Microbiological Research

Metacaspases are cysteine proteases and are found in every group of organisms except animals. They are structural orthologs of caspases, which orchestrate apoptosis in animals. Caspases, metacaspases, paracaspases and orthocaspases are class C14 proteases with a similar active site (the hemoglobinase fold) and a catalytic cysteine residue. However, metacaspases cleave after arginine and lysine instead of aspartate and some researchers point out that a name ending with caspase is unsuitable. Also, most metacaspases are activated by calcium, they do not target the same proteins as caspases and they cut multiply instead of once. The Candida albicans metacaspase Mca1p mediates cell death in response to various stresses but also has pro-life functions. Many plant and protist metacaspases lack cell death roles and are involved in development, differentiation and immunity. This article highlights an interesting phenomenon, whereby the promotion of stress sensitivity and reduction of virulence, by Mca1p in C. albicans cells from exponential culture are abolished and reversed, respectively in cells from stationary-phase culture. This hints at the proteases's dual nature and at the conditions that might initiate a switch between pro-life and pro-death functions.

Bacteria utilize specialized protein secretion systems, particularly the type VI secretion system (T6SS), to dominate interbacterial competitions and kill the prey bacteria in a contact-dependent manner. Here, we report that disruption of the thioesterase gene encoded in the gramibactin (a bacterial siderophore) biosynthetic gene cluster activates the T6SS in the Burkholderia gladioli strain NGJ1, even in the absence of prey. The thioesterase mutant exhibits enhanced antibacterial activity, and ectopic expression of the gene complements the phenotype. Notably, thio mutant as well as thioesterase-complement strains show enhanced sensitivity to oxidative stress. The qRT-PCR analysis showed that the thio mutant had upregulated expression of RpoN1, a sigma factor, which positively regulates T6SS functions. We demonstrate that rpoN1 mutant is hypersensitive to oxidative stress and defective in T6SS activation, whereas overexpression of RpoN1 enhances oxidative stress tolerance in NGJ1 and activates T6SS. We propose that, during interbacterial conflicts encountered during plant colonization, NGJ1 faces an oxidative-stress-enriched environment that modulates thioesterase expression, induces RpoN1, and activates T6SS. Overall, our study exemplifies the broader physiological role of Thioesterase in maintaining intracellular ROS homeostasis in bacteria and stimulating antibacterial activity.

Extremophilic fungi are a relatively unexplored source of stress-resilient microbes for sustainable agriculture. Here, we describe Talaromyces pseudofuniculosus FAR, isolated from the highly acidic site of Piccolo Inferno in southern Italy and identified by ITS and {beta}-tubulin (BenA) sequencing. Despite its origin from an extreme environment, T. pseudofuniculosus FRA remained fully functional under non-stress conditions and displayed tolerance to salinity and heavy metals while maintaining key plant growth-promoting traits, including phosphate solubilization, auxin-related compound production and hydrolytic activities. In soybean (Glycine max L.), the live fungus promoted plant growth under both non-stress conditions and agriculturally relevant salinity and copper stress, and successfully colonized the roots. A cell-free extracellular extract (exCFS) promoted early seedling development in vitro, but its effects were not sustained in pot experiments. GC-MS analysis provided a qualitative characterization of the extracellular metabolite profile and revealed condition-dependent differences between control, NaCl and CuSO4; growth conditions, indicating environment-dependent modulation of fungal metabolism. Overall, these findings highlight the physiological and metabolic plasticity of T. pseudofuniculosus FRA and support its potential as a fungal biostimulant for saline- and metal-impacted agricultural soils.

Pectobacterium, a member of the soft rot Pectobacteriaceae (SRP), can cause highly destructive soft rot and blackleg diseases on many important crop and other host plants, particularly potato (Solanum tuberosum). Pectobacterium and its sister genus Dickeya have common virulence strategies, yet a highly diverse "open" pangenome containing many unique and hypothetical proteins. Here, we used randomly-barcoded transposon insertion-site sequencing (RB-TnSeq) on six Pectobacterium species to identify genes involved in bacterial survival and growth in potato tubers, as well as in minimal media conditions containing different single carbon sources. The strains evaluated varied broadly in genomic content as well as host plant and year of isolation. We focused on identifying conditionally essential genes across all strains tested, as well as genes that were important for growth in one or few of the tested strains. These results highlight the importance of many common genes involved in metabolism and maintaining cellular homeostasis, as well as shared and rare virulence traits including polygalacturonate catabolism, genes involved in polysaccharide biosynthesis, immunity proteins, and (for P. brasiliense) self-tolerance of the antibiotic carbapenem. This large dataset of conditionally essential genes, generated across Pectobacterium species during soft rot infection of the agriculturally important plant host potato, can serve as a rich dataset for further hypothesis generation and identification of important and/or novel in planta fitness factors.

The human gut microbiota has been explored for the last two decades and studies have uncovered its major role in the degradation of complex glycans from the diet as well as the host tissues. Strikingly, until very recently, no similar investigation has been carried out on the human oral cavity microbiota, which is increasingly recognized has an important parameter in gastrointestinal health. Prevotellaceae are an important and under-investigated family of bacteria in both the gut and the oral cavity microbiota. The oral cavity is particularly rich in glycosaminoglycans which could provide an abundant carbon source for the inhabiting microorganisms, and play a significant role in host-pathogen interactions. Hence, we analyzed Polysaccharide Utilization Loci in oral microbiota species and we identified a glycosaminoglycan-degrading Polysaccharide Utilization Locus in Segatella oris. We produced and characterized the four enzymes, confirmed their ability to degrade glycosaminoglycans, and we focused our efforts on the biochemical and structural characterization of the first reported Prevotellaceae endosulfatase.

Four rhizobial strains isolated from root nodules of tropical leguminous plants in Costa Rica were characterized. Strain LEGMi-135b was isolated from Lonchocarpus felipei root nodules, LEGMi-12c from Cojoba arborea, LEGMi-198b from Calliandra haematocephala and Rhizobium altiplani LEGMi-166a from Mimosa pigra. Phylogenetic analyses based on the 16S rRNA gene and four concatenated housekeeping genes (recA, atpD, GATase and gyrB) placed these strains within the genera Martinezella (LEGMi-12c, LEGMi-135b, and LEGMi-198b) and Rhizobium (LEGMi-166a). Genome-based analyses showed that strain LEGMi-135b exhibited an average nucleotide identity (ANI) value of 94% and a digital DNA-DNA hybridization (dDDH) value of 53.2% compared with its closest related species, Martinezella hainanensis CCBAU 57015T, supporting its classification as a distinct species. LEGMI-12c exhibited ANI and dDDH values of 98% and 87%, respectively, compared with Martinezella hainanensis CCBAU 57015T. LEGMi-198b showed the highest similarity to Rhizobium cerradonense CNPSo 3464, with ANI and dDDH values of 98% and 81.4%, respectively; however, this taxon has not been validly published. LEGMi-166a exhibited ANI and dDDH values of 100% and 99.3%, respectively, compared with Rhizobium altiplani BR 10423, confirming its assignment to this species. Additionally, differentiation of strains LEGMi-135b and LEGMi-198b from their closest phylogenetic relatives was supported by phenotypic, physiological, and biochemical analyses. Based on the combined genomic, phenotypic, and biochemical evidence, we propose the establishment of the novel rhizobial species Martinezella legmii sp. nov., with strain LEGMi-135b designated as the type strain (LEGMi-135b), and Martinezella ivanii sp. nov., with strain LEGMi-198b designated as the type strain (LEGMi-198b). This study expands our understanding of rhizobial diversity associated with nitrogen-fixing symbioses, particularly among tropical leguminous plants from Costa Rica

Ugba, a traditional alkaline-fermented condiment produced from African oil bean seeds (Pentaclethra macrophylla Benth.), remains poorly characterised at genome resolution. This study characterised a single spontaneous ugba fermentation using genome-resolved shotgun metagenomics across Early (0-24 h), Mid (48-96 h), and Late (120-144 h) composite stages. Thirty metagenome-assembled genomes (MAGs) were recovered and grouped according to their temporal abundance patterns. The Early-stage retained MAG population was dominated by Bacteroidota-associated MAGs, which exhibited the highest carbohydrate-active enzyme density and broad glycoside hydrolase repertoires, consistent with substantial genome-encoded potential for utilisation of the plant-derived seed matrix. The Mid stage showed marked restructuring of MAG composition, while Core and Late-associated MAGs increased towards fermentation maturation. Functional differentiation was also evident among Corynebacterium MAGs: C. nuruki encoded a complete urease system, whereas C. phoceense_A encoded glutamate dehydrogenase and nitrate-reduction genes. These stage-associated changes coincided with progressive alkalinisation from pH 7.15 to 8.01. Together, the results show that temporal restructuring of reconstructed microbial populations was accompanied by differentiation in genome-encoded functional potential. This study provides a genome-resolved characterisation of microbial composition and functional potential during spontaneous ugba fermentation and expands current understanding of the microbial organisation of this traditional African fermented food.

During phage infection, phages reprogram their host to secure energy, building blocks for their genome, and proteins, as well as the transcription and translation machinery required to replicate their genome and produce phage particles. This also includes reprogramming the host transcriptome to modify host gene expression and align it with the needs of the phage during infection. In this study, we present a putative early-expressed protein, Gp204 of Mosigvirus AV110, renamed Host transcription modulator (Htm), that has a broad impact on the extended-spectrum beta-lactamase (ESBL)- associated E. coli transcriptome. Overexpression of Htm decreased the growth rate of ESBL-E. coli and caused DNA condensation. Htm is structurally similar to bacterial enhancer-binding proteins, binds ssDNA, and alters the expression levels of over 700 E. coli genes when expressed. The majority of these genes were upregulated and are involved in multiple pathways, such as galactose metabolism and pentose and glucuronate interconversions, which may promote the synthesis of purines, pyrimidines, and histidine, all of which are required for the replication, transcription, and translation of the phage genome. Furthermore, energy production is likely to be upregulated by increased availability of acetyl-CoA, resulting from, among other factors, fatty acid degradation. Htm also downregulated the expression of the structural genes involved in flagellar assembly and multiple two-component systems. In conclusion, Htm could promote phage progeny production by increasing the availability of nucleotides, amino acids, and energy, while inhibiting non-essential processes, such as flagella formation.

Background Group B Streptococcus (GBS) is a leading cause of bacterial sepsis globally. Late-onset (LO) GBS disease is preceded by colonization within the gastrointestinal tract in vulnerable neonates. Previous studies demonstrate that Candida albicans (C. albicans) promotes GBS colonization of host tissues including the bladder and vagina. However, its impact on GBS colonization of the intestinal tract is unknown. Methods We established a new animal model to study GBS and C. albicans co-colonization model in the context of the newborn host to examine C. albicans impact on GBS and further evaluated fluconazole prophylactic treatment to these same outcomes. Physical interactions between organisms in vivo were imaged and GBS gene expression was evaluated. Results C. albicans increased GBS burden in the neonatal gut and GBS was physically colocalized with C. albicans hyphae as evidenced by the colocalization analysis. Fluconazole treatment of co-colonized mice decreased extraintestinal GBS dissemination. C. albicans presence upregulated genes encoding proteins responsible for GBS adhesion and invasion of intestinal epithelial cells. Conclusion C. albicans enhances GBS burden and pathogenicity in the neonatal gut. These findings highlight the need for further investigation into C. albicans as a potential risk factor for GBS colonization and LO disease in human infants.

Spontaneous vegetable fermentation is driven by the indigenous microbiota associated with the raw ingredients. The process often involves an initial heterofermentative phase followed by a predominantly homofermentative phase, both ensured by lactic acid bacteria (LAB). While microbial dynamics during fermentation are well characterized, phage communities remain poorly understood, despite evidence linking phage activity to LAB succession. We monitored viral and bacterial succession during sauerkraut and carrot fermentations using a metagenomics approach to characterize community dynamics. To further assess the impact of phage PLE001 on bacterial community dynamics, we developed a simplified carrot fermentation model comprising representative strains of Rahnella aquatilis, Leuconostoc mesenteroides and Lactiplantibacillus plantarum. Two consortia were assembled: one containing the PLE001-susceptible strain L. mesenteroides CIM3004, and the other containing also its phage-resistant derivative, CIM3004 BIM. We observed major shifts in the viral community during the first week of fermentation, coinciding with changes in bacterial structure during hetero- and homofermentative phases transition. In the simplified model, phage PLE001 reduced the population of the host during the heterofermentative phase but did not affect overall fermentation dynamics nor metabolite production. These results suggest that bacteriophages contribute to bacterial succession during fermentation, while communities remain resilient to phage infection, preserving fermentation stability.

A subgroup of membrane-anchored bacterial Alkaline Phosphatase superfamily members transfers phospholipid headgroups from phospholipids to diverse acceptor molecules. As part of type II and hybrid type I/II cellulose biosynthesis gene clusters BcsG transfers phosphorylethanolamine pEtN from phospholipid to the emerging 1,4 {beta}-D-glucan chain synthesized by the BcsABC cellulose biosynthesis nanomachine. BcsG homologs throughout the phylogenetic tree possess a uniquely structured tripartite linker that connects the fifth transmembrane domain with the periplasmic catalytic domain. A N-terminal unstructured sequence of highly variable length and biased amino acid composition, the N-linker, is followed by an -helix and an unstructured sequence of high similarity and constant length leading into the first {beta}-strand of the catalytic domain. With BcsG from Salmonella typhimurium synthesizing pEtN cellulose as a model, linkers were shown to be promiscuous and linker length positively correlated with transfer efficiency of the pEtN headgroup and protein stability. As the donor substrate, the phosphatidylethanolamine content of the membranes affects pEtN transfer efficiency. With Alkaline Phosphatase superfamily members to be promiscuous enzymes the catalytic domain of OpgE transfers pEtN to osmoregulated periplasmic glucan. BcsG-OpgE hybrid proteins cause colony morphology alterations thus potentially transferring pEtN to the BcsA synthesized glucan chain. With BcsG with shortest linkers not associated with a cellulose biosynthesis gene cluster, multiple hypotheses can be proposed that select for different length of the N-linker in BcsG homologs located within a cellulose biosynthesis gene cluster in other Pseudomonadati.

Adherent Invasive Escherichia coli (AIEC) colonize the ileal mucosa of Crohn's disease patients. Among bacterial virulence factors potentially implicated, type 6 secretion systems (T6SS) are molecular machineries sending effectors in prokaryotic or eukaryotic target cells. Two T6SS have been reported in AIEC LF82 reference strain. Here, we show that T6SS genes are more prevalent in Crohn's disease patients' microbiota compared to healthy controls, and that T6SS are more frequent in AIEC pathovar compared to E. coli in general and associated with FimH pathoadaptive polymorphism. The use of an in vitro gastrointestinal system coupled with T6SS promoter reporter assay highlighted expression of LF82 T6SS under gastrointestinal conditions, with a major role for bile salts. In mice infected with AIEC LF82 versus T6SS-deleted mutants, T6SS showed a functional impact in modulating the composition of mucosa-associated gut microbiota. Thus, AIEC may use their T6SS to compete with endogenous microbiota to colonize host mucosa.

Pectobacterium comprises Gram-negative, pectinolytic plant pathogens that cause soft rot and blackleg diseases on numerous economically important crops. During sampling in 2018 in taro (Colocasia esculenta) fields in Hawaii, two bacterial strains PL152T and PL155 were isolated from taro. Peliminary molecular characterization placed both strains in the genus Pectobacterium. However, their taxanomic postions could not be resoved at the species level. A polyphasic taxonomic approach incorporating phenotypic characterization, 16S rRNA gene sequence analysis, multilocus sequence analysis (MLSA), whole genome based phylogeny, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH) was therefore employed to determine their taxonomic status. Phylogenetic and phylogenomic analyses consistently placed PL152 and PL155 together in a distinct and well supported lineage within the genus Pectobacterium, separate from currently recognized species. The two strains exhibited high genomic relatedness to each other, whereas the average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values, were lower than the accepted species delineation thresholds of 95% and 70%, respectively, supported the delineation of a novel bacterial species. The genomic characteristic of PL152 included a genome size of 5.02 Mb and a DNA G+C content of 52%. Collectively, based on a polyphasic approach including phylogenetic, phlyogenomic, biochemical, and phenotypic evidences supports the classification of strains PL152 and PL155 as representatives of a novel species within the genus Pectobacterium, for which the name Pectobacterium pacificum sp. nov. is proposed. Strain PL152 (ICMP 2436274, LMG 32536) is designated as the type strain.

Background Fungi in plant root systems are key to understanding the dynamics and functioning of terrestrial ecosystems. Whereas our knowledge of below-ground plant-fungus symbioses has derived mainly from studies of mycorrhizal and pathogenic fungi, recent studies have uncovered an unexpected diversity of ecological niches occupied by root-associated fungi. The fungal order Helotiales is conspicuous in this respect because this predominantly root-associated taxon encompasses a broad ecological spectrum, ranging from saprotrophic and pathogenic fungi to endophytic and mycorrhizal fungi with plant growth-promoting functions. In this study, we integrated a worldwide dataset of 1,996 Helotiales Species Hypotheses (i.e., species-level taxa tentatively defined based on DNA sequences) detected in roots of 362 plant genera representing 117 families and 47 orders, and systematically quantified the specificity of Helotiales-plant associations. Results The analysis of 37,688 unique occurrence records of Helotiales fungi across 1,940 global sampling locations revealed highly structured associations between Helotiales genera and plant lineages, exemplified by the strong specificity of Hyaloscypha, Oidiodendron, and Phialocephala for Pinales and that of Glutinomyces for Fagales, even after statistically controlling background biogeographic patterns. At the Species-Hypothesis-level analysis, major clades within the Helotiales phylogeny consistently exhibited high specificity for Pinaceae, whereas highly specific associations with other ectomycorrhizal, arbuscular mycorrhizal, and nonmycorrhizal plants were restricted to small terminal clades of the fungal phylogeny. Conclusions The integration of ecological specificity and phylogenetic analyses suggests the possibility that Helotiales fungi established close associations with Pinaceae early in their evolutionary history and subsequently underwent host shifts to diverse angiosperm lineages. Given the emerging evidence that Helotiales fungi play pivotal ecological and physiological roles in plant root systems, these statistical insights into Helotiales-plant associations provide a fundamental basis for understanding the evolutionary history and functioning of terrestrial ecosystems.

Abstract The Bacillus cereus group is a complex of closely related species, which vary in terms of their geographic spread, industrial utility, and pathogenic potential. In addition to encompassing well-known species represented by thousands of genomes (e.g., bioterrorism agent B. anthracis, foodborne pathogen B. cereus, biopesticide B. thuringiensis), the B. cereus group houses "rare" species discovered/first sequenced over a decade ago. Until recently, four rare species (B. manliponensis, B. bingmayongensis, B. gaemokensis, "B. clarus") were each represented by a single unique genome; as such, virtually nothing is known about these species beyond their type strains. We hypothesized that additional representatives of rare B. cereus group species may already be present in public databases as yet-unassembled metagenomes. To this end, we used species-specific markers to identify 7 metagenomic samples in NCBI's Sequence Read Archive, which harbored B. manliponensis, B. bingmayongensis, or B. gaemokensis. Metagenomic assembly and binning yielded an isolate-quality B. manliponensis metagenome-assembled genome (MAG) from avocado orchard soil in Israel, plus a partial B. bingmayongensis MAG from commercial spinach spiked with Shiga toxin-producing Escherichia coli. For rare species represented by >1 unique genome, we compared our novel MAGs to all available genomes and identified strain- and lineage-specific genomic elements (e.g., sphingomyelinase C and hemolysin BL homologs, variably present in B. manliponensis and B. bingmayongensis, respectively). Altogether, our study indicates that some rare B. cereus group species may be more common and geographically widespread than previously thought, and highlights the utility of culture-independent methods for recovering rare/under-isolated taxa. Impact Statement Individual B. cereus group strains vary in terms of their pathogenic potential and industrial utility. While some strains cause illness in humans and/or animals (e.g., anthrax, emetic intoxication, diarrheal toxicoinfection, non-gastrointestinal illnesses), other strains have important biotechnological applications (e.g., biocontrol, plant growth promotion, production of antimicrobials and other natural products). Because many virulence- and biotechnological-related phenotypes are strain-dependent (rather than species-dependent), phenotypes associated with an individual strain (e.g., a species type strain) cannot be extrapolated to an entire B. cereus group species. Within this context, rare B. cereus group species represent a major gap in our understanding of B. cereus group evolution, ecology, and pathogenicity, as until 2021, each rare species was represented solely by its type strain genome. Here, we aggregate all publicly available genomes/MAGs of rare B. cereus group species and supplement them with their first novel representatives since 2024. Our efforts bring the total number of unique B. manliponensis and B. bingmayongensis genomes/MAGs to >2, allowing us to conduct the first-ever phylogeographic survey and strain-level assessment of functional potential for each species. Overall, our study represents a critical first step toward understanding rare B. cereus group species beyond their type strains.

Protein-protein interactions (PPIs) influence critical biological processes in pathogenic microorganisms, such as the human fungal pathogen, Cryptococcus neoformans. Fungal thermotolerance and stress response pathways are key virulence determinants that directly impact pathogen adaptation and survival and the infection process. To establish a comprehensive baseline of PPIs in C. neoformans and explore these interactions to infer functional roles for uncharacterized proteins, we applied size exclusion chromatography coupled with mass spectrometry to the secreted and cellular proteomes of the fungi. As a result, 216 and 1699 unique proteins were identified across 24 secretome and proteome fractions, respectively. The predicted secretome networks included expected proteins associated with vesicles and virulence, indicating a role in extracellular defense. Whereas the cryptococcal proteome highlighted interactions among proteins with defined roles in fungal virulence for protein stability and thermotolerance, including two previously uncharacterized proteins, CNAG_00287 and CNAG_05199, putatively involved in complex formation with heat-shock proteins (HSP). Based on sequence and structure homology, we propose that CNAG_00287 is a tetratricopeptide repeat-containing co-chaperone that modulates Hsp 70 activity and CNAG_05199 functions as a Hsp70. We validated the thermotolerance role of CNAG_00287 in heat-related stress, as its absence significantly impaired fungal growth in nutrient-limited media at 37 {degrees}C. Together, this work resolves virulence-associated PPIs within C. neoformans and reveals new molecular regulators of thermotolerance that underpin fungal pathogenicity.

Serine protease inhibitors (serpins) are extremely rare in prokaryotes, and their role in immune evasion remains unclear. We identified a bifunctional TLR2-binding serpin (miropin) produced by Tannerella forsythia that broadly suppresses neutrophil antimicrobial capacity. Miropin binding to TLR2 primed neutrophils, enhancing phagocytic capacity yet, counterintuitively, promoting bacterial persistence. Mechanistically, like other serpins, miropin acted as an irreversible suicide inhibitor of neutrophil serine proteases (NSPs), blocking their activity upon granule mobilization and NETosis. Unexpectedly, miropin, independent of its protease-inhibitory activity, bound and inhibited myeloperoxidase (MPO), thereby blocking the oxidative burst. This simultaneous suppression of oxidative and non-oxidative bactericidal mechanisms promoted bacterial survival in vivo without significantly altering the tissue inflammatory response. Thus, we identified novel moonlighting roles (TLR2 signaling and MPO inhibition) for an unorthodox prokaryotic serpin and provided context for how T. forsythia and other oral pathogens survive within the subgingival niche, an environment highly enriched in activated neutrophils.

Jeongeupia sacculi (HS-3) is a novel {beta}-proteobacterium with a unique multicellular life cycle. The genetic tractability and mechanisms underlying differentiation are unknown. Here, we adapt a method of homologous recombination for genetic modification in HS-3 and generate a disruption mutant of rpoS, which encodes the alternative sigma factor{sigma} S commonly associated with general stress response or major behavioral switches. We then characterize phenotypes associated with rpoS disruption. Under low-nutrient conditions, rpoS disruption reduced Congo red binding, consistent with rpoS acting as a regulator of low-nutrient response by upregulating production of biofilm-associated materials. Surprisingly, rpoS disruption did not significantly alter survival in response to other stressors in general stress response assays tested. Our work establishes a method for genetic manipulation in HS-3 and enables future investigation into the life cycle of HS-3, including whether rpoS has a role in differentiation triggered by low-nutrient response.

Microbial metabolism in extreme environments has long been shrouded in profound mystery. Distinct from the direct assimilation of exogenous organic carbon, microorganisms predominantly exploit inorganic nitrogen substrates as their primary nitrogen sources. As a critical nitrogen assimilation pathway, assimilatory nitrite reduction plays a vital role in microbial nitrogen acquisition, especially in extreme environments with poor nutrient availability. In this study, we identified the coexistence of two isoforms of NADH-dependent nitrite reductase in single genomes. NIR- and NIR-{beta}, named based on their phylogenetic relationships, exhibit significant differences in protein sequence, genomic context and evolutionary relationship, a phenomenon that has rarely been reported before. They also show distinct expression patterns, which may allow bacteria to achieve efficient nitrogen conversion in nutrient-poor environments. This finding calls for a revision of the conventional model of nitrogen cycling gene combinations inferred from common model microorganisms and holds numerous implications for how we understand and study the environmental adaptation mechanisms of extremophiles.

The opportunistic pathogen Pseudomonas aeruginosa can store carbon by polymerizing (R)-3-hydroxy fatty acids into polyhydroxyalkanoates (PHA) and forming large intracellular carbonosomes. These structures primarily function as stores of excess carbon that can be employed during nutrient imbalances, though they have been increasingly reported to have secondary functions in stress responses. A range of Proteobacteria can form carbonosomes, and they have been well characterised in strains used in bioplastic production and wastewater treatment. However, the occurrence, regulation and functions of carbonosomes in pathogenic bacteria remain poorly understood. Using flow cytometry and fluorescence microscopy following staining of carbonosomes with BODIPY 493/503, we characterised carbonosome abundance and dynamics in growth-arrested P. aeruginosa starved of nitrogen but provided with excess carbon. We found that carbonosomes substantially impact the energetic and biosynthetic output of growth-arrested P. aeruginosa, affecting protein biosynthesis, flagellar motility, and ATP/NADPH pools. The production and use of carbonosomes was found to confer a strong fitness advantage to growth-arrested P. aeruginosa during fluctuating nutrient conditions. Small carbonosomes were observed during the lag phase of growth in rich media and the ability to produce these structures was conserved across several different laboratory and clinical strains, although with different dynamics and maximal amounts. For example, the Liverpool epidemic strain LESB58 showed very little BODIPY staining while the closely related LESB65 strain showed high levels of staining. Crucially, we found that growth-arrested cells depolymerize carbonosomes upon tobramycin exposure and that carbonosome depolymerization significantly increases P. aeruginosa tobramycin sensitivity. Together, our observations suggest that carbonosomes significantly impact the physiology, metabolism, and antibiotic susceptibility of P. aeruginosa.