although sensitive to erythromycin, the JVH47 strain exhibited higher erythromycin resistance and harbored antibiotic resistance genes. This study underscores the ecological versatility of Exiguobacterium and its potential role as a reservoir for antibiotic resistance genes. While rarely associated with human infections, its ability to survive in extreme conditions and form biofilms raises concerns for immunocompromised individuals. These findings highlight the need for careful consideration of Exiguobacterium in biotechnological applications and its implications under the One Health framework.

The Bacillus genus comprises physiologically versatile, endospore-forming bacteria widely distributed in natural environments. In this study, we report the isolation and genomic characterization of strain Bva_UNVM-123, recovered from agricultural soil in Pergamino, Argentina. Whole-genome sequencing using Illumina technology yielded a 5.1 Mbp draft genome assembled in 67 contigs with a GC content of 36%. Comparative genomic analyses using the TYGS server and digital DNADNA hybridization (dDDH) values supported its classification as a potentially novel species within the Bacillus sensu lato (s.l.) group. Genome annotation revealed 4,866 protein-coding genes, including multiple determinants conferring resistance to antibiotics (e.g., fosfomycin, tetracycline, beta-lactams) and toxic heavy metals (e.g., arsenic, cadmium, mercury), supporting its potential application in bioremediation. Additionally, PathogenFinder predicted a low probability of human pathogenicity (0.207), reinforcing its safety for environmental use. Functional classification based on Swiss-Prot further supported a metabolically versatile profile and revealed the presence of resistance-related categories associated with environmental adaptation. This study adds to the growing knowledge of environmental Bacillus species and their biotechnological potential

Currently, there are three recognized rhizobial genera belonging to the beta branch of the proteobacteria; Trinickia, Paraburkholderia, and Cupriavidus. These beta-rhizobia have been found associated with legume species mainly within the Mimosoideae and Papillonoideae. Most diversity, evolutionary, and functional studies have focused on Paraburkholderia, whereas few have addressed the diversity and evolution of symbiosis in the Cupriavidus genus. The present work aimed to provide an actual view of the symbiotic Cupriavidus diversity and to analyse the origin and evolution of their symbiotic genes. Using whole-genome information for phylogenetic reconstruction, we showed that the described symbiotic Cupriavidus strains belong to five distinct lineages, although they are intermixed with non-symbiotic species. The high synteny and sequence conservation of symbiotic genes suggest a common origin of acquisition for all rhizobial Cupriavidus described so far. However, we observed very low sequence conservation among (mega)plasmids carrying the symbiotic island, excluding the existence of a conserved symbiotic plasmid within beta-rhizobia. We can conclude that up to now there are five rhizobial species within the Cupriavidus genus, and we predict the description of new symbiotic species in the near future.

Aeromonas taxonomy has long been complicated by overlapping phenotypic, biochemical, and protein profiles. Here, we establish a robust genome-based framework for Aeromonas species delineation. We analyzed genome-wide average nucleotide identity (ANI) across 3,782 genomes representing the 33 currently recognized Aeromonas species and identified 95.2% ANI as the species boundary. This threshold defined 34 ANI-based species, comprising 30 corresponding to existing Aeromonas species and four potential novel species, while three existing species were merged into closely related species. A genus-level core genome of 673 loci was defined using 392 representative genomes, and core-genome phylogenetic analysis of 1,505 genomes showed consistency with ANI-based clustering. Digital DNA-DNA hybridization (dDDH) values between clusters were [≤] 55%, further supporting species separation. We also found that nine Aeromonas species had intraspecies dDDH values below 70%, cautioning the use of this cutoff for species delineation. We provide the tool Aeromonas ANI species typer (AeromonasANIStyper), an ANI-based species typing tool that assigns query genomes using ANI similarity to medoid genomes and achieved 100% accuracy. This framework supports standardized genome-based Aeromonas taxonomy and future surveillance. ImportanceAeromonas species have gained increased attention as emerging human enteric pathogens. Aeromonas taxonomy has long been complicated by overlapping phenotypic, biochemical, and protein profiles. Although an average nucleotide identity (ANI) threshold was proposed previously for the Aeromonas species delineation, the growing number and diversity of genomes requires a more rigorous reassessment of species boundaries. By analyzing 3,782 Aeromonas genomes, we identified 95.2 % ANI as an optimal cutoff for defining Aeromonas species. Using this threshold, we delineated 34 ANI-defined species, which were further validated by core genome phylogeny and digital DNA-DNA hybridization (dDDH). In addition, we clarified previously misidentified species. Importantly, we provide the Aeromonas ANI species typer (AeromonasANIStyper), a tool that accurately assigns Aeromonas genomes to ANI-defined species. These findings clarify Aeromonas species identification and support improved pathogen surveillance, evolutionary and epidemiological studies.

ImportanceMolecular confirmation of Listeria monocytogenes typically employs a multiplex PCR method that targets both genus-specific prs and species-specific hlyA genes. This study assessed the specificity of this assay within Nigeria, where local microbial diversity may influence performance outcomes. MethodsOut of eight phenotypically presumptive L. monocytogenes food isolates tested, six produced the expected prs and hlyA amplicons, with five classified as serogroup 1/2b. However, all six isolates tested negative for the crucial virulence regulator prfA, necessitating further investigation. ResultsDefinitive 16S rRNA gene sequencing revealed that only two of the six PCR-positive isolates were identified as L. monocytogenes, while the remaining four were identified as Enterococcus faecium. This results in a false-positive rate of 66.7% (4/6) for the assay in this particular context. Phylogenetic analysis corroborated the taxonomic distinction, exhibiting a robust clustering of the four E. faecium isolates with reference strains. In contrast, the two confirmed L. monocytogenes isolates formed a separate sub-clade, indicating regional divergence and further underscoring the assays inability to differentiate between L. monocytogenes and Enterococcus species. ConclusionThese findings highlight a significant lack of specificity, as the prs/hlyA primers exhibited cross-reactivity with non-target E. faecium. The anomalous negative result for prfA served as a critical diagnostic indicator. Consequently, the positive outcomes from this widely utilized confirmatory assay should be regarded as presumptive and necessitate additional verification.

Summer squash (Cucurbita pepo) is a popular vegetable in Mississippi. These are harvested during the tender and immature stages. This vegetable is known to be a good source of vitamins A and C, as well as potassium, and iron. Small farms typically sell summer squash directly to consumers through local farmers markets. In this study, we isolated and identified a soft rot causing bacteria, Pectobacterium brasiliense strain 25ASUB12 (GenBank: PX884501), from symptomatic fruit of field-grown summer squash in Mississippi. We deployed both phenotypic and molecular techniques to identify this important pathogen which has a wide host range, including cucumbers, potatoes, and tomatoes.

Amino acid catabolism is a vital metabolic process in bacteria, providing energy, carbon and potentially nitrogen as resources, and affecting global cycles of these elements. The ability of a bacterium to catabolize an amino acid is often inferred from the presence of the relevant catabolic pathways in its genome, yet the "gene=function" inference is not straightforward. Here, we use growth assays in 96 well plates on individual amino acids and their combinations to directly measure the ability of a model marine bacterium, Alteromonas macleodii ATCC 27126, to utilize these resources for growth. With the exception of aspartate and glutamate, which did not support growth in any of our experiments, ATCC 27126 grew on all other amino acids. However, the probability of growth, together with growth yield and rate, differed depending on the entry point of the catabolic pathway to central carbon metabolism, with robust growth occurring only on amino acids catabolized into pyruvate or acetyl CoA. Growth on combinations of two amino acids revealed reproducible patterns, the clearest being inhibition of growth on other amino acids by asparagine, aspartate and their degradation product, oxaloacetate. Finally, growth was different in test tubes compared with 96 well plates. Our results reveal hidden complexity in amino acid utilization and suggest a "TCA-centric" viewpoint for amino acid utilization, perhaps reflecting the high metabolic flexibility of pyruvate and specific regulatory aspects of the TCA cycle in Alteromonas.

AbstractEmergomyces africanus is a thermally dimorphic fungal pathogen endemic to Southern Africa which can cause fatal systemic infections in persons with advanced HIV disease. Its mechanisms of pathogenesis are not well understood. Characterisation of virulence traits in this pathogen requires appropriate molecular tools for genetic manipulation. Molecular technologies developed for the transformation of H. capsulatum were adapted for use in E. africanus. Agrobacterium-mediated transformation was used to generate a reporter strain expressing green fluorescent protein (GFP). The E. africanus GFP reporter strain facilitated the study of yeast interaction with macrophages in vitro and allowed the identification of infected phagocyte cell types in the mouse lung by flow cytometry. E. africanus could also maintain episomal plasmids with telomere-like sequences, to introduce expression constructs without genome modification. Using this plasmid system, RNA interference constructs were used to knock down the expression of cell wall (1,3)-glucan by targeting the transcripts of the -glucan synthase (AGS1). An episomal CRISPR/Cas9 system was evaluated for E. africanus, which effectively disrupted GFP in a reporter strain and enabled the generation of a URA5 uracil auxotroph. These tools and strains will facilitate future studies to elucidate the mechanisms of pathogenesis of E. africanus. ImportanceEmergomyces africanus is an opportunistic fungal pathogen affecting persons with advanced HIV disease in South Africa. The biology and pathogenesis of E. africanus are not well understood, as the importance of the disease caused by this fungus (emergomycosis) has only been recognised in recent years and molecular studies have been impaired by the lack of genetic technologies. In this work, we describe tools and methods for the genetic modification of this pathogen, which will accelerate future studies investigating how the fungus causes disease in the human host. These essential tools include (1) the ability to create fluorescent reporter strains, such as the green fluorescent protein E. africanus strain described here, which facilitates tracking the spread of the fungus during infection and enhances microscopy studies, (2) methods for knocking down gene expression in E. africanus, and (3) the permanent disruption of genes through CRISPR/Cas9 gene editing.

Despite the successful cultivation of many microbes from rich bacterial communities inhabiting alkaline soda lakes, members of the bacterial phylum Verrucomicrobiota have so far been detected only through metagenomics. Here, we used alginate as a selective substrate to enrich and isolate two strains of haloalkaliphilic Verrucomicrobiota. The isolates share identical 16S rRNA gene sequences representing a new genus lineage, and, together with other metagenome assembled genomes, a new family within Opitutales. Cells of strains AB-alg1T (from soda lakes) and AB-alg4 (from soda solonchak soils) are small and motile cocci forming submerged colonies in soft alginate agar. They are saccharolytic heterotrophs growing aerobically on polysaccharides (alginate, starch and inulin) and sugars (glucose, fructose, mannose, sucrose, melezitose, maltose and cellobiose). They also grow anaerobically by fermentation of alginate and D-mannose and by coupling incomplete denitrification to oxidation of alginate. Both isolates are obligately alkaliphilic and moderately salt-tolerant. The dominant membrane phospholipids include phosphatidylcholines and diphosphatidylglycerols (cardiolipins). The genome of AB-alg1T features polysaccharide lyases of the PL6, 7, 15, 17, 38, and 39 families for depolymerization of alginate. Based on distinct phenotype and phylogeny, we propose classification of strains AB-alg1T (JCM 35393T=UQM 41574T) and AB-alg4 as Verruconatronum alginivorum gen. nov., sp. nov. within a new family Verruconatronumaceae. ImportanceThe presented isolates are the first isolated representatives of an environmental family of Opitutales, part of the core microbiome of alkaline soda lakes. These bacteria feed on polysaccharides. We present the key enzymatic machinery for the polysaccharide breakdown. These enzymes are high-pH tolerant and have potential for industry applications, for example in washing powders and biomass waste recycling.

Carbapenem-resistant (CR) bacteria have emerged and been spreading beyond healthcare-associated facilities into the environment. It is recognized that toilet bowl water in patient rooms of healthcare-associated facilities can be one of internal reservoirs of CR bacteria. In accordance with this idea, toilet bowl water samples were collected from patient rooms in a tertiary healthcare-associated facility in North Macedonia, and meropenem (MEM)-resistant bacterial isolates were obtained from the toilet bowl water. In this study, because a MEM-resistant C. braakii isolate, that was one of MEM-resistant opportunistic pathogens, was obtained from the toilet water, whole-genome sequencing (WGS) of this isolate was performed to obtain genetic characteristics of the blaNDM-1-positive C. braakii isolate. By the WGS, four contigs were constructed, the longest contig, contig 1 (5,189,681 bp), contained blaCTX-M with some additional antimicrobial-resistance genes (ARGs). Interestingly, blaNDM-1 was detected in contig 2 (177,260 bp) and contig 3 (64,168 bp). Plasmid replicon of contig 2 was IncA/C2 but plasmid replicon of contig 3 was IncN and different from one of contig 2. Genetic structures surrounding blaNDM-1 were different between these two blaNDM-1-positive plasmids implying transfer or insertion of blaNDM-1 had occurred by IS or other mechanism. Further molecular epidemiology will be needed to explain the mechanism that allowed the C. braakii isolate to possess two structurally different blaNDM-1 plasmids.

Leaf diseases pose a serious threat to faba bean production. Leaf blotch of faba bean, caused by Alternaria spp., has become increasingly widespread and destructive in several countries. Leaf diseases pose a serious threat to faba bean production. The infection of plant by pathogens can be influenced by various factors associated with the host plant, environmental conditions and presence of other microorganisms. The phyllosphere and endosphere play a critical role in plant health and disease development. This study aimed to evaluate the factors shaping the structure and diversity of fungal communities associated with faba beans. Plant samples were collected in 2004 from two intensively managed faba bean production fields in the central region of Latvia. Fungal assemblages were characterized using an ITS region metabarcoding approach based on Illumina MiSeq sequencing. Among the assigned amplicon sequence variant (AVS), 65% belonged to the phylum Ascomycota, while approximately 4% were classified as Basidiomycota. Alternaria and Cladosporium were the dominant genera across samples. The alfa and beta diversities of fungal communities was higher during flowering of faba beans to compare with ripening. The higher abundance of Basidiomycota yeasts were observed during flowering, in contrast, Cladosporium genus was significantly more abundant during ripening. Alternaria DNA was found on leaves that showed no symptoms of the disease. The diversity and composition of fungal communities were significantly influenced by sampling time and presence of leaf blotch, caused by Alternaria spp.